Engineered membrane hybridization exosome entrapped with complete large-fragment gene editing system and preparation method of engineered membrane hybridization exosome

Abstract

The invention discloses an engineered membrane hybridization exosome entrapped with a complete large-fragment gene editing system and a preparation method thereof. The engineered membrane hybridization exosome is prepared by fusing cationic lipid nanoparticles entrapped with related plasmids and an engineered exosome modified with MyoAAV 1A characteristic peptide, lNP entrapping a dCas9.hyPB large-fragment gene editing system is fused with an engineered exosome modified by MyoAAV 1A through membrane fusion, the average particle size is 145-155 nm, the potential is-10 <-8 > mV, good biocompatibility is achieved, targeted delivery of muscle cells can be achieved, and accurate insertion of large-fragment DNA is achieved at a specific site. According to the invention, the toxic and side effects of the existing product are reduced or eliminated, the drug targeting is improved, the liver accumulation is reduced, the drug administration difficulty and frequency are reduced, the treatment effect is improved, and the treatment cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to an engineered membrane hybrid exosome encapsulating a complete large fragment gene editing system, and also discloses a preparation method of the exosome, and belongs to the technical field of biological pharmacy. BACKGROUND

[0002] Monogenic disorders mainly refer to diseases caused by single-point mutations or gene sequence changes on a pair of alleles. At present, most monogenic diseases have no radical cure, and mainly adopt supportive treatment methods, that is, corresponding treatment and nursing are given to patients according to disease phenotypes, so as to achieve the purpose of relieving symptoms, including glucocorticoids, signal pathway modulators and analgesic drugs. However, long-term treatment for these phenotypes will also bring serious adverse reactions to patients, such as abnormal glucose metabolism, myocardial infarction, increased risk of fracture, etc.

[0003] With the development of genetic engineering technology, gene therapy programs are gradually becoming a new direction of treatment research for monogenic diseases. The purpose is to restore the normal expression of proteins that have lost function due to mutations, so as to achieve the purpose of complete cure. How to establish an accurate editing system for large fragment DNA and construct a large-carrying, efficient and safe targeted delivery vector for it is a key problem that needs to be solved in this program.

[0004] The existing DNA large fragment editing system mainly includes CRISPR / Cas system and transposon system. The unique sgRNA guiding property of CRISPR / Cas system enables it to conveniently and accurately edit the site of interest, and certain progress has been made in the treatment research of some diseases caused by single-point mutations. However, due to the need for double-strand break mechanism in its action process, it may introduce unexpected chromosomal changes such as ectopic and inversion. Transposon, also known as jumping gene, is a DNA fragment that can move on the genome. PB transposon is the most active transposon currently applied to mammals, and hyPB is its high-activity mutant. They can carry more than 10 kb of gene fragments, bypass the DSB mechanism, specifically recognize TTAA sites on the genome and insert the carried fragments, without leaving additional traces, and have natural advantages in safety and efficiency. However, due to the wide existence of TTAA sequence in biological genomes, the off-target rate is high. In previous studies, researchers tried to combine the directional guiding function of sgRNA with the high carrying capacity and efficient cutting and integration ability of PB. Although the efficiency is low (0.32%), this study shows that the fused dCas9.PB transposase molecule can perform site-specific and directional transposition.

[0005] Among existing gene editing delivery systems, virus-associated vectors, such as adeno-associated viruses (AAVs), have strong diffusion capabilities and a wide range of infective hosts. However, their payload capacity is limited, and in most cases, only truncated target genes can be delivered, with a high risk of immunogenicity. Among non-viral vectors, lipid nanoparticles offer higher payload capacity but suffer from poor stability and difficulty in targeted modification. In contrast, targeted modification of exosomes is easier to achieve, and exosomes exhibit strong in vivo circulation stability, but their encapsulation and delivery efficiency for large nucleic acid fragments is relatively low. Summary of the Invention

[0006] This invention provides an engineered membrane hybridization exosome carrying a complete large-fragment gene editing system and its preparation method. The method is simple to operate and suitable for industrial production. By fusing LNPs carrying the dCas9.hyPB large-fragment gene editing system with engineered exosomes modified with MyoAAV 1A through membrane fusion, it can target muscle cells throughout the body and achieve precise insertion of large DNA fragments at specific sites.

