CN114452407A

CN114452407A - Gene editing delivery system and preparation method and application thereof

Info

Publication number: CN114452407A
Application number: CN202210130435.2A
Authority: CN
Inventors: 李明强; 陶玉; 孔慧敏; 卓陈雅; 易可; 郑春雄; 钟庆国
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2022-05-10

Abstract

The application belongs to the technical field of medicines, and particularly relates to a gene editing and delivering system, and a preparation method and application thereof. The gene editing nano delivery system of the present application comprises: outer layer of membrane fusion liposome and inner core of calcium precipitation hybrid gene editing plasmid; the outer layer of the membrane fusion liposome is wrapped on the surface of the inner core of the calcium precipitation hybrid gene editing plasmid. The application provides a method for realizing high-efficiency delivery of a gene editing system in a cell through membrane fusion mediation, an endocytosis path can be directly bypassed, so that the risk of degradation of lysosomes after endosome embedding is avoided, high-efficiency CRISPR/Cas9 plasmid delivery is expected to be realized, and the technical problem that the current gene editing system is difficult to deliver into the cell is effectively solved; by modifying and modifying the nano-carrier and the CRISPR/Cas9 system, accurate gene editing and treatment of liver-related diseases such as hepatitis, liver cancer, liver failure, liver fibrosis and the like can be realized.

Description

Gene editing delivery system and preparation method and application thereof

Technical Field

The application belongs to the technical field of medicines, and particularly relates to a gene editing and delivering system, and a preparation method and application thereof.

Background

The CRISPR/Cas9 gene editing technology can realize precise fixed-point gene editing by editing a target gene sequence, and is widely used for resisting diseases closely related to human health, such as viruses, bacteria, cancers, hereditary diseases and the like. Currently, viral vectors are mainly used for gene editing, but the following limitations mainly include: (1) the load is low, and the CRISPR/Cas9 system (CRISPR/Cas9 plasmid-10000 bp) with larger capacity is difficult to load; (2) immunogenicity, inflammation, potential carcinogenicity; (3) low universality; (4) lack of organ/cell targeting specificity. The non-viral nano-vector can overcome the limitations of the viral vector and provides a vector platform with clinical application prospect. However, the non-viral vectors disclosed in the prior art are complex in preparation process and high in toxicity; intracellular delivery is mediated primarily through endocytic pathways, but the low efficiency of lysosomal escape makes gene editing systems susceptible to degradation, with only a small fraction of the loaded gene editing system being able to function, resulting in overall inefficiency.

Disclosure of Invention

In view of the above, the application provides a gene editing nano-delivery system mediated by membrane fusion, and a preparation method and an application thereof, which can realize direct membrane fusion to deliver the gene editing system into cells, bypass endocytosis, thereby avoiding lysosome degradation risk, are expected to realize efficient CRISPR/Cas9 plasmid delivery, and effectively solve the technical problem that the current gene editing system is difficult to deliver into cells; by modifying and modifying the nano-vector and the CRISPR/Cas9 system, accurate gene editing and treatment of liver cell related diseases in the liver are realized.

The present application provides in a first aspect a gene editing nano-delivery system mediated by membrane fusion, comprising:

outer layer of membrane fusion liposome and inner core of calcium precipitation hybrid gene editing plasmid; the outer layer of the membrane fusion liposome is wrapped on the surface of the calcium carbonate hybrid gene editing plasmid inner core;

the preparation method of the outer layer of the membrane-fused liposome comprises the following steps: preparing an outer layer of a membrane fusion liposome by a thin film hydration method by using phospholipid structure lipid, cationic functional lipid and polyethylene glycol (PEG) functionalized lipid;

the preparation method of the calcium precipitation hybrid gene editing plasmid kernel comprises the steps of mixing soluble calcium salt, soluble carbonate and/or soluble phosphate with the gene editing plasmid, coprecipitating and coagulating to form the calcium precipitation hybrid gene editing plasmid kernel; the gene editing system employs the CRISPR/Cas9 plasmid containing a liver-specific promoter.

In particular, the gene editing nano delivery system provided by the application can realize intracellular delivery of the gene editing system through membrane fusion.

Specifically, the phospholipid structure lipid at the outer layer of the membrane fusion liposome is a structural component, and the cationic functional lipid and the PEG functional lipid are functional components.

In another embodiment, the phospholipid structure lipid is selected from dimyristoylphosphatidylcholine DMPC or/and dioleoylphosphatidylethanolamine DOPE.

In another embodiment, the cationic functional lipid is selected from trimethyl-2, 3-dioleoyloxypropylammonium bromide DOTAP or/and trimethyl-2, 3-dioleoyloxypropylammonium chloride DOTMA.

In another embodiment, the PEG functionalized lipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG, distearoylphosphatidylethanolamine-polyethylene glycol-galactose DSPE-PEG-Gal, and distearoylphosphatidylethanolamine-polyethylene glycol-N-acetylgalactosamine DSPE-PEG-GalNac.

In the outer layer of the membrane-fused liposome, the molar ratio of the cationic functional lipid is 19-20%, the molar ratio of the PEG functional lipid is 3.8-5%, and the molar ratio of the phospholipid structure lipid is 75-76.2%.

Specifically, the outer layer of the membrane-fused liposome comprises dimyristoyl phosphatidylcholine DMPC, trimethyl-2, 3-dioleoyloxypropylammonium bromide DOTAP and distearoyl phosphatidylethanolamine-polyethylene glycol DSPE-PEG.

Specifically, in the membrane fusion liposome, the molar ratio of the DOTAP is 19-20%, the molar ratio of the DSPE-PEG is 3.8-5%, and the molar ratio of the DMPC phospholipid is 75-76.2%.

In another embodiment, the soluble calcium salt is selected from CaCl₂Or/and CaHPO₄The soluble carbonate is selected from Na₂CO₃Or/and NaHCO₃The soluble phosphate is selected from NaH₂PO₄Or/and Na₂HPO₄。

Specifically, the gene editing plasmid can be an existing editing system capable of accurately editing a target gene, and can also be an editing system capable of accurately editing a target gene in the future.

In another embodiment, the gene editing plasmid is selected from one or more of a CRISPR/Cas9 gene editing system, a CRISPR/dCas9 gene editing system, a CRISPR/Cas13 gene editing system, a CRISPR/Cpf1 gene editing system, a meganuclease gene editing system, a zinc finger nuclease ZFNs gene editing system, and a transcription activator-like effector nuclease TALEN gene editing system.

Preferably, the gene editing plasmid is in the form of a plasmid, mRNA or protein of the CRISPR/Cas9 gene editing system, and more preferably in the form of a plasmid of the CRISPR/Cas9 gene editing system.

