Genetically engineered exosome capable of efficiently delivering Cas9 ribonucleoprotein complex and application of genetically engineered exosome

Abstract

The invention provides a genetically engineered exosome capable of efficiently delivering a Cas9 ribonucleoprotein complex, and belongs to the technical field of genetic engineering.The genetically engineered exosome comprises an exosome body, Cas9 / sgRNA and an anti-inflammatory factor IL-10, the two ends of a characteristic protein CD63 of the exosome body are connected with com nucleic acid aptamer binding proteins respectively, and the Cas9 / sgRNA and the anti-inflammatory factor IL-10 are connected with Cas9 / sgRNA. The sgRNA scaffold ST2 loop ring of the Cas9 / sgRNA is modified by adopting a com nucleic acid aptamer, and the com nucleic acid aptamer binding protein and the com nucleic acid aptamer are mutually combined; wherein the nucleotide sequence of the com nucleic acid aptamer is as shown in SEQ ID NO. 1, and the amino acid sequence of the com nucleic acid aptamer binding protein is as shown in SEQ ID NO. 2. The genetically engineered exosome can efficiently enrich a Cas9 ribonucleoprotein complex, is beneficial to realizing in-vivo effective gene editing, and improves the gene treatment effect of osteoarthritis. The invention also provides an application of the gene engineering exosome for efficiently delivering the Cas9 ribonucleoprotein complex in preparation of a medicine for preventing or treating osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a genetically engineered exosome for efficiently delivering a Cas9 ribonucleoprotein complex and an application thereof. Background Art

[0002] In the development of osteoarthritis, dysregulated gene expression is an important cause of disrupting immune homeostasis, aggravating local inflammation and bone damage. Therefore, regulating gene expression through gene therapy strategies is expected to open up new avenues for the treatment of osteoarthritis. However, the commonly used gene delivery methods in clinical practice have defects in immune response and off-target effects.

[0003] Exosomes are a type of cell-derived extracellular vesicles that can serve as a delivery vehicle to mediate the delivery of Cas9 RNP. However, how to improve the loading efficiency of Cas9 RNP in exosomes to achieve effective gene editing in vivo has always been a difficult point in this research. In addition, exosomes also have the problem of short retention time in vivo. Summary of the invention

[0004] In order to solve the problem of low loading efficiency of existing Cas9 RNP delivery vehicles, the present invention provides a genetically engineered exosome that efficiently delivers Cas9 ribonucleoprotein complexes. The genetically engineered exosomes can efficiently enrich Cas9 ribonucleoprotein complexes, which is conducive to achieving effective gene editing in vivo and improving the efficacy of gene therapy for osteoarthritis.

[0005] The present invention also provides a use of genetically engineered exosomes that efficiently deliver Cas9 ribonucleoprotein complexes in the preparation of drugs for preventing or treating osteoarthritis.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a genetically engineered exosome for efficiently delivering a Cas9 ribonucleoprotein complex, wherein the genetically engineered exosome comprises an exosome and a Cas9 / sgRNA, wherein both ends of a characteristic protein CD63 of the exosome are respectively connected to a com nucleic acid aptamer binding protein, the sgRNA scaffold ST2 loop of the Cas9 / sgRNA is modified with a com nucleic acid aptamer, and the com nucleic acid aptamer binding protein binds to the com nucleic acid aptamer;

[0008] The nucleotide sequence of the com nucleic acid aptamer is shown in SEQ ID NO.1, and the amino acid sequence of the com nucleic acid aptamer binding protein is shown in SEQ ID NO.2.

[0009] Optionally, in the Cas9 / sgRNA, the nucleotide sequence of the sgRNA after modification by the com nucleic acid adaptor is as shown in SEQ ID NO.3.

[0010] Optionally, the exosomes are derived from 293T cells, and the exosomes are loaded with anti-inflammatory factor IL-10.