[0007] The engineered membrane hybridization exosome carrying a complete large-fragment gene editing system described in this invention is prepared by membrane fusion of cationic lipid nanoparticles carrying relevant plasmids and engineered exosomes modified with MyoAAV 1A characteristic peptides. The prepared cationic lipid nanoparticles have an average particle size of 100-110 nm and an average potential of 10-20 mV. They also have good encapsulation and protection capabilities for DNA and transfection capabilities comparable to the transfection reagent PEI. After encapsulating the dCas9.hyPB large fragment gene editing system, the particle size becomes 130-140 nm and the average potential is around 10 mV. The engineered exosomes modified with MyoAAV 1A characteristic peptide prepared had an average particle size of 135-145 nm and an average potential of around -38 mV. The exosome characteristic proteins CD63, HSP70, and TSG101 could be directly detected on their surface, and MyoAAV 1A peptide could be detected indirectly. They also showed obvious C2C12 cell targeting. The engineered membrane hybridization exosomes carrying the complete large-fragment gene editing system prepared have an average particle size between 145 and 155 nm and a potential of about -9 mV. This indicates that the characteristic proteins of exosomes HSP70, CD63, and TSG101 can be directly detected, and the MyoAAV 1A peptide can be indirectly detected. They have good biocompatibility and can achieve targeted delivery to muscle cells throughout the body, enabling precise insertion of large DNA fragments at specific sites.

[0008] The method for preparing engineered membrane hybridization exosomes carrying a complete large-fragment gene editing system, as described in this invention, is as follows: Lipids and related plasmids were dissolved separately in solvents. Under magnetic stirring, the organic phase was uniformly injected into the aqueous phase, and the solvent was evaporated at room temperature to obtain lipid nanoparticles. After transfecting cells with engineered exosome-assisted plasmids, engineered exosomes were isolated from the cell supernatant. The lipid nanoparticles and engineered exosomes were mixed in a certain ratio and incubated for fusion to obtain engineered membrane hybridization exosomes loaded with a complete large-fragment gene editing system.

[0009] The relevant plasmids include the dCas9.hyPB fusion protein expression plasmid, transposon donor plasmid, and transposon receptor plasmid.

[0010] The lipid nanoparticles include natural neutral lipids, synthetic neutral phospholipids, synthetic negatively charged modified phospholipids, and synthetic cationic lipids.

[0011] The natural neutral lipids include cholesterol (Chol).

[0012] The synthetic neutral phospholipids include 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC).

[0013] The synthetic negatively charged modified phospholipids include DSPE-PEG2000-Mal.

[0014] The synthetic cationic lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methyl 4-(N,N-dimethylamino)butyrate (Dlin-MC3-DMA).

[0015] The engineered exosomes include membrane-bound exosomes (Mexo) expressing the MyoAAV 1A characteristic peptide.

[0016] The solvents include, but are not limited to, one or a mixture of two or more of the following: water, methanol, ethanol, dichloromethane, chloroform, and diethyl ether.

[0017] In the preparation method, the molar ratio of DOTAP, cholesterol, and DOPC is 50:37.5:10; the molar ratio of DOTAP to DSPE-PEG2000-Mal is 50:1 to 50:4.

[0018] In the preparation method, the nitrogen-to-phosphorus ratio of DNA to DOTAP is 8:1 to 6:1.

[0019] In the preparation method, the magnetic stirring speed is 300 rpm to 1500 rpm, the solvent evaporation time is 10 min to 40 min, and the initial lipid concentration in the organic phase is 5 to 20 mM.

[0020] In the preparation method, the fusion ratio of lipid nanoparticles to engineered exosomes is 1:5 to 5:1, and the fusion time is 0.5h to 4h.

[0021] The positive effects of this invention are as follows: Engineered membrane hybridization exosomes carrying a complete large-fragment gene editing system are prepared by fusing cationic lipid nanoparticles carrying relevant plasmids with engineered exosomes modified with the MyoAAV 1A characteristic peptide. The average particle size is between 145 and 155 nm, the potential is around -9 mV, and they exhibit good biocompatibility. This allows for targeted delivery to muscle cells and precise editing of gene sites of interest. Establishing a precise editing system for large-fragment DNA is a way to construct a high-capacity, efficient, and safe targeted delivery vector to restore the normal expression of proteins that have lost function due to mutations, thereby achieving a complete cure. This invention reduces or eliminates the toxic side effects of existing products, improves drug targeting, reduces liver accumulation, and lowers the difficulty and frequency of drug administration. Simultaneously, it enables complete sequence replacement of mutated genes, significantly improving treatment efficacy and reducing treatment costs to some extent. Attached Figure Description