Preferably, the CRISPR/Cas9 plasmid is an optimized targeting expression plasmid, and the promoter of the plasmid is modified into a chimeric promoter ApoE.HCR.hAAT with hepatocyte specificity, and the chimeric promoter is formed by combining an apolipoprotein gene enhancer ApoE.HCR and a human alpha 1-antitrypsin promoter.

Specifically, the gene editing plasmid can carry out site-directed editing on a target gene in a cell. The target gene may be a target gene causing a disease, such as a virus fragment, and the like, particularly a hepatotrophic virus: hepatitis B Virus (HBV).

In another embodiment, the outer layer of the membrane fusion liposome further comprises an organ targeting ligand or/and a tumor targeting ligand. In particular, the PEG-functionalized lipid may be linked to the organ targeting ligand or/and the tumor targeting ligand.

In another embodiment, the organ-targeting ligand is a liver-targeting ligand modification; the liver targeting ligand modifier is selected from one or more of galactose, N-acetylgalactosamine, lactose, asialofetuin and HBV pre-S1 peptide.

In another embodiment, the PEG functionalized lipid is selected from one or more of distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), distearoyl phosphatidyl ethanolamine-polyethylene glycol-galactose (DSPE-PEG-Gal), distearoyl phosphatidyl ethanolamine-polyethylene glycol-N-acetylgalactosamine (DSPE-PEG-GalNac).

In a second aspect, the present application provides a method for preparing a gene editing nano-delivery system based on membrane fusion, comprising:

step 1, dropwise adding a soluble carbonate mixed solution B into a soluble mixed solution A which is oscillated or stirred at a constant speed to obtain a calcium precipitation hybrid gene editing plasmid solution; wherein the soluble mixed solution A comprises a soluble calcium salt, a gene editing plasmid and a first solvent; the soluble carbonate/and soluble phosphate mixed solution B includes a soluble carbonate/and soluble phosphate and a first solvent.

Mixing phospholipid structure lipid, cation functional lipid, PEG functional lipid and a second solvent by a thin film hydration method, and volatilizing the second solvent to obtain a lipid thin film of the membrane fusion liposome;

and 2, adding the calcium precipitation hybrid gene editing plasmid solution into the membrane fusion liposome film, continuously oscillating until the lipid film is uniformly dispersed in the mixed solution, and passing the obtained solution through a filter membrane with gradient pore size to obtain the gene editing nano delivery system.

Specifically, the first solvent is water (which may be double distilled water, ultrapure water, purified water, etc.); the second solvent is organic solvent such as chloroform, dichloromethane and the like.

Specifically, phospholipid structure lipid, cationic functional lipid DOTAP and PEG functional lipid are mixed with a second solvent according to a preset molar ratio.

In another embodiment, the filter membrane comprises a filter membrane with a pore size of 800nm, a filter membrane with a pore size of 600nm, a filter membrane with a pore size of 400nm and a filter membrane with a pore size of 200 nm; the obtained solution sequentially passes through a filter membrane with the aperture of 800nm, a filter membrane with the aperture of 600nm, a filter membrane with the aperture of 400nm and a filter membrane with the aperture of 200nm according to the gradient sequence to obtain the membrane fusion gene editing nano-delivery system with uniform particle size distribution.

The gene editing nano delivery system provided by the third aspect of the application or the gene editing nano delivery system prepared by the preparation method is applied to delivering gene editing plasmids, mRNA or proteins in cells.

Specifically, the gene editing nano delivery system is controlled to play a corresponding role by controlling the editing target of the gene editing plasmid in the gene editing nano delivery system, for example, the gene editing plasmid for cutting and editing hepatitis virus is adopted, so that the gene editing nano delivery system for eliminating hepatitis virus is obtained.

In particular, in the application, the cell can be a somatic cell such as a hepatocyte.

Specifically, the gene editing nano delivery system provided by the application can be applied to gene therapy drugs, and gene editing plasmids are delivered to cells through a membrane fusion way, so that the gene editing plasmids can conveniently play a gene editing role in the cells, and a disease treatment effect is achieved.

In the field of nucleic acid delivery, the CRISPR/Cas9 gene editing system with a large molecular weight has the problems of difficult entrapment and low delivery efficiency. The application realizes that the calcium precipitation encapsulates the gene editing plasmid with larger molecular weight to form a solid core, and the liposome shell which can realize the membrane fusion effect is coated on the surface of the solid core. The liposome-calcium precipitation hybrid nanocomposite vector has the characteristics of cell membrane fusion and direct delivery of gene-editing plasmids with biological activity into cells, and can also modify specific organ targeting ligands or/and tumor targeting ligands (such as liver targeting ligands) on the membrane fusion liposome; in addition, the promoter of the CRISPR/Cas9 plasmid is a liver-specific promoter, and can realize accurate regulation and control on the targeting property and the spatial activity of a gene editing system.

In conclusion, the gene editing nano-delivery system mediated by the membrane fusion effect is a hybrid nano-complex carrier with a targeted cell membrane fusion liposome-calcium precipitation core-shell structure, which can realize efficient membrane fusion direct delivery, can bypass an endocytosis-lysosome approach, avoids the degradation risk in cells, and is used for delivering gene editing plasmids (such as CRISPR/Cas9 plasmids) with larger molecular weight to realize in vitro and in vivo gene editing. The gene editing nano delivery system of the present application has the following features: (1) kernel: calcium carbonate/and calcium phosphate of the calcium precipitation hybrid gene-editing plasmid can effectively compress and encapsulate the gene-editing plasmid; (2) a housing: the membrane fusion liposome component directly enters cells in a membrane fusion mode, so that the risk that endocytosis is degraded after entering lysosomes is avoided; (3) surface modification can be carried out on the outer shell of the membrane fusion liposome (such as modification of a liver targeting ligand); the promoter of the CRISPR/Cas9 plasmid can be transformed into an ApoE.HCR.hAAT promoter with specificity of liver cells, so that accurate gene editing with specificity of liver is realized.

The gene editing effect of the gene editing plasmid loaded nano composite carrier is realized in different cell and mouse animal models, the gene of a target virus can be knocked out, and viral infection diseases (such as HBV hepatitis B virus) are expected to be completely cured. In addition, the liposome material with membrane fusion property and targeted modification endows the carrier system with the properties of efficient delivery and specific targeting; the CRISPR/Cas9 plasmid with hepatocyte-specific promoter drive confers the property of specific expression. In summary, the present application provides strong evidence that the membrane-fused liposome-calcium precipitated "core-shell" structured hybrid nanocomposite vector of the present application is a highly efficient gene editing system delivery tool.