[0011] Alternatively, the present invention provides a genetically engineered exosome that efficiently delivers Cas9 ribonucleoprotein complexes, wherein the genetically engineered exosomes are secreted by 293T cells transfected with plasmids pY4 and pY5;

[0012] The pY4 is pY4-SB-EF1α-spCas9-Flag-IL-10-BleoR-PA, and the plasmid map is as follows Fig.12 As shown;

[0013] The pY5 is pY5-SB-U6-sgMMP-13-CMV-VSVG-com-CD63-com-PuroR-PA, and the plasmid map is as follows Fig.13 shown.

[0014] Based on the same inventive concept, the present invention provides a use of genetically engineered exosomes that efficiently deliver Cas9 ribonucleoprotein complexes in the preparation of drugs for preventing or treating osteoarthritis.

[0015] Based on the same inventive concept, the present invention provides a genetically engineered exosome hydrogel complex, the complex comprising GelMA-TA hydrogel microspheres and genetically engineered exosomes encapsulated in the GelMA-TA hydrogel microspheres, the genetically engineered exosomes being the above-mentioned genetically engineered exosomes that efficiently deliver Cas9 ribonucleoprotein complexes;

[0016] The GelMA-TA hydrogel microspheres are obtained by modifying tannic acid onto the surface of the GelMA hydrogel microspheres through Michael addition reaction.

[0017] Based on the same inventive concept, the present invention provides an application of a genetically engineered exosome hydrogel complex as or in the preparation of a drug for preventing or treating osteoarthritis.

[0018] Based on the same inventive concept, the present invention also provides a method for preparing genetically engineered exosomes that efficiently deliver Cas9 ribonucleoprotein complexes, the preparation method comprising:

[0019] Construction of genetic engineering plasmids pY4 and pY5;

[0020] Transfecting the pY4 and the pY5 into 293T cells to screen out stable monoclonal cell lines;

[0021] Cultivating the stably transfected monoclonal cell line, and then extracting genetically engineered exosomes;

[0022] Wherein, the pY4 is pY4-SB-EF1α-spCas9-Flag-IL-10-BleoR-PA, and the plasmid map is as follows Fig.12 As shown;

[0023] The pY5 is pY5-SB-U6-sgMMP-13-CMV-VSVG-com-CD63-com-PuroR-PA, and the plasmid map is as follows Fig.13 shown.

[0024] Furthermore, the transfection of the pY4 and the pY5 into 293T cells specifically includes:

[0025] The pY4 and the pY5 were transfected into 293T cells at a molar ratio of 1:1.

[0026] Furthermore, the stable monoclonal cell line is cultured and then genetically engineered exosomes are extracted, specifically comprising:

[0027] The stably transformed monoclonal cell line is cultured, and then the cell supernatant is collected, and the genetically engineered exosomes in the cell supernatant are extracted by ultracentrifugation.

[0028] Specifically, SEQ ID NO.1~3 are as follows:

[0029] SEQ ID NO.1: CUGAAUGCCUGCGAGCAUCCCAC;

[0030] SEQ ID NO.2:

[0031] MKSI RCKNCNKLLFKADSFDHI EI RCPRCKRVRYHI IMLNACEHPTEKHCGKREKITHSSDETVRY;

[0032] SEQ ID NO.3:

[0033] GGGACCATTTGAGTGTTCGAGTTTGAGAGCTAGAAATAGCAAGTTCAAATAAGGCTAGTCCGTTATCAACTTGGCTGA ATGCCTGCGAGCATCCCACCCAAGTGGCACCGAGTCGGTGCTTTTTT.

[0034] In SEQ ID NO.3, the bold sequence is the targeting sequence of sgRNA (sgMMP-13), and the sequence after the targeting sequence is the scaffold sequence modified by the com nucleic acid aptamer.

[0035] In the present invention, the nucleotide sequence of pY4 is shown as SEQ ID NO.4, and the nucleotide sequence of pY5 is shown as SEQ ID NO.5.