[0022] Figure 1 Transfection capability test of the LNP of this invention (scale bar: 50 μm). Figure 2 This is a Western blot image of exosome characteristic proteins in Mexo from this invention; Figure 3 The following are the SDS-PAGE results of several different exosomes of this invention; Figure 4 These are Western blotting images of exogenous peptides in the engineered exosomes of this invention. Figure 5 For the verification of DNA@MELN membrane fusion of the present invention (scale bar = 200 μm); Figure 6 A comparison of particle size and potential of DNA@MELN, DNA@LNP and Mexo of the present invention (A) Particle size; (B) Potential (n = 3); Figure 7 These are Western blot images of Mexo and DNA@MELN proteins from this invention. Figure 8 The results of SDS-PAGE of Mexo, DNA@MELN, and cells in this invention are as follows; Figure 9 Image showing the uptake distribution of DNA@MELN in different cells according to the present invention (scale bar = 200 μm); Figure 10The uptake of DNA@MELN by different cells in this invention (A) and its quantitative analysis (B) (n = 3, ***p < 0.001); Figure 11 The effect of different carriers on cell viability in this invention (n = 5) (*p < 0.05, ****p < 0.0001); Figure 12 This is a schematic diagram of the dCas9.hyPB gene editing system of the present invention; Figure 13 This is a verification of the precise writing capability of the dCas9.hyPB@MELN of this invention (scale bar = 50 μm). Detailed Implementation

[0023] The specific implementation methods of the present invention are illustrated by the following examples, but these do not limit the scope of protection of the present invention in any way.

[0024] Abbreviations given in the examples: LNP is the name of lipid nanoparticles prepared by the ethanol injection method. When the cationic lipid component is not specified, LNP refers to lipid nanoparticles with DOTAP as the cationic lipid component; Mexo is the name of engineered exosomes modified with MyoAAV1A peptide; MELN is the name of engineered exosome-liposome membrane hybridization vectors; DNA@LNP is an LNP encapsulated with DNA; DNA@MELN is a MELN encapsulated with DNA; dCas9.hyPB is a MELN containing dCas9.hyPB@MELN, which encapsulates the dCas9.hyPB fusion protein expression plasmid, transposon donor plasmid, and transposon acceptor plasmid. Example 1

[0025] The addition of PEG lipids and cholesterol can prolong the half-life of cationic lipids, reduce immune clearance, and extend in vivo circulation time. However, excessive addition of PEG lipids can affect the encapsulation and release of liposomal drugs. Therefore, it is necessary to screen the ratio of DOTAP to DSPE-PEG2000-Mal. Under the condition that the ratios of other components and preparation conditions remain constant, the ratio of DOTAP to DSPE-PEG2000-Mal was adjusted and characterized.

[0026] (1) Preparation of organic phase: DOTAP, DOPC, cholesterol and DSPE-PEG2000-Mal were dissolved in anhydrous ethanol to prepare a 100mM stock solution (concentration) and ultrasonically dispersed evenly; the stock solution was taken according to the molar ratio of DOTAP:cholesterol:DOPC=50:37.5:10, and the molar ratio of DOTAP to DSPE-PEG2000-Mal was set to 50:1, 50:2, 50:3 and 50:4. The lipid solutions were mixed and diluted with anhydrous ethanol to 10mM and ultrasonically dispersed evenly.

[0027] (2) Preparation of aqueous phase: Mix 33 mL of citric acid solution and 17 mL of sodium citrate solution, slowly add NaOH solution, adjust the pH value to 4, filter through a 0.22 μm filter membrane and sterilize at 121 °C and 101 kPa for 20 min to obtain citric acid buffer. Add different plasmid components to the aqueous phase as needed.

[0028] (3) Take 1 ml of aqueous phase and place it in a small beaker. Set the rotation speed to 600 rpm. Use a 1 ml syringe to inject 100 μL of organic phase into the aqueous phase at a speed of 1 ml / min. Stir at room temperature for 20 min to evaporate the ethanol.

[0029] (4) Add 3 times the volume of citrate buffer and use an ultrafiltration tube with a cutoff size of 30 kD to ultrafilter to 1 / 4 of the loading volume to remove unloaded DNA and lipids; add 3 times the volume of PBS solution and use an ultrafiltration tube with a cutoff size of 30 kD to ultrafilter to 1 / 4 of the loading volume. Repeat twice to replace the solvent of LNP with PBS. After sterilization by filtration through a 0.22 μm filter membrane, store at 4 ℃ for later use.