Drawings

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

Fig. 1 is a schematic structural diagram of a gene editing nano-delivery system (galactose modified membrane-fused calcium carbonate nanoparticle @ CRISPR/Cas9 plasmid) provided in an embodiment of the present application and a pathway of membrane-fusion delivering the gene editing plasmid; the gene editing and delivering system realizes targeting effect by combining galactose components in the DSPE-PEG-Gal with a sialoglycoprotein receptor on the surface of a hepatocyte, membrane fusion liposome in the gene editing and delivering system is fused with the cell membrane of the hepatocyte, and calcium precipitation hybridization gene editing plasmid in the gene editing and delivering system is delivered into the hepatocyte;

FIG. 2 is an in vivo schematic view of a gene-editing nano-delivery system for HBV gene-editing therapy provided by an embodiment of the present application; the constructed gene editing nano delivery system is injected into the body from tail vein in a disease model of mouse HBV virus replication, and the system realizes liver target delivery by the action of galactose component in DSPE-PEG-Gal and sialic acid glycoprotein receptor on the surface of liver cells; after reaching the target cell, the membrane fusion effect of the system can directly release the cargo with gene editing activity, and an endocytosis path is bypassed, so that the risk of lysosome degradation is avoided; meanwhile, the system further realizes the expression of the liver-specific CRISPR/Cas9 through a liver-specific promoter, and effectively edits two targets (gRNA1-P and gRNA-XP) of the HBV virus to achieve the treatment effect;

FIG. 3 is a study of the optimization of the lipid component ratio for membrane fusion of DMPC/DOTAP/DSPE-PEG liposomes provided in the examples of the present application. Marking a liposome layer by using a DiD dye, marking cell nucleus by using DAPI, observing fluorescence co-localization taken by cells under different DOTAP proportions by using a fluorescence microscope, verifying the optimal proportion of membrane fusion, and screening a proper outer layer of the membrane fusion liposome;

fig. 4 is data of particle size (fig. 4A), potential (fig. 4B) and loading efficiency (fig. 4C) of membrane fusion gene nano-delivery systems loaded with different amounts of EGFP plasmid p1 (reporter plasmid) provided in the examples of the present application;

FIG. 5 is an electron microscope image (FIG. 5B) showing the cell transfection efficiency (FIG. 5A) of membrane fusion gene nano-delivery system loaded with different amounts of EGFP plasmid p1 and the corresponding optimized screened system provided in the examples of the present application, wherein the A image is calculated by the expression intensity of EGFP on p1 plasmid in cells, and a commercial transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific), abbreviated as Lipo, is used as a positive control. The B picture is the CaCO of the inner core after the optimized load (liposome to plasmid mass ratio 25/1)₃Electron micrographs corresponding to/p 1 and MFC/p1 after encapsulation of the membrane-fused liposome shell;

fig. 6 is a verification of an endocytosis inhibition experiment of the membrane fusion gene nano-delivery system loaded with the EGFP plasmid p1 provided in the present application embodiment, where the MFC nano-delivery system is labeled with a DiD dye, a is a flow experiment result taken up by cells after being treated with different endocytosis inhibitors, and B is a statistical mean fluorescence intensity MFI numerical result after repeated experiments;

FIG. 7 is an experimental verification of fluorescence co-localization during the cell uptake process of EGFP plasmid p1 loaded membrane fusion gene nano delivery system provided in the present application, which determines the co-localization of the nano delivery system with the nucleus and lysosome during the cell uptake process, wherein A is a confocal photograph, B is co-localization analysis of DAPI blue and YOYO-1 green fluorescence, and C is co-localization analysis of LysoTracker red and YOYO-1 green fluorescence;

FIG. 8 is a test of transfection efficiency of CRISPR/Cas9 plasmid p2 containing liver-specific promoter in different cells provided in the examples of the present application, wherein A is a ratio change of transfection efficiency of plasmid p2 containing liver-cell specific promoter and pX333 plasmid in different cells (HeLa and 293T are non-liver cell lines, HepG2 is a liver cell line), and B is a fluorescence microscope picture of plasmid transfection of p2 and pX333 in HeLa and 293T, HepG2 cells;

fig. 9 is a representation of cell transfection efficiency of different gene editing nano delivery systems loaded with CRISPR/Cas9 plasmid p2 provided in the present embodiment, where a and B are fluorescence intensity of EGFP green fluorescent protein expressed by the intracellular gene editing plasmid and corresponding laser confocal microscopy images after HepG2 liver cancer cells are transfected by the different gene editing nano delivery systems, respectively;

fig. 10 shows the design and sequencing verification of grnas targeting HBV provided in the examples of the present application. A and B are sequence design and sequencing-by-synthesis data of gRNA1-P and gRNA2-XP respectively;

fig. 11 is an in vitro test of anti-HBV hepatitis virus therapeutic effect and therapeutic gene editing of different gene editing nano-delivery systems loaded with CRISPR/Cas9 plasmid p3 targeting HBV provided in the examples of the present application. A is T7E1 mutation cutting detection of HBV gene editing in vitro by delivering the plasmid p3 through MFC, GNMFC and GMFC, B is the gene sequencing result of con control and GMFC @ p3, and C is an in vitro index for analyzing HBV cccDNA (reflecting virus replication level) after being delivered through the plasmid p3 through MFC, GNMFC and GMFC by qPCR detection;

fig. 12 is a graph showing in vivo efficacy tracking evaluation of anti-HBV hepatitis virus of different gene editing nano delivery systems loaded with CRISPR/Cas9 plasmid p3 of targeted HBV provided in this application, establishing a mouse HBV replication model, injecting different gene editing systems into a mouse for treatment by tail vein injection, performing blood sampling tracking detection on mice in PBS control group and treatment group delivered with plasmid p3 by MFC, GNMFC and GMFC on

days

1, 2,3, 5 and 7 of treatment, where a is serum HBsAg level monitoring, B is serum HBeAg level monitoring, and C is serum HBVDNA level monitoring;

fig. 13 is an in vivo test of short-term and long-term anti-HBV hepatitis virus efficacy of different gene editing nano delivery systems loaded with CRISPR/Cas9 plasmid p3 of targeted HBV provided in the present application embodiment, a mouse HBV replication model is established, and different gene editing systems are injected into a mouse body for treatment by tail vein injection. A to D are HBsAg, HBeAg, HBVDNA and HBV RNA indexes in liver in vivo after plasmid p3 is delivered and treated by MFC, GNMFC and GMFC, respectively, and E is HBsAg immunofluorescence observation of liver slices after 2 days and 7 days of delivery and treatment of plasmid p3 by MFC, GNMFC and GMFC;

fig. 14 is a verification of in vivo gene editing results of anti-HBV hepatitis virus of different gene editing nano delivery systems of CRISPR/Cas9 plasmid p3 carrying targeted HBV provided in this application embodiment, where the plasmid p3 is subjected to gene extraction through the liver obtained from an HBV replication mouse model after MFC, GNMFC and GMFC delivery treatment, a is PCR sequencing of a target sequence of gene editing, B is T7E1 mutation cleavage detection, and C is TIDE-related Indel analysis;

fig. 15 is an in vivo compatibility evaluation test of gene editing nano delivery system of different load-targeted HBV CRISPR/Cas9 plasmid p3 provided in the examples of the present application. After 7 days of treatment, a is H & E staining of organs (heart, liver, spleen, lung, kidney) after treatment with plasmid p3 delivered by MFC, GNMFC and GMFC, B is liver and kidney function index (ALT, AST, CREA, UREA levels of serum).