[0036] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0037] 1. The present invention provides a genetically engineered exosome that efficiently delivers Cas9 ribonucleoprotein complexes, which is obtained by secretion of 293T cells transfected with plasmids pY4 and pY5, wherein pY4 is pY4-SB-EF1α-spCas9-Flag-IL-10-BleoR-PA, so that the cells express spCas9 and I L-10, pY5 is pY5-SB-U6-sgMMP-13-CMV-VSVG-com-CD63-com-PuroR-PA, which makes the cells express sgMMP-13 modified by com nucleic acid aptamer and CD63 protein modified by com nucleic acid aptamer binding protein. The exosome characteristic protein CD63 is expressed on the surface of 293T cells. The com nucleic acid aptamer binding protein, namely com-CD63-com, is fused to both ends of CD63, which can attract sgMMP-13 modified by com nucleic acid aptamer into exosomes. sgMMP-13 then guides Cas9 protein into exosomes, thereby achieving efficient enrichment of Cas9 / sgMMP-13 ribonucleoprotein complex. Cas9 / sgMMP-13 can target the MMP-13 gene in cartilage for gene editing, inhibit the degradation of articular cartilage matrix and cartilage loss, which is conducive to achieving effective gene editing in vivo and improving the efficacy of gene therapy for osteoarthritis.

[0038] 2. The present invention discloses a genetically engineered exosome that efficiently delivers Cas9 ribonucleoprotein complex. After 293T cells are transformed by synthetic biology technology, the exosomes secreted by the cells can achieve efficient enrichment of Cas9 / sgMMP-13 ribonucleoprotein complex. At the same time, the engineered exosomes carry the anti-inflammatory factor IL-10 during the production process, and finally obtain Cas9 / IL-10Exo. Cas9 / IL-10Exo has a gene editing effect on cartilage matrix metalloproteinase MMP-13. At the same time, IL-10 inhibits the inflammation level in the joint to achieve a synergistic treatment effect for osteoarthritis, showing broad application prospects in the future treatment of osteoarthritis. In addition, Cas9 / IL-10Exo can also be encapsulated into tannic acid-modified hydrogel microspheres GelMA-TA through microfluidic technology to achieve a longer-lasting sustained release effect of exosomes in vivo.

[0039] 3. The present invention provides an application of a genetically engineered exosome that efficiently delivers a Cas9 ribonucleoprotein complex in the preparation of a drug for preventing or treating osteoarthritis. Through genetic engineering technology, 293T cells are designed to synthesize an engineered exosome, so that the exosome can efficiently enrich the Cas9 / sgMMP-13 ribonucleic acid protein complex. Cas9 / sgMMP-13 can target the MMP-13 gene in OA cartilage and inhibit the degradation of articular cartilage matrix. The anti-inflammatory factor IL-10 carried synchronously in the engineered exosome can act on joint synovial macrophages and promote the polarization of macrophages to the anti-inflammatory phenotype M2. After the engineered exosomes are encapsulated in the tannic acid-modified hydrogel microspheres Ge lMA-TA, a longer-lasting sustained release effect of the exosomes can be achieved in the body, thereby improving the efficacy of gene therapy for osteoarthritis, and providing a new research idea for gene therapy for osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 Schematic diagram of genetic engineering plasmid design and engineered exosome synthesis: A: represents sgRNA and nucleic acid aptamer modification; B: engineered plasmid linear map; C: engineered plasmid transfection and exosome extraction process; D: GelMA-TA encapsulation of exosomes process.

[0042] Figure 2The results of Cas9, sgMMP-13 and IL-10 content detection in engineered exosomes: Nat ive Exo represents exosomes secreted by natural 293T cells, IL-10Exo represents exosomes secreted by 293T cells after transfection with pY4 plasmid only, Cas9 / IL-10 represents exosomes secreted by 293T cells after transfection with pY4 and pY5, Maker represents the standard molecular weight of DNA; wherein, A: Cas9 protein enrichment effect in exosomes; B: sgMMP-13 expression in exosomes; C: Elisa detection of IL-10 content in exosomes.