[0030] (5) Take the LNPs containing different proportions of DSPE-PEG2000-Mal obtained in step (4), dilute them and add them to the sample cell of the ZS90 particle size potential analyzer. The detection temperature is 25℃ and the equilibration time is 60s. Each sample is tested in parallel 3 times to measure the particle size, dispersibility index and potential of LNPs.

[0031] (6) Preparation of engineered exosomes: HEK 293T cells were transfected with engineered exosome helper plasmids. Cell supernatant was collected and impurities were removed by gradient centrifugation: centrifugation at 300×g for 5 min removed floating cells; the supernatant was transferred to a new centrifuge tube and centrifuged at 2,000×g for 5 min to remove cell debris; the supernatant was transferred to a new centrifuge tube and centrifuged at 10,000×g for 30 min to remove smaller impurity particles. The treated cell supernatant was concentrated using a 30 kDa ultrafiltration tube and centrifuged at 121,000×g at 4 ℃ for 3 h using an ultra-high speed refrigerated centrifuge. The supernatant was discarded, and the precipitate at the bottom of the centrifuge tube was resuspended with 100 μL PBS to obtain an exosome solution. 1 μL of the obtained exosome solution was used to quantify the protein content using a BCA protein quantification kit. Western blotting was used to detect the exosome characteristic proteins and MyoAAV 1A characteristic peptide on the surface of engineered exosomes. (7) Preparation of engineered membrane hybridization exosomes: Since LNP contains DNA, the fusion vector was prepared by co-incubation. DNA@LNP and exosomes were mixed at a mass ratio of 5:1 and incubated at 37 °C for 2 h to obtain DNA@MELN. (8) Take the DNA@MELN obtained in step (7), dilute it and add it to the sample cell of the ZS90 particle size potential analyzer. The detection temperature is 25℃ and the equilibration time is 60s. Each sample is detected in parallel 3 times to measure the particle size, dispersibility index and potential of LNP. Use protein immunoblotting to detect the exosome characteristic proteins and MyoAAV 1A characteristic peptide on the surface of engineered membrane hybrid exosomes.

[0032] Experimental results: The particle size, potential, and PDI of particles containing different proportions of DOTAP and DSPE-PEG2000-Mal in Example 1 were analyzed using a ZS90 particle size potential analyzer. The particle size, potential, and PDI are shown in Table 1. With the increase of DSPE-PEG2000-Mal input, the particle size showed a trend of first decreasing and then increasing.

[0033] Table 1 Effect of the molar ratio of DOTAP to DSPE-PEG2000-Mal on the particle size of Dlin-MC3-DMA-LNP (mean ± SD, n = 3)

[0034] Summary: Based on the experimental results, 50:3 was selected as the final molar ratio of DOTAP to DSPE-PEG2000-Mal in LNP. Example 2

[0035] Evaluation of the transfection capacity of DOTAP-LNP and Dlin-MC3-DMA-LNP: Due to the electrostatic interaction between the positively charged lipid membrane and the negatively charged cell membrane, cationic lipid nanoparticles are more readily internalized by cells than anionic lipid nanoparticles. The type of cationic lipid in LNPs has a significant impact on their transfection capacity. Two cationic lipids, DOTAP and Dlin-MC3-DMA, were selected to synthesize corresponding LNPs for parallel testing (in formulation screening, they are collectively referred to as cationic lipid components).

[0036] DOTAP-LNP and Dlin-MC3-DMA-LNP, which contain green fluorescent protein expression plasmids, were prepared by dissolving them in aqueous solution. After quantification using picogreen dye, they were transfected into HEK 293T cells and C2C12 cells, respectively, and the expression of green fluorescent protein was observed under a fluorescence microscope.

[0037] Experimental results: like Figure 1 As shown, 24 h after transfection, DOTAP-LNP exhibited transfection efficiency comparable to the common transfection reagent PEI, while Dlin-MC3-DMA-LNP showed almost no transfection ability. Because the conditions for Dlin-MC3-DMA lipids to function are quite demanding, Dlin-MC3-DMA-LNP cannot function well when cell conditions are poor.

[0038] In summary, based on the experimental results, DOTAP was selected as the cationic lipid component in this invention. Example 3

[0039] Determination of LNP encapsulation efficiency: (1) LNP sample preparation: Triton X-100 was prepared into a 4% demulsifier using 1×TE working solution. 100 μL was added to a 96-well black-bottom microplate, 1 μL of the prepared DNA@LNP was added, and the mixture was incubated at room temperature for 5 min to complete the demulsification.