Detailed Description

The application provides a gene editing nano delivery system mediated by membrane fusion, a preparation method and application thereof, which are used for overcoming the technical defect that the gene editing system is difficult to deliver into cells in the prior art.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The raw materials and reagents used in the following examples were commercially available or self-made.

The plasmids used in the following examples include EGFP plasmid p1, CRISPR/Cas9 plasmids p2 and p3 are p2 plasmid which is based on commercial pX333 modified gene and apoe.hcr.haat hepatocyte specific promoter, and p3 plasmid which introduces two targeted HBV grnas on the basis of p 2.

Example 1

The embodiment of the application provides a liposome capable of realizing the membrane fusion effect, and the liposome can be used as a shell component to form a gene nano delivery system mediated by membrane fusion. The membrane fusion liposome comprises cationic liposome DOTAP, DMPC and DSPE-PEG, and is formed into the liposome by a thin film hydration method after being mixed according to the proportion, and the specific preparation method comprises the following steps:

(1) preparing a membrane fusion liposome film by adopting a film hydration method: DSPE-PEG, DOTAP and DMPC dissolved in chloroform were mixed in a round bottom flask in molar ratios of 3.8:0:96.2 (0% DOTAP), 3.8:10:86.2 (10% DOTAP), 3.8:20:76.2 (20% DOTAP), 3.8:30:66.2 (30% DOTAP), 3.8:40:56.2 (40% DOTAP), and the chloroform was evaporated to form a lipid film at the bottom of the flask, labeled 0% DOTAP, 10% DOTAP, 20% DOTAP, 30% DOTAP and 40% DOTAP.

(2) Adding pure ddH₂Adding O into a round-bottom flask for forming a lipid film, shaking and uniformly mixing, and sequentially passing through filter membranes with the aperture of 800nm, 600nm, 400nm and 200nm according to a gradient sequence to finally prepare the liposome containing different proportions of DOTAP.

The prepared liposome is marked by DiD fluorescent dye, a cell uptake experiment is carried out, and the running-in condition of the liposome and a cell membrane is observed by a fluorescence microscope, and the result is shown in figure 3, and the membrane fusion effect can be realized by optimally screening the liposome under the condition of 20 percent of DOTAP proportion.

Example 2

The embodiment of the application provides a first gene nano delivery system mediated by membrane fusion, which structurally comprises a membrane fusion liposome shell and a calcium carbonate precipitation hybrid gene plasmid kernel, wherein the membrane fusion liposome shell is wrapped on the surface of the calcium precipitation hybrid gene editing plasmid kernel; the outer layer of the membrane fusion liposome comprises cationic liposome DOTAP, DMPC and DSPE-PEG; the preparation method of calcium carbonate hybrid gene editing plasmid comprises mixing Ca²⁺、CO₃ ^2-Mixing with EGFP plasmid (reporter plasmid), co-precipitating, and coagulating to form calcium carbonate hybrid plasmid kernel; the specific preparation method of the gene nano delivery system comprises the following steps:

(1) 16 mu LCaCl₂(0.5M) solution, 2. mu.g of EGFP plasmid p1 with ddH₂O is mixed to form 50 mu LA solution, and B solution is mixed with 16 mu LNa₂CO₃(0.01M) solutionLiquid and ddH₂Mixed and diluted with O to form (50. mu.L); dropwise adding the solution B into the solution A, and standing the obtained mixed solution C at room temperature for 15 min.

(2) Preparing a membrane fusion liposome film by adopting a film hydration method: DSPE-PEG, DOTAP and DMPC dissolved in chloroform were mixed in a round bottom flask in a molar ratio of 3.8:20:76.2, and chloroform was evaporated to form a lipid film on the bottom of the flask.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film, shaking and uniformly mixing, and sequentially passing through filter membranes with the aperture of 800nm, 600nm, 400nm and 200nm according to a gradient sequence to finally prepare a gene delivery system MFC @ p 1; wherein, the mass ratio of the membrane fusion liposome (the total mass of DSPE-PEG, DOTAP and DMPC) to the loaded EGFP plasmid in the gene nano delivery system is controlled to be 5:1, 7.5:1, 12.5:1, 17.5:1, 25:1, 32.5:1, 40:1 and 50:1 (marked as MFC @ p1 w/w ═ lipid/p1 in figure 4) by controlling the addition amount of the C liquid.

MFC @ p1 prepared in the above-mentioned manner and having different membrane fusion liposome/plasmid weight ratios was examined using a Zeta potentiometer and a NanoDrop microspectrophotometer, and the results are shown in FIG. 4, CaCO in FIG. 4₃@ p is CaCO carrying EGFP plasmid p1 prepared in step (1)₃The results show that the particle size of MFC @ p1 is below 200nm, the potential increases with the mass ratio of membrane fusion liposome/plasmid, and the plasmid loading efficiency can reach above 80%.

When the gene nano-delivery system of example 2 was used in cell transfection experiments, p1 plasmid could express EGFP green fluorescent gene, FIG. 5A shows the result of HepG2 hepatocyte transfection with the gene nano-delivery system of example 2, CaCO₃@ p1 is CaCO carrying the plasmid prepared in step 1 of example 2₃The transfection results of (1), con is a blank control, Lipo @ p1 is the transfection result of a commercial transfection reagent-loaded plasmid p1, MFC @ p1 is the transfection result of a gene nano-delivery system with membrane-fused liposomes (DSPE-PEG, DOTAP and DMPC total mass) and EGFP plasmid p1 in a mass ratio of 5:1, 7.5:1, 12.5:1, 17.5:1, 25:1, 32.5:1, 40:1, 50:1 (labeled MFC @ p1 w/w ═ lipid/p1 in fig. 5), preferably with the best transfection efficiency ratio (MFC @ p1 w/w ═ 25).

The selected nano delivery system optimized by the cell transfection experiment in example 2 was observed by an electron microscope, and FIG. 5B shows the inner core CaCO of the gene nano delivery system in example 2₃Electron micrographs corresponding to/p 1 and MFC/p1 after encapsulation of the membrane-fused liposome shell.