[0043] Figure 3 The engineered exosomes produce gene editing effects on MMP-13 and promote the polarization of macrophages to M2: A: After co-incubation of engineered exosomes Cas9 / IL-10Exo with cells, MMP-13 undergoes insertion and deletion mutations; B: IL-10 in the engineered exosomes promotes the expression of CD206 on the surface of macrophages, inducing macrophages to polarize to M2.

[0044] Figure 4 The distribution diagram of fluorescently labeled engineered exosomes encapsulated in GelMA-TA hydrogel microspheres.

[0045] Figure 5 The in vitro degradation and release diagram of engineered exosomes after encapsulation in GelMA-TA hydrogel microspheres: A: release curve of exosomes from GelMA / GelMA-TA microspheres; B: degradation rate of GelMA / GelMA-TA before and after exosome encapsulation.

[0046] Figure 6 This is a diagram showing the retention effect of fluorescently labeled engineered exosomes encapsulated with GelMA-TA microspheres in the joint cavity before and after: A: the retention time of exosomes encapsulated with GelMA-TA in the rat joint cavity; B: the fluorescence change trend of exosomes in the rat joint cavity; C: the fluorescence distribution of various tissues and organs of the rat after the in vivo fluorescence observation.

[0047] Figure 7 This is a comparison of the Micro-CT results of the rat knee joints in each group after 4 weeks of treatment in Example 4. G1 refers to natural exosomes Nat ive Exo@GelMA-TA, G2 refers to IL-10Exo@GelMA-TA, and G3 refers to Cas9 / IL-10Exo@GelMA-TA.

[0048] Figure 8This is a comparison of the HE staining results of the rat knee joint repair after 4 weeks of treatment in each group in Example 4, G1 refers to natural exosomes Nat ive Exo@GelMA-TA, G2 refers to IL-10Exo@GelMA-TA, and G3 refers to Cas9 / IL-10Exo@GelMA-TA.

[0049] Fig. 9 This is a comparison of the SO / FG staining results of the rat knee joint repair after 4 weeks of treatment in each group in Example 4: G1 refers to natural exosomes Nat ive Exo@GelMA-TA, G2 refers to IL-10Exo@GelMA-TA, and G3 refers to Cas9 / IL-10Exo@GelMA-TA.

[0050] Fig.10 This is a comparison of the immunohistochemical staining results of MMP-13 gene after rat knee joint repair in each group after 4 weeks of treatment in Example 4: G1 refers to natural exosomes Nat ive Exo@GelMA-TA, G2 refers to IL-10Exo@GelMA-TA, and G3 refers to Cas9 / IL-10Exo@GelMA-TA.

[0051] Fig.11 The OARSI (Osteoarthritis Research Association International) scores and MMP-13 immunohistochemistry quantitative results of each group after 4 weeks of treatment in Example 4: the groups were healthy articular cartilage tissue (Sham), untreated OA joints (PBS), hydrogel microspheres encapsulating natural exosomes (Exo@GelMA-TA: G1), hydrogel microspheres encapsulating IL-10 exosomes (IL-10Exo@GelMA-TA: G2), and hydrogel microspheres containing Cas9 / IL-10Exo exosomes (Cas9 / IL-10Exo@GelMA-TA: G3). Among them, A: the knee joints of rats in each group were scored according to the Osteoarthritis Research Association International scoring criteria; B: the expression of MMP-13 in the knee cartilage tissue of rats.

[0052] Fig.12 This is a map of the plasmid pY4 of the present invention.