[0040] (2) Preparation of free nucleic acid samples: Add 100 μL of 1×TE working solution and 1 μL of prepared DNA@LNP to a 96-well black-bottom microplate and mix well.

[0041] (3) Determination of encapsulation efficiency: Add 100 μL of working solution to the sample wells, incubate at room temperature in the dark for 5 min, and then read the fluorescence intensity (Ex=480 nm, Em=520 nm) using a microplate reader. Calculate the encapsulation efficiency according to the following formula:

[0042] Where EE represents the encapsulation efficiency; C(t) represents the total DNA concentration measured after demulsification; and C(f) represents the free DNA concentration measured before demulsification.

[0043] Experimental results: Table 2 Encapsulation efficiency of LNP on DNA

[0044] In summary, based on the theoretical N:P ratio of 6:1, the encapsulation rate of LNP on DNA was 84.6 ± 2.6%, demonstrating that the LNP in this invention has good DNA encapsulation ability. Example 4

[0045] Preparation of Mexo: (1) Plasmid transfection: Calculate the amount of plasmid required for cell transfection according to a concentration of 1 μg / mL. After transfecting HEK293T cells, continue culturing for 48 hours.

[0046] (2) Collection of cell supernatant: After collecting the cell supernatant, use gradient centrifugation to remove impurities: centrifuge at 300×g for 5 min to remove floating cells; take the supernatant and put it into a new centrifuge tube, centrifuge at 2,000×g for 5 min to remove cell debris; take the supernatant and put it into a new centrifuge tube, centrifuge at 10,000×g for 30 min to remove smaller impurity particles.

[0047] (3) Extraction of Mexo: The treated cell supernatant was concentrated using a 30 kDa ultrafiltration tube and centrifuged at 121,000×g and 4 °C for 3 h using an ultra-high speed refrigerated centrifuge. The supernatant was discarded and the precipitate at the bottom of the centrifuge tube was resuspended with 100 μL PBS to obtain the exosome solution.

[0048] (4) Identification of Mexo: A suitable amount of exosome solution was diluted and added to the sample cell of the ZS90 particle size potentiometer. The detection temperature was 25℃ and the equilibration time was 60s. Each sample was tested in parallel 3 times to measure the particle size, dispersibility index and potential of Mexo. The protein content of the obtained exosomes was quantified and the characteristic proteins were identified.

[0049] Experimental results: As shown in Table 3, the particle size of the empty exosomes was 126.0 ± 2.6 nm, and the potential was -5.8 ± 0.6 mV, consistent with the characteristics of exosomes. The particle size of the exosomes modified with the MyoAAV1A characteristic peptide was 142.3 ± 2.6 nm, and the potential was -37.9 ± 1.9 mV. Compared with the empty exosomes, the introduction of the negatively charged MyoAAV1A characteristic peptide slightly increased the particle size of the exosomes and significantly decreased the potential, which is consistent with expectations.

[0050] Table 3. Exosome particle size, PDI, and potential (mean ± SD, n = 3)

[0051] Immunoblotting detection of exosome characteristic proteins was performed on the extracted engineered exosomes, such as... Figure 2 As shown in Figure 3, immunoblotting results indicated that three characteristic proteins of exosomes could be detected in the extracted exosomes, consistent with the characteristics of exosomes. After SDS-PAGE electrophoresis of several different exosomes, the gels were stained with Coomassie brilliant blue. The results are shown in Figure 3. The protein abundance in the exosomes before and after modification was consistent, and characteristic exosome proteins were detected in all cases, indicating that the engineered exosomes obtained after peptide modification still retained the biological functions of exosomes.

[0052] Using FLAG-tags as a control, the expression of the exogenous characteristic peptide MyoAAV1A on the surface of exosomes was investigated. Figure 4 According to Western blotting results, HA-tag expression was detected on the surfaces of both engineered exosomes, indicating that the plasmid was successfully transfected into cells and the exogenous protein was successfully expressed on the exosome surface. In the control group, FLAG-tag expression was detected on the Fexo membrane at the same location as the HA-tag, indicating a high correlation between the two tags, further demonstrating that the HA-tag can be used as a basis for detecting the expression of exogenous characteristic peptides. The Western blotting results from both groups showed that the MyoAAV1A peptide was successfully linked to the transmembrane protein Lamp2B and expressed on the exosome membrane surface. Furthermore, the peptide-Lamp2B fusion protein did not affect the ability of the characteristic peptide to interact with its binding chaperone.