Example 3

The embodiment of the application provides a second gene nano delivery system mediated by membrane fusion, which structurally comprises a membrane fusion liposome with the structural lipid of DOPE and a calcium carbonate precipitation hybrid gene plasmid, wherein the membrane fusion liposome is wrapped on the surface of the calcium precipitation hybrid gene editing plasmid; the outer layer of the membrane fusion liposome comprises cationic liposome DOTAP, DOTAP and DSPE-PEG; the preparation method of calcium carbonate hybrid gene editing plasmid comprises mixing Ca²⁺、CO₃ ^2-Mixing with EGFP plasmid (reporter plasmid), co-precipitating, and coagulating to form inner core calcium carbonate hybrid plasmid inner core; the specific preparation method of the gene nano delivery system comprises the following steps:

(1) 16 mu LCaCl₂(0.5M) solution, 2. mu.g of EGFP plasmid p1 with ddH₂Mixing O to obtain 50 μ LA solution and B solution containing 16 μ LNa₂CO₃(0.01M) solution with ddH₂Mixed and diluted with O to form (50. mu.L); dropwise adding the solution B into the solution A, and standing the obtained mixed solution C at room temperature for 15 min.

(2) Preparing a membrane fusion liposome film by adopting a film hydration method: DSPE-PEG, DOTAP and DOPE dissolved in chloroform were mixed in a round bottom flask in a molar ratio of 3.8:20:76.2, and chloroform was volatilized to form a lipid film at the bottom of the flask.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film, shaking and uniformly mixing, and sequentially passing through filter membranes with the aperture of 800nm, 600nm, 400nm and 200nm according to a gradient sequence to finally prepare a gene delivery system MFC @ p 1; wherein, the mass ratio of the membrane fusion liposome (DSPE-PEG, DOTAP and DOPE total mass) to the supported EGFP plasmid in the gene nano delivery system is controlled to be 25:1 by controlling the addition amount of the C liquid, and MFC (DOPE/DOTAP/DSPE-PEG/CaCO) is prepared₃)@p1。

The above-mentioned products were measured using a nanometer particle size and Zeta potentiometer, NanoDrop microspectrophotometerThe obtained MFC (DOPE/DOTAP/DSPE-PEG/CaCO)₃) @ p1, the particle size measured was about 205nm, the potential was about +8.2mV, and the plasmid loading efficiency was about 70%.

Example 4

The embodiment of the application provides a third gene delivery system mediated by membrane fusion, which structurally comprises DMPC membrane fusion liposome and carbonate/calcium phosphate precipitation hybrid gene plasmid as structural lipids, wherein the membrane fusion liposome is wrapped on the surface of the calcium precipitation hybrid gene editing plasmid; the outer layer of the membrane fusion liposome comprises cationic liposome DOTAP, DOTAP and DSPE-PEG; the preparation method of the carbonic acid/calcium phosphate hybrid gene editing plasmid comprises the following steps of mixing Ca²⁺、CO₃ ^2-、PO₄ ^3-Mixing with EGFP plasmid (reporter plasmid), co-precipitating and coagulating to form inner core calcium carbonate hybrid plasmid; the specific preparation method of the gene nano delivery system comprises the following steps:

(1) 16 mu LCaCl₂(0.5M) solution, 2. mu.g EGFP plasmid p1 with ddH₂O is mixed to form 50 mu LA solution, and B solution is mixed with 15 mu LNa₂CO₃(0.01M) solution, 1. mu. LNa₂HPO₄(0.01M) solution with ddH₂Mixed and diluted with O to form (50. mu.L); dropwise adding the solution B into the solution A, and standing the obtained mixed solution C at room temperature for 15 min.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film, shaking and uniformly mixing, and sequentially passing through filter membranes with the aperture of 800nm, 600nm, 400nm and 200nm according to a gradient sequence to finally prepare a gene delivery system MFC @ p 1; wherein the mass ratio of the membrane fusion liposome (DSPE-PEG, DOTAP and DMPC total mass) to the loaded EGFP plasmid in the gene nano delivery system is controlled to be 25:1 by controlling the addition amount of the C liquid, so as to prepare the MFCP @ p 1.

And (3) detecting the mass ratio of the prepared membrane fusion liposome to the plasmid by using a nano-particle size, Zeta potentiometer and a NanoDrop micro spectrophotometer to be 25:1 MFCP @ p1, the particle size was found to be about 220nm, the potential was about +9.4mV, and the plasmid loading efficiency was 89.5%.

Example 5

The present application provides performance testing of the intracellular delivery pathway of example 2, specifically including:

1. verification of endocytosis-independent membrane fusion pathway of gene editing nano-delivery system mediated by membrane fusion:

as shown in fig. 6A and B, the cellular uptake and gene transfection effects of different endocytosis inhibitors (amiloride, chlorpromazine, nystatin) on plasmid-loaded MFC @ p1 of example 1 were analyzed using flow cytometry. The results show that the delivery of MFC @ p1 is substantially unaffected by inhibition of the endocytic pathway, which is an energy-dependent membrane fusion delivery modality. At 37 ℃, the intracellular uptake efficiency and transfection efficiency of the membrane fusion mediated gene editing nano-delivery system of the present application were not affected by endocytosis inhibitors, but only by low temperature (4 ℃).

2. Comparative experiments were followed for intracellular delivery pathways of gene nano-delivery system (MFC @ p1) and non-membrane fusion delivery system (NMFC @ p1, DOTAP ratio 0) mediated by membrane fusion:

observation using a laser confocal microscope: (1) the location of the DiD-labeled membrane-fused/non-membrane-fused liposome-calcium precipitation hybrid complex vector and YOYO-1-labeled CRISPR/Cas9 plasmid in the cell and their changes over time (DAPI-labeled nuclei); (2) the plasmid (YOYO-1 marker) co-localizes with lysosomes (Lysotracker Red marker) and nuclei (DAPI marker) during delivery. The results are shown in the confocal laser microscopy image of FIG. 7A, the co-localization analysis of DAPI blue and YOYO-1 green fluorescence in FIG. 7B, and the co-localization analysis of LysoTracker red and YOYO-1 green fluorescence in FIG. 7C, showing that MFC @ p1 of example 2 can deliver plasmid by membrane fusion, allowing the plasmid to bypass the endocytosis of lysosomes and enter the nucleus; in contrast, NMFC @ p1 showed the same endocytic delivery pattern as most non-viral vectors, with poor efficiency of entry into the nucleus.

Example 6

The embodiments of the present application provide engineered CRISPR/Cas9 plasmids with liver-specific promoters. The method specifically comprises the following steps:

(1) the EGFP gene fragment was inserted into a commercial CRISPR/Cas plasmid pX333, so that the plasmid can express EGFP green fluorescence for convenient observation and comparison of transfection effects.

(2) Plasmid p2 was prepared by substituting apoe.hcr.haat for the original plasmid promoter, which has hepatocyte specificity.

The modified p2 plasmid of this example was used for transfection experiments of hepatic and non-hepatic cells. As shown in FIG. 8, p2 was transfected more efficiently in the hepatocyte cell line than pX333, whereas the transfection efficiency in the non-hepatocyte cell line was reduced, and specifically enhanced expression in the hepatocyte was achieved.