[0053] Fig.13 This is a map of the plasmid pY5 of the present invention. DETAILED DESCRIPTION

[0054] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0055] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0056] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0057] The overall idea of ​​the present invention is as follows:

[0058] The discovery of synthetic biology has promoted the modular construction of recombinant proteins, enabling cells to perform functions as designed, which provides a new idea for the engineering of exosomes. In the present invention, we use the idea of ​​synthetic biology to introduce the constructed gene route (plasmid pY4, pY5) into cells, so that the cells can secrete exosomes after performing functions as designed, and the cells can efficiently enrich Cas9 / sgMMP-13RNP into exosomes: com nucleic acid aptamers and nucleic acid aptamer binding protein interaction technology are used to achieve efficient enrichment of Cas9 / sgMMP-13 ribonucleoprotein complexes in exosomes. Based on the pathological environment of osteoarthritis studied, it is designed to load I L-10 synchronously into engineered exosomes to obtain engineered exosomes Cas9 / I L-10Exo. In order to improve the in vivo utilization of exosomes, we finally chose to encapsulate them into tannic acid-modified hydrogel microspheres (GelMA-TA) to achieve the long-term release of engineered exosomes Cas9 / I L-10Exo in the joint cavity for the treatment of osteoarthritis.

[0059] The present invention utilizes the interaction between com aptamers and aptamer binding proteins to design an engineered plasmid, so that after the plasmid is transfected, the cells can efficiently enrich the Cas9 RNP complex targeting the MMP-13 gene into exosomes according to the design, and simultaneously utilize the exosomes to synchronously carry the anti-inflammatory factor IL-10 to achieve a synergistic treatment effect for osteoarthritis, and then after in vitro detection of the gene editing activity and anti-inflammatory effect of the engineered exosomes, Cas9 / IL-10Exo is selected to be encapsulated into GelMA hydrogel microspheres, and the microspheres are modified with tannic acid (TA). The presence of TA can enhance the adhesion of the hydrogel microspheres to the tissue, and at the same time has a good free radical scavenging (ROS) effect. The encapsulated engineered exosomes Cas9 / I L-10Exo@GelMA-TA were injected in situ into the knee joints of osteoarthritis rats. Cas9 / I L-10Exo@GelMA-TA was able to adhere to the rat joint cavity, allowing Cas9 / I L-10Exo to be slowly released from the GelMA-TA hydrogel microspheres. The CRISPR / Cas9 RNP complex in Cas9 / I L-10Exo can enter damaged chondrocytes, target the MMP-13 gene, inhibit the degradation of the extracellular matrix and cartilage loss, and at the same time, the anti-inflammatory factor I L-10 in the exosomes can act on macrophages in the synovium of the joints, inducing macrophages to polarize to the anti-inflammatory phenotype M2, thereby achieving a comprehensive therapeutic effect for osteoarthritis.

[0060] The genetically engineered exosomes for efficiently delivering Cas9 ribonucleoprotein complexes and their applications of the present invention are described in detail below in combination with the examples and experimental data.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Example 1

[0063] This embodiment provides a method for preparing genetically engineered exosomes that efficiently deliver Cas9 ribonucleoprotein complexes (eg, Figure 1 ), as follows:

[0064] (1) Plasmid design and synthesis

[0065] The second loop of the sgRNA scaffold (scaffold) with the highest activity targeting rat MMP-13, i.e., ST2 loop, was modified with com nucleic acid aptamer, and U6 was used as the typical promoter of sgRNA. The CD63 amino acid sequence was downloaded from the NCBI website, and the com nucleic acid aptamer binding protein was fused to the N-terminus and C-terminus, respectively. The com binding protein and CD63 were connected by amino acid linker. CMV was selected as the promoter of com-CD63-com protein expression. The U6-sgMMP-13-CMV-com-CD63-com sequence (sgMMP-13 refers to the sequence modified by com nucleic acid aptamer) was obtained by Snap gene simulation. The p229 Sleeping Beauty plasmid was used as a vector (p229 is a transposable vector. Placing the target fragment on the Sleeping Beauty vector can enable the target sequence to be integrated into the genome). After Kpn I-SmaI cutting, the target fragment U6-sgMMP-13-CMV-VSVG-com-CD63-com was inserted. pY4-SB-EF1α-spCas9-Flag-P2A-IL-10-BleoR-PA was obtained by Infusion cloning technology.