[0053] In summary, the engineered exosome Mexo prepared retains the biological functions of exosomes while also maintaining the interaction ability between its surface characteristic peptides and their binding partners. Example 5

[0054] DNA@MELN preparation and identification: DNA@LNP was labeled with DiO dye and Mexo was labeled with DiI dye. DNA@LNP and exosomes were mixed at a mass ratio of 5:1 and incubated at 37 °C for 2 h to obtain DNA@MELN. Membrane fusion was confirmed under a fluorescence microscope.

[0055] A suitable amount of DNA@MELN solution was diluted to 1 ml and added to the sample cell of a ZS90 particle size and potential analyzer. The detection temperature was 25℃, and the equilibration time was 60 s. Each sample was analyzed in triplicate to measure the particle size, dispersibility index, and potential of the DNA@MELN. Characteristic proteins in the DNA@MELN were then identified.

[0056] Experimental results: The fluorescence channels of DiO and DiI dyes were detected separately, and the results are as follows: Figure 5 As shown, after overlaying the images, the overlap of the two fluorescent spots was observed to be over 90%. Fluorescence imaging indicates that the two carriers have fused, and the fusion ratio is appropriate.

[0057] Under the same conditions, three batches of DNA@MELN were prepared, and their particle size, PDI, and potential were detected. The results are shown in Table 4 and 5. Figure 6 As shown, the particle size of DNA@MELN is 152.2 ± 3.9 nm, and the potential is approximately -8.7 ± 1.3 mV. Compared with DNA@LNP and Mexo, the particle size of DNA@MELN is slightly increased, and the potential is between the two, further proving that the two have fused.

[0058] Table 4. Particle size characterization of DNA@MELN (mean ± SD, n = 3)

[0059] The results of the Western blot experiment for DNA@MELN are as follows: Figure 7 As shown, characteristic proteins of exosomes can be detected on the fused vector, indicating that the prepared fusion vector retains the function of exosomes. Coomassie brilliant blue staining results are shown below. Figure 8 Compared to total cellular protein, the extracted exosome protein abundance was lower. The fused DNA@MELN protein content was lower, but its abundance was similar to that of exosomes, further indicating that the fused vector retained the modification of exosomes with biologically targeted peptides.

[0060] In summary, DNA@MELN was prepared by co-incubating engineered exosomes Mexo with DNA@LNP to achieve membrane fusion. Western blot experiments confirmed that the resulting fusion vector MELN retained the biological characteristics of exosomes and the labeling of the target peptide after membrane fusion. Example 6

[0061] Targeting of DNA@MELN on C2C12 cells: DNA@MELN prepared using DiO dye was administered to HEK 293T cells, HUVEC cells, and C2C12 cells at the same concentration and incubation time. The uptake results were qualitatively analyzed under a fluorescence microscope, and quantitatively analyzed by flow cytometry.

[0062] Experimental results: DNA@MELN was labeled with DiI dye and added to three different cell types at the same concentration and for the same time. The targeted uptake of DNA@MELN by different cells was observed. Figure 9 and Figure 10 As shown, uptake was low in HEK 293T and HUVEC cells, but significantly increased in C2C12 cells. The same results were obtained in flow cytometry quantification experiments, indicating that the prepared DNA@MELN is targeted to C2C12 cells.

[0063] In summary, qualitative and quantitative uptake experiments showed that DNA@MELN has a tendency to target and deliver its contents to C2C12 cells. Example 7

[0064] Cytotoxicity evaluation of DNA@MELN: (1) Drug administration: The prepared material was diluted with DMEM high-glucose basal medium according to the preset concentration gradient (0, 5, 10, 20, 30, 50, 100, 200 μg / mL) to prepare drug-containing medium. At the same time, PEI-DMEM was prepared as a positive control according to the same concentration gradient. The old medium in the well plate was carefully aspirated and the prepared drug-containing medium was added. Six parallel wells were set up for each group and the plates were put back into the incubator for 24 h.