Example 7

The embodiment of the application provides a liver-specific membrane fusion-mediated gene editing nano delivery system MFC @ p2, and the specific preparation method comprises the following steps:

(1) 16 mu LCaCl₂(0.5M) solution, 2. mu.g of CRISPR/Cas9 plasmid p2 and ddH prepared in example 6₂O is mixed to form 50 mu LA solution, and B solution is mixed with 16 mu LNa₂CO₃(0.01M) solution with ddH₂Mixed and diluted with O to form (50. mu.L); dropwise adding the solution B into the solution A, and standing the obtained mixed solution C at room temperature for 15 min.

(2) DSPE-PEG, DOTAP and DMPC dissolved in chloroform were mixed in a round bottom flask in a molar ratio of 3.8:20:76.2, and chloroform was evaporated to form a lipid film on the bottom of the flask.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film by a film hydration method, shaking and uniformly mixing, controlling the mass ratio of a membrane fusion liposome (DSPE-PEG, DOTAP and DMPC total mass) to a loaded CRISPR/Cas9 plasmid p2 in a gene editing delivery system to be 25:1 by controlling the addition amount of the solution C, and sequentially passing through filter membranes with the pore diameters of 800nm, 600nm, 400nm and 200nm to finally prepare the gene editing nano delivery system MFC @ p 2.

Example 8

The embodiment of the application provides a dual liver specificity membrane fusion mediated gene editing delivery system GMFC @ p2, and the specific preparation method comprises the following steps:

(2) DSPE-PEG-Gal (galactose ligand on DSPE-PEG-Gal has specific targeting effect on liver cells), DOTAP and DMPC dissolved in chloroform were mixed in a round-bottomed flask in a molar ratio of 3.8:20:76.2, and chloroform was volatilized to form a lipid film on the bottom of the flask.

(3) Adding the obtained solution C into a round-bottomed flask for forming a lipid film by a film hydration method, shaking and uniformly mixing, controlling the mass ratio of a membrane fusion liposome (DSPE-PEG, DOTAP and DMPC total mass) to a loaded CRISPR/Cas9 plasmid p2 in a gene editing delivery system to be 25:1 by controlling the addition amount of the solution C, and sequentially passing through filter membranes with the pore diameters of 800nm, 600nm, 400nm and 200nm to finally prepare the dual liver specificity membrane fusion mediated gene editing nano delivery system GMFC @ p 2.

The gene editing nano delivery system loaded with the CRISPR/Cas9 plasmid p2 of example 8 was subjected to physicochemical property detection including electron microscopy analysis and particle size distribution analysis, and as a result, the particle size distribution of the system was uniform, about 200 nm.

Example 9

The embodiment of the application provides CRISPR/Cas9gRNA design of highly conserved sequences (P region and XP region) of targeted HBV viruses, and two gRNAs are introduced into the plasmid P2 constructed in the embodiment 6 to prepare the CRISPR/Cas9 plasmid P3 of targeted HBV. Fig. 10 shows the designed sequences of the two grnas and the sequencing of the clones at the corresponding positions on the inserted plasmids.

Example 10

The embodiment of the application provides a liver specificity membrane fusion mediated gene editing nano delivery system MFC @ p3 targeting HBV, and the specific preparation method comprises the following steps:

(1) 16 mu LCaCl₂(0.5M) solution, 2. mu.g of the HBV-targeted CRISPR/Cas9 plasmid p3 and ddH prepared in example 9₂O is mixed to form 50 mu LA solution, and B solution is mixed with 16 mu LNa₂CO₃(0.01M) solution with ddH₂Mixed and diluted with O to form (50. mu.L); dropwise adding the solution B into the solution A, and standing the obtained mixed solution C at room temperature for 15 min.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film by a film hydration method, shaking and uniformly mixing, controlling the mass ratio of a membrane fusion liposome (DSPE-PEG, DOTAP and DMPC total mass) to the loaded CRISPR/Cas9 plasmid p3 in a gene editing delivery system to be 25:1 by controlling the addition amount of the solution C, and sequentially passing through filter membranes with the pore diameters of 800nm, 600nm, 400nm and 200nm to finally prepare the MFC @ p3 gene editing delivery system.

Example 11

The embodiment of the application provides a dual liver-specific membrane fusion-mediated gene editing and delivering system GMFC @ p3 targeting HBV, and the specific preparation method comprises the following steps:

(2) DSPE-PEG-Gal (galactose ligand on DSPE-PEG-Gal has specific targeting effect on liver cells), DOTAP and DMPC dissolved in chloroform were mixed in a round bottom flask in a molar ratio of 3.8:20:76.2, and chloroform was volatilized to form a lipid film on the bottom of the flask.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film by a film hydration method, shaking and uniformly mixing, controlling the mass ratio of a membrane fusion liposome (DSPE-PEG, DOTAP and DMPC) in a gene editing and delivering system to the loaded CRISPR/Cas9 plasmid p3 to be 25:1 by controlling the addition amount of the solution C, and sequentially passing through filter membranes with the pore diameters of 800nm, 600nm, 400nm and 200nm to finally prepare the dual liver-specific membrane fusion mediated gene editing and nano delivering system GMFC @ p3 of the targeted HBV.

Comparative example

The application of the comparative example provides non-membrane fusion mediated gene editing delivery systems GNMFC @ p2 and GNMFC @ p3, and the specific preparation method comprises the following steps:

(2) DSPE-PEG-Gal (galactose ligand on DSPE-PEG-Gal has specific targeting effect on liver cells) dissolved in chloroform and DMPC are mixed in a round-bottomed flask according to a molar ratio of 3.8:0:96.2, and chloroform is volatilized to form a lipid film at the bottom of the flask.

(3) Adding the obtained solution C into a round-bottom flask for forming a lipid film by a film hydration method, shaking and uniformly mixing, controlling the mass ratio of a membrane fusion liposome (DSPE-PEG, DOTAP and DMPC) in a gene editing and delivering system to the loaded CRISPR/Cas9 plasmid p2 to be 25:1 by controlling the addition amount of the solution C, and sequentially passing through filter membranes with the pore diameters of 800nm, 600nm, 400nm and 200nm to finally prepare the dual liver-specific membrane fusion mediated gene editing and nano delivering system GMFC @ p2 of the targeted HBV.

(4) Similarly, the CRISPR/Cas9 plasmid p2 was replaced with the HBV-targeted CRISPR/Cas9 plasmid p3 prepared in example 9, and the HBV-targeted dual liver-specific non-membrane fusion gene editing nano delivery system GNMFC @ p3 was prepared.