[0066] (2) Cell transfection and stable monoclonal cell line screening

[0067] First, replace the cell culture medium with an incomplete medium without serum and antibiotics. Take two clean sterile centrifuge tubes and add 125 μL of each Med i um, then add 2.5μg plasmid DNA to one of the tubes. When two plasmids are co-transfected, transfect them at a ratio of 1:1, and mix them gently with a gun; add 5μL Lipo293 transfection reagent to the other tube, and mix them gently with a gun. Pay special attention not to vortex or centrifuge. Gently add the culture solution containing DNA to the culture solution containing Lipo293 transfection reagent with a gun, gently invert the centrifuge tube or mix them gently with a gun, and let it stand at room temperature for 15 minutes. Slowly add the prepared transfection reagent to the 293T cell well plate in good condition, mark it, and gently shake the culture solution to make the transfection reagent evenly distributed in the culture medium. After 6 hours, aspirate the liquid in the well plate and replace it with DMEM complete culture medium with serum and antibiotics.

[0068] After transfection, the cells were digested and re-plated into the well plate, so that the cell fusion degree was about 25% when the screening began. Bleomycin and puromycin were added to make the final concentrations of 400μg / mL and 3μg / mL respectively. The cell culture medium was replaced every two days, and the drug was added again for screening. The cells that survived after one week were the cells that stably expressed the target gene (i.e., the stably transfected monoclonal cell line). When the cells were full, they were used to verify the expression and perform monoclonal screening.

[0069] (3) Engineering exosome extraction

[0070] After the cells adhered, the culture medium was removed and an appropriate amount of PBS buffer was added to completely remove the fetal bovine serum in the culture medium. 10 mL of DMEM complete culture medium prepared with exosome-free serum was added to the cells in a 10 cm culture dish and incubated at 37 °C with 5% CO 2 Culture in a cell culture incubator, and the cell fusion degree is above 70%. Collect the cell supernatant after 48 hours. First, centrifuge the collected cell supernatant at 300xg for 10min, then centrifuge at 3000xg for 10min, and then centrifuge at 10,000xg for 30min. Finally, filter the collected supernatant with a 0.22μm filter membrane. Store the collected liquid in a -80℃ refrigerator for later use or extract exosomes by high-speed centrifugation after ultrafiltration concentration. Concentrate about 5 times with a 100kda ultrafiltration tube, and centrifuge at 120,000xg for 70min after concentration. Then resuspend in PBS and continue to centrifuge at 120,000xg for 70min to remove impurities. Finally, resuspend with a small amount of PBS to obtain the exosome sample. The extracted exosomes are measured by the BCA method and stored at -80℃.

[0071] The method for preparing exosome-free serum is as follows: normal serum is placed in an ultracentrifuge tube, centrifuged at 120,000 x g and 4°C for 14 hours, and after centrifugation, the liquid is found to be divided into three layers, the first layer is colorless and clear, the second layer is light yellow and clear, and the third layer is not clear and dark yellow. Exosomes in serum are enriched at the bottom and wall of the tube. When the serum is drawn, only the clarified part of the serum is drawn, and the wall and bottom of the tube are avoided. Only 70-80% of the clarified serum is drawn. The whole process is gentle and slow to obtain exosome-free serum.

[0072] The levels of Cas9 and sgMMP-13 in engineered exosomes were determined by Western blot and q-PCR techniques, and the content of IL-10 in engineered exosomes was detected by Eli sa. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that compared with other control groups, the engineered exosome Cas9 / IL-10 achieved efficient enrichment of Cas9 protein ( Figure 2 A), and strong expression of sgRNA was detected ( Figure 2 B), ELISA was used to detect the IL-10 content in exosomes ( Figure 2 C), confirming that engineered exosomes effectively enriched IL-10.

[0073] (4) In vitro functional verification of engineered exosomes

[0074] The T7E1 experiment was used to evaluate the editing efficiency of engineered exosomes on target genomic sites. Rat chondrosarcoma cells were plated in 6-well plates, with 200,000 cells per well. After the cells adhered, the cell culture medium was replaced with serum-free or low-serum DMEM culture medium one hour in advance. 200 μg of exosome samples were taken from each group and incubated with cells. After 48 hours, the cell DNA was collected and the target site sequence was amplified by specific primers. The amplified fragments were recovered by the gel recovery kit, and 200 μg of the recovered DNA fragments were taken and operated according to the instructions of the T7EI kit. The sites where gene editing occurs can be specifically recognized and sheared by the T7EI enzyme.