[0065] (2) MTT assay: Use a disposable syringe to completely aspirate the drug-containing culture medium from the well plate, inject 100 μL of fresh DMEM basal medium into the wells, and leave one row of uninoculated wells containing only DMEM basal medium as blank wells. All subsequent steps should be performed in the dark. Add 10 μL of MTT solution to each well, return the well plate to 37 ℃ and continue incubation for 4 h. Carefully aspirate the liquid from the well plate with a syringe, avoiding aspirating the purple crystals at the bottom of the well plate as much as possible. Add 100 μL of DMSO to the wells and let it stand at room temperature for 10 min to allow the purple crystals to completely dissolve in the DMSO. Use an MD multi-mode microplate reader to detect the absorbance of the sample at 570 nm. To ensure that the purple crystals in the sample have completely dissolved and mixed, shake the plate for 10 s before detection using the microplate reader's shaking function.

[0066] Experimental results: The traditional transfection reagent PEI, at a concentration of 20 μg / mL, caused cell viability to drop to 50%, exhibiting strong cytotoxicity. For example... Figure 11 As shown, at concentrations below 100 μg / mL, the cell survival rate in the DNA@MELN group reached over 80%, and at a concentration of 200 μg / mL, the cell survival rate reached over 72%. When DNA@LNP was administered alone, the cell survival rate reached over 70% at concentrations below 50 μg / mL, but it exhibited strong cytotoxicity at higher concentrations.

[0067] In summary, compared with traditional transfection reagents such as PEI and DNA@LNP alone, DNA@MELN in this invention can maintain good biocompatibility even at high concentrations. Example 8

[0068] dCas9.hyPB@MELN's fixed-point transpose: Four plasmids—dCas9.hyPB expression plasmid containing two sgRNAs, transposon donor plasmid, and transposon acceptor plasmid—were mixed in a mass ratio of 1:1:4:4. After being loaded into LNPs and fused with engineered exosomes Mexo, the mixture was quantified using picogreen dye to obtain the dCas9.hyPB@MELN system. This system was then delivered into C2C12 cells, and the expression of fluorescent proteins in the cells was observed.

[0069] Experimental results: like Figure 12 As shown, the transposable receptor in the dCas9.hyPB@MELN system is an EGFP expression vector with its promoter deleted, which normally does not autonomously express fluorescent protein. When a pre-designed precise transposition occurs, the PGK promoter on the transposable donor inserts upstream of the EGFP protein's ORF, initiating fluorescent protein expression. Therefore, when fluorescent protein is observed in the cell, it can be considered that a pre-planned precise transposition event has occurred. Figure 13 As shown, a weak green fluorescence could be observed in some cells 48 h after transfection.

[0070] In summary, after treating cells with the dCas9.hyPB@MELN system, normal expression of EGFP protein was restored, indicating that the expected precise transposition event occurred in the cells, further demonstrating that the dCas9.hyPB@MELN constructed in this invention has the ability to write precisely.

[0071] The engineered membrane hybrid exosome of the present invention, which encapsulates a complete large-fragment gene editing system, is prepared by fusing cationic lipid nanoparticles carrying relevant plasmids with engineered exosomes modified with MyoAAV 1A characteristic peptides. The average particle size is between 145 and 155 nm, the potential is around -9 mV, and it has good biocompatibility. It can achieve targeted delivery to muscle cells and precise editing of specific gene sites.

Claims

1. An engineered membrane hybrid exosome encapsulating a complete large fragment gene editing system, characterized by: being prepared by membrane fusion of cationic lipid nanoparticles encapsulating related plasmids and engineered exosomes modified with MyoAAV 1A signature peptides; having an average particle size of 145-155 nm and an average potential of -10 to -8 mV, being directly detectable for exosome characteristic proteins HSP70, CD63, TSG101, and indirectly detectable for MyoAAV 1A peptides, having good biocompatibility, and being capable of targeted delivery to systemic muscle cells and precise insertion of large fragment DNA at specific sites.

2. A preparation method of the engineered membrane hybrid exosome encapsulating a complete large fragment gene editing system according to claim 1, characterized by: dissolving lipids and related plasmids with solvents, respectively, and uniformly injecting the organic phase into the aqueous phase under magnetic stirring, and volatilizing the solvent at room temperature to obtain lipid nanoparticles; transfecting cells with the engineered exosome auxiliary plasmid, and separating the engineered exosomes from the cell supernatant; mixing the lipid nanoparticles and the engineered exosomes in a certain proportion, and incubating and fusing to obtain the engineered membrane hybrid exosome encapsulating a complete large fragment gene editing system; the related plasmids include a dCas9.hyPB fusion protein expression plasmid, a transposition donor plasmid, and a transposition acceptor plasmid; the lipid nanoparticles include natural neutral lipids, synthetic neutral phospholipids, synthetic negatively charged modified phospholipids, and synthetic cationic lipids; the natural neutral lipids include cholesterol (Chol); the synthetic neutral phospholipids include 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC); the synthetic negatively charged modified phospholipids include DSPE-PEG2000-Mal; the synthetic cationic lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 4-(N,N-dimethylamino) butyric acid (dilin-MC3-DMA); the engineered exosomes include exosomes (Mexo) expressing MyoAAV 1A signature peptides on the membrane; the solvents include, but are not limited to, one or a mixture of two or more of water, methanol, ethanol, dichloromethane, chloroform, and diethyl ether.