Example 12

The examples of the present application provide the nanocarrier delivery performance evaluations of example 7, example 8, and comparative examples, specifically:

the gene-editing nano-delivery systems loaded with plasmid p2 of examples 7, 8, and comparative examples were transfected with HepG2 hepatocytes, respectively, and the results of flow cytometry and fluorescence microscopy were examined, and fig. 9 shows the fluorescence intensity of green fluorescent protein EGFP for the different gene-editing nano-delivery systems, where con is the blank control result, Free p2 is the transfection result of pure p2 plasmid, Lipo @ p2 is the transfection result of p2 plasmid loaded with a commercial transfection reagent, MFC @ p2 is the transfection result of MFC @ p2 of example 4, GNMFC @ p2 is the transfection result of MFC @ p2 of comparative example, and MFC @ gmp 2 is the result of GMFC @ gnp 2 of example 5. Experimental results prove that the gene editing nano delivery system mediated by membrane fusion can obviously improve the intracellular delivery performance and effectively improve the transfection efficiency of the gene editing system.

Example 13

The embodiment of the application provides an in vitro gene editing efficiency and curative effect evaluation experiment of a membrane fusion mediated gene editing nano delivery system, which specifically comprises the following steps:

(1) example 6 was prepared separately: membrane fusion mediated gene editing nano-delivery system carrying CRISPR/Cas plasmid p3 editing HBV hepatitis virus (MFC @ p3), example 7: liver-targeted modified membrane fusion mediated gene editing nano-delivery system carrying p3 editing HBV hepatitis virus (GMFC @ p3), comparative example: liver-targeting modified non-membrane-fused gene-editing nano-delivery system (GNMFC @ p3) carrying CRISPR/Cas9 plasmid p3 for editing HBV hepatitis virus.

(2) Constructing an in vitro HBV replication model: the pBB4.5-1.2 XHBV plasmid is transfected into HepG2 liver cancer cells by a commercial transfection reagent to construct a HepG2 liver cell model infected by the HBV virus.

(3) The MFC, GNMFC and GMFC loaded with plasmid p3 prepared in (1) were added to the constructed HBV-infected HepG2 cells, respectively, for transfection. After 6h, the cells were washed with PBS and the culture medium was replaced, and the cell culture was continued.

(4) After 48h of culture, genome in cells was extracted, and through T7E1 mismatch enzyme cleavage (fig. 11A) and gene sequencing analysis (fig. 11B), it was observed that the nanocarrier of example 7 showed the best gene editing effect and had a gene editing efficiency of over 20%, and the editing efficiencies of two targets of HBV reached 23.9% and 26.5%, respectively.

(5) After 48h of culture, the genome in the cells was extracted and the cccDNA concentration in the HBV viral genome was determined by qRT-PCR (fig. 11C). The results show that the gene editing plasmids of example 6 and example 7 significantly reduce cccDNA index of HBV replication model in vitro, and example 7 shows more than 85% of therapeutic effect.

Example 14

The embodiment of the application provides an in vivo efficacy evaluation experiment of a gene editing nano delivery system mediated by membrane fusion, which specifically comprises the following steps:

(1) construction of in vivo HBV replication models: pBB4.5-1.2 XHBV plasmid is injected into C57BL/6 mice of 6-8 weeks old through high pressure tail vein, and the mice with consistent HBV virus expression level are selected for grouping and subsequent experiments are carried out.

(2) The gene-editing nano-delivery system GNMFC @ p3 carrying CRISPR/Cas9 plasmid p3 editing HBV hepatitis virus of example 10, example 11, and comparative example were prepared separately and injected via tail vein into a mouse model of HBV infection.

(3) Mice from each group were bled on

days

1, 2,3, 5, and 7 of the treatment and tested for HBsAg, HBeAg, and HBVDNA levels in the HBV viral genome at different time points by the Elisa method (fig. 12).

(4) Meanwhile, a batch of mice is sacrificed on

days

2 and 7 after administration, and liver tissues are obtained for detection:

1) detecting the concentration of HBsAg (FIG. 13A) and HBeAg (FIG. 13B) in HBV viral genome by Elisa method, and detecting the concentration of HBVDNA (FIG. 13C) and HBVRNA (FIG. 13D) in HBV viral genome by qRT-PCR method;

2) determination of HBsAg antigen Change in HBV-replicating mouse model by immunofluorescence Picture (FIG. 13E)

From the results of in vivo efficacy, it is known that in the HBV-replicated mouse model, the gene editing system successfully reduces the expression level of HBV-related indices, demonstrating an effective antiviral therapeutic effect.

Example 15

The embodiment of the application provides in vivo gene editing efficiency verification of a membrane fusion mediated gene editing nano delivery system, which specifically comprises the following steps:

(2) Gene editing nano-delivery systems carrying CRISPR/Cas9 plasmid p3 editing HBV hepatitis virus of example 10, example 11 and comparative example were prepared separately and injected via tail vein into HBV-infected mouse models.

(3) At day 2 after the administration of the treatment, a batch of mice was sacrificed, liver tissues were obtained, genes were extracted for detection, and gene editing of the anti-HBV targets was detected by T7E1 mismatch enzymatic cleavage (fig. 14A), gene PCR sequencing (fig. 14B), and TIDE analysis (fig. 14C).

From the results of in vivo gene editing, it was found that the gene editing system successfully achieved > 10% in vivo gene editing efficiency in a mouse model of HBV replication, demonstrating an effective level of in vivo editing.

Example 16

The embodiment of the application provides an in vivo safety evaluation experiment of a gene editing nano delivery system mediated by membrane fusion, which specifically comprises the following steps:

(2) Gene-editing nano-delivery systems carrying CRISPR/Cas9 plasmid p3 editing HBV hepatitis virus of example 6, example 7, and comparative example were prepared separately and injected via tail vein into a mouse model of HBV infection. Subsequently, a batch of mice was sacrificed on

days

2 and 7 after administration, and heart, liver, spleen, lung, kidney tissues and serum were obtained, histopathological analysis was performed by H & E staining (fig. 15A), and body safety of the liver-specific gene-editing nano-drug of this example at the time of in vivo treatment was judged by detecting liver function indices glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), kidney function indices UREA and CREA from the collected serum (fig. 15B). According to the results obtained, the gene editing delivery system of the present application has good biocompatibility without causing significant cytotoxicity.