[0075] BMDM were selected and induced to differentiate into macrophages by M-CSF. In order to detect the effect of IL-10 in exosomes on the phenotypic changes of macrophages, BMDM were stimulated with LPS (100 ng / mL) and IL-4 (20 ng / mL) at 100 μg for 48 hours. Flow cytometry was used to detect cell polarization. The results are shown in Figure 3 .from Figure 3 It can be seen that engineered exosomes containing IL-10 can promote the expression of CD206 on the surface of macrophages, drive macrophages to polarize toward M2, and thus effectively inhibit inflammation.

[0076] Example 2

[0077] The engineered exosomes prepared in Example 1 were labeled with a cell membrane red fluorescent probe (Di I), 200 μg of the labeled exosomes were added to a 10% GelMA (methacrylated gelatin) solution and mixed evenly, and 0.5% of the photoinitiator LAP was added to the GelMA solution. The hydrogel microspheres were synthesized using microfluidics technology, and the distribution of exosomes in the microspheres was observed using a fluorescence microscope.

[0078] Among them, the specific operation method of synthesizing hydrogel microspheres using microfluidic technology is:

[0079] Prepare the aqueous phase: add Di I-labeled exosomes to 10% GelMA solution and mix well, filter with a 0.22μm syringe filter, and then add 0.5% LAP photoinitiator. Prepare the oil phase: take 20mL of liquid paraffin and add 1% Span80. Connect the microfluidic device, and after observing the appearance of microspheres, use UV irradiation to promote the cross-linking of microspheres into gel. Use acetone to remove the oil phase on the surface of the microspheres, and then use PBS to wash away the acetone, and rinse multiple times until the oil phase is completely washed away.

[0080] The synthesis method of GelMA gel is as follows:

[0081] Prepare 0.25 M carbonate buffer: weigh 7.95 g sodium carbonate and 14.65 g sodium bicarbonate and dissolve them in 1 L deionized water, and adjust the pH to 9.

[0082] Synthesis of GelMA gel: Weigh 10g gelatin and add 100mL of prepared carbonate buffer, heat to 90℃, stir continuously at 500rpm until the gelatin is completely dissolved, add 1mL of methacrylic anhydride when the temperature drops to 50℃, start the reaction for 3h, then add 200mL of preheated deionized water to dilute and stop the reaction. Then dialyze for 3 days and freeze-dry for storage. (10% GelMA solution is the solution obtained by dissolving GelMA gel freeze-dried powder in water)

[0083] The results are as follows Figure 4 As shown: After the engineered exosomes were labeled with Di I, they were successfully encapsulated into hydrogel microspheres using microfluidic technology. Under a fluorescence microscope, it was observed that the hydrogel was filled with red exosomes, proving that the exosome-loaded hydrogel was successfully prepared. TA modification of the hydrogel microspheres did not significantly affect the exosomes in the hydrogel.

[0084] Among them, the specific operation method of TA modified hydrogel microspheres is:

[0085] Under alkaline conditions, the hydrogel was modified by utilizing the Michael addition reaction between the amine group on GelMA and the catechol double bond on TA: 0.5 g of Cas9 / IL-10Exo@GelMA microspheres were added to the pH = 8.5 tannic acid solution, and the reaction was slowly shaken in a horizontal shaker for 3 hours, while keeping the pH constant during the reaction. After the reaction, the microspheres were washed with PBS to wash away the unchelated tannic acid on the surface of the microspheres, and the Cas9 / IL-10Exo@GelMA-TA microspheres were obtained and stored in PBS at 4°C.