3. The preparation method of the engineered membrane hybrid exosome encapsulating a complete large fragment gene editing system according to claim 2, comprising the following steps: (1) Preparation of the organic phase: dissolve DOTAP, DOPC, cholesterol, and DSPE-PEG2000-Mal in anhydrous ethanol, respectively, to prepare 100 mM concentration mother liquor, and ultrasonically disperse uniformly; take the mother liquor according to the molar ratio of DOTAP: cholesterol: DOPC: DSPE-PEG2000-Mal = 50:37.5:10:3, mix the lipid solution, and dilute with anhydrous ethanol to 10 mM, and ultrasonically disperse uniformly; (2) Preparation of the aqueous phase: mix 33 mL of the citric acid solution and 17 mL of the sodium citrate solution, slowly add the NaOH solution, adjust the pH value to 4, filter through a 0.22 μm filter membrane, sterilize at 121 ℃ and 101 kPa for 20 min, and obtain a citric acid buffer solution. Add different plasmid components to the aqueous phase as needed; (3) Take 1 mL of the aqueous phase into a small beaker, set the rotation speed to 600 rpm, use a 1 ml syringe to inject 100 μL of the organic phase into the aqueous phase at a speed of 1 ml / min, stir at room temperature for 20 min, and volatilize ethanol; (4) Add 3 volumes of the citric acid buffer solution, use an ultrafiltration tube with a 30 kD cutoff to ultrafiltrate to 1 / 4 of the loading volume, remove the unloaded DNA and lipids; add 3 volumes of PBS solution, use an ultrafiltration tube with a 30 kD cutoff to ultrafiltrate to 1 / 4 of the loading volume, repeat 2 times, replace the solvent of the DNA@LNP with PBS, filter through a 0.22 μm filter membrane to remove bacteria, and store at 4 ℃ for standby; (5) Take the DNA@LNP obtained in step (4), dilute, and add to the sample pool of the ZS90 particle size potential analyzer, the detection temperature is 25 ℃, the equilibrium time is 60 s, each sample is detected in parallel for 3 times, and the particle size, dispersibility index and potential of the LNP are measured; (6) Preparation of engineered exosomes: use an engineered exosome auxiliary plasmid to transfect HEK 293T cells, collect the cell supernatant, remove impurities by gradient centrifugation: 300xg centrifugation for 5 min to remove floating cells; take the supernatant into a new centrifuge tube, 2,000xg centrifugation for 5 min to remove cell debris; take the supernatant into a new centrifuge tube, 10,000xg centrifugation for 30 min to remove smaller impurity particles; concentrate the treated cell supernatant with a 30 kDa ultrafiltration tube, use an ultra-high-speed refrigerated centrifuge to centrifuge at 121,000xg and 4 ℃ for 3 h, discard the supernatant, resuspend the sediment at the bottom of the centrifuge tube with 100 μL of PBS, and obtain an exosome solution; take 1 μL of the obtained exosome solution, use a BCA protein quantification kit to quantitate the protein content; use Western blotting to detect the exosome characteristic proteins and MyoAAV 1A characteristic peptides on the surface of the engineered exosomes; (7) Preparation of engineered membrane hybrid exosomes: since the LNP encapsulates DNA, a co-incubation method is used to prepare the fusion carrier; mix the DNA@LNP and the exosomes according to a mass ratio of 5:1, incubate at 37 ℃ for 2 h, and obtain DNA@MELN; (8) Take the DNA@MELN obtained in step (7), dilute, and add to the sample pool of the ZS90 particle size potential analyzer, the detection temperature is 25 ℃, the equilibrium time is 60 s, each sample is detected in parallel for 3 times, and the particle size, dispersibility index and potential of the LNP are measured; use Western blotting to detect the exosome characteristic proteins and MyoAAV 1A characteristic peptides on the surface of the engineered membrane hybrid exosomes.

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