The gene editing nano delivery system realizes cell targeting and direct membrane fusion intracellular delivery, avoids the risk of degradation by lysosomes after endocytosis uptake, and greatly optimizes delivery efficiency and gene editing efficiency. In order to realize membrane fusion instead of endocytosis, the gene editing nano delivery system needs to optimize and screen out a proper proportion of membrane fusion liposomes (phospholipid structural components, cationic functional lipids and PEG functional lipids), and the test results show that the intracellular uptake efficiency and transfection efficiency of the membrane fusion mediated gene editing delivery system are not influenced by endocytosis inhibitors and are only influenced by low temperature (energy dependence), which indicates that the membrane fusion liposomes can realize cell membrane fusion so as to more efficiently deliver gene editing systems (such as plasmids, mRNA and proteins of CRISPR/Cas 9), and the process is energy-dependent. Compared with the membrane-fused liposome, the application verifies that the lipid components of the membrane-fused liposome can be embedded and fused on the cell membrane of an ingested cell, and in the whole ingesting process, the CRISPR/Cas9 plasmid does not enter a lysosome through endocytosis, and the strategy of escaping from the lysosome is bypassed, but directly enters the cytoplasm and further enters the nucleus. Thus, it was also demonstrated that the gene editing delivery system mediated by membrane fusion of the present application can achieve efficient direct cytosolic delivery, superior to the delivery system without membrane fusion.

Calcium precipitation in the membrane fusion mediated gene editing nano delivery system can compress a large-volume CRISPR/Cas9 plasmid with the length of more than 10000bp for effective encapsulation and loading, and an inner core is provided for a membrane fusion liposome; the membrane fusion mediated gene editing delivery system of the present application is advantageous herein in that nucleic acid drugs of large size can be efficiently delivered. The calcium carbonate of the examples of this application is CaCl₂With Na₂CO₃Solid inner core particles formed by precipitation with CRISPR/Cas9 plasmid, and then an outer coating film is coated with a fusogenic liposome layer to obtain the gene editing with a core-shell structureA nano-delivery system. The experimental result proves that calcium carbonate precipitation can effectively load CRISPR/Cas9 plasmid, and the package of the membrane fusion liposome layer further optimizes the delivery efficiency.

The membrane fusion mediated gene editing nano delivery system of the present application can be modified by further outer liver targeting ligands; the carried CRISPR/Cas9 plasmid can be transformed into a hepatocyte specific promoter to drive expression, so that liver targeting specificity is realized, and accurate gene editing and treatment on liver related diseases are realized.

The foregoing is only a preferred embodiment of the present application and it should be noted that, as will be apparent to those skilled in the art, numerous modifications and adaptations can be made without departing from the principles of the present application and such modifications and adaptations are intended to be considered within the scope of the present application.

Claims

1. The gene editing nano delivery system based on membrane fusion is characterized by comprising the following components:

outer layer of membrane fusion liposome and inner core of calcium precipitation hybrid gene editing plasmid; the outer layer of the membrane fusion liposome is wrapped on the surface of the inner core of the calcium precipitation hybrid gene editing plasmid;

the preparation method of the outer layer of the membrane-fused liposome comprises the following steps: preparing an outer layer of the membrane fusion liposome by a thin film hydration method by using phospholipid structure lipid, cationic functional lipid and polyethylene glycol functional lipid;

the preparation method of the calcium precipitation hybrid gene editing plasmid kernel comprises the following steps: mixing soluble calcium salt, soluble carbonate and/or soluble phosphate with the gene editing plasmid, co-precipitating and condensing to form a calcium precipitation hybrid gene editing plasmid kernel; the gene editing system employs a CRISPR/Cas9 plasmid containing a liver-specific promoter.

2. The gene editing nano delivery system according to claim 1, wherein the phospholipid structure lipid is selected from dimyristoylphosphatidylcholine DMPC or/and dioleoylphosphatidylethanolamine DOPE.

3. The gene editing nano delivery system according to claim 1, characterized in that the cationic functional lipid is selected from trimethyl-2, 3-dioleoyloxypropylammonium bromide DOTAP or/and trimethyl-2, 3-dioleoyloxypropylammonium chloride DOTMA; the polyethylene glycol functionalized lipid is selected from one or more of distearoyl phosphatidyl ethanolamine-polyethylene glycol DSPE-PEG, distearoyl phosphatidyl ethanolamine-polyethylene glycol-galactose DSPE-PEG-Gal and distearoyl phosphatidyl ethanolamine-polyethylene glycol-N-acetyl galactosamine DSPE-PEG-GalNac;

in the outer layer of the membrane-fused liposome, the molar ratio of the cationic functional lipid is 19-20%, the molar ratio of the polyethylene glycol functional lipid is 3.8-5%, and the molar ratio of the phospholipid structure lipid is 75-76.2%.

4. The gene editing nano delivery system of claim 1, wherein the soluble calcium salt is selected from CaCl₂Or/and CaHPO₄The soluble carbonate is selected from Na₂CO₃Or/and NaHCO₃The soluble phosphate is selected from NaH₂PO₄Or/and Na₂HPO₄。

5. The gene-editing nano-delivery system of claim 1, wherein the gene-editing plasmid is selected from one or more of CRISPR/Cas9 gene editing system, CRISPR/dCas9 gene editing system, CRISPR/Cas13 gene editing system, CRISPR/Cpf1 gene editing system, meganuclease gene editing system, zinc finger nuclease ZFNs gene editing system, transcription activator-like effector nuclease TALEN gene editing system.

6. The gene editing nano-delivery system according to claim 1, characterized in that the CRISPR/Cas9 gene editing system employs a modified plasmid containing a liver-specific promoter.

7. The gene editing nano delivery system of claim 1, wherein the outer layer of the membrane fusion liposome further comprises an organ targeting ligand or/and a tumor targeting ligand.

8. The gene editing nano delivery system of claim 7, wherein the organ targeting ligand is a liver targeting ligand modifier; the liver targeting ligand modifier is selected from one or more of galactose, N-acetylgalactosamine, lactose, asialofetuin and HBV pre-S1 peptide.

9. The preparation method of the gene editing nano delivery system based on the membrane fusion effect is characterized by comprising the following preparation steps:

step 1, dropwise adding a mixed solution B of soluble carbonate and/or soluble phosphate into a mixed solution A of soluble calcium salt and plasmid which is oscillated or stirred at a constant speed to obtain a calcium precipitation hybrid gene editing plasmid solution; wherein the soluble mixed solution A comprises soluble calcium salt, gene editing plasmid and a first solvent; the soluble carbonate mixed solution B comprises soluble carbonate and/or soluble phosphate and a first solvent;

mixing phospholipid structure lipid, cation functional lipid, polyethylene glycol functional lipid and a second solvent, and volatilizing the second solvent to obtain a lipid film of the membrane fusion liposome;

and 2, adding the calcium carbonate hybrid gene editing plasmid solution into the membrane fusion liposome film, carrying out ultrasonic oscillation until the lipid film is uniformly dispersed in the mixed solution, and passing the obtained solution through a filter membrane with gradient pore size to obtain the gene editing nano delivery system.

10. Use of the gene-editing nano-delivery system according to any one of claims 1 to 8 or the gene-editing nano-delivery system produced by the production method according to claim 9 for delivering gene-editing plasmids, mRNA or proteins into cells.