[0086] Exo@GelMA-TA was dispersed in 2 μg / mL type II collagenase solution and placed in a shaker at 60 rpm and 37 °C. The supernatant was removed at predetermined time intervals, and the hydrogel microspheres were gently rinsed twice with deionized water. The samples were then freeze-dried to obtain the residual weight and subsequently redispersed in fresh collagenase solution. Each group was repeated three times. The microsphere degradation rate was calculated as follows:

[0087] 100 mg of Exo@GelMA and Exo@GelMA-TA were placed in a transwell (8 μm pores) and immersed in PBS (pH 7.4) at 37°C and 60 rpm vibration until drug release was complete. The supernatant was collected after incubation for 1, 3, 5, 7, 9, 11, 13, and 15 days. The concentration of the collected supernatant was detected using a BCA protein detection kit, and the release amount and ratio of each test were recorded to draw the exosome release curve. The results are shown in Figure 2. Figure 5 .from Figure 5 It can be seen that in the simulated environment in vitro, the Exo@GelMA-TA microspheres were completely released in about two weeks ( Figure 5 A). The Exo release efficiency of Exo@GelMA and Exo@GelMA-TA was about 80%, but compared with Exo@GelMA, Exo@GelMA-TA showed a slow release trend; in the in vitro degradation experiment, the degradation rate of GelMA alone was faster, while the GelMA microspheres modified with TA showed a more uniform degradation trend ( Figure 5 B). Therefore, Exo@GelMA-TA microspheres exhibited slow and stable release and degradation characteristics.

[0088] Example 3

[0089] The exosomes prepared in Example 1 were labeled with DiD fluorescent dye, and DiD-Exo@GelMA-TA was prepared by microfluidic technology. The experimental groups were set as: DiD, DiD-Exo, DiD-Exo@GelMA-TA, and they were injected into the rat joint cavity in situ after anesthesia with isoflurane. At different time points, the fluorescence changes of injected DID, DiD-Exo, and DiD-Exo@GelMA-TA were observed by the in vivo fluorescence imaging system IVIS at an excitation wavelength of 754nm and an emission wavelength of 778nm to determine the retention of exosomes in the rat knee joint.

[0090] After the observation, the joints and other organs were removed to observe the retention of exosomes in the knee joint and whether there was distribution in other organs.

[0091] The results are as follows Figure 6 As shown: Encapsulating engineered exosomes into hydrogel microspheres can significantly prolong the retention time of exosomes in the joints, and the exosomes are only retained in the joints.

[0092] Example 4

[0093] The hydrogel microspheres Cas9 / IL-10Exo@GelMA-TA loaded with engineered exosomes prepared in Example 2 were injected into the joints of osteoarthritis rats once a week (each injection volume was 100 μl Exo@GelMA-TA), and samples were collected 4 weeks after injection. The harvested bone joints were subjected to Micro-CT joint analysis. The bone joints were then decalcified and embedded for sectioning, and HE and safranin fast green histological staining was performed to observe the joint repair, and MMP-13 tissue immunostaining was performed to observe the expression of MMP-13 in cartilage tissue.

[0094] The results are as follows Figure 7-11 As shown: The recovery of the knee joints of rats after 4 weeks of treatment was observed. Micro-CT results showed that in the osteoarthritis PBS treatment group, bone joints were destroyed, obvious osteophytes were produced (red arrows indicate osteophytes) and the joint surface was uneven. In comparison, the Sham group presented a complete knee joint with a smooth and complete joint surface. In the treatment group, the osteophytes in the Cas9 / IL-10Exo@GelMA-TA treatment group were significantly reduced, and the joint surface was relatively smooth and complete. Pathological examination further proved that the bone and joint recovery of Cas9 / IL-10Exo@GelMA-TA was good. Safranin fast green staining SO&FG proved that Cas9 / IL-10Exo@GelMA-TA had a better cartilage recovery effect than other treatment groups. Finally, immunohistochemistry was used to observe that the expression level of MMP-13 in the cartilage tissue after Cas9 / IL-10Exo@GelMA-TA treatment was significantly reduced. The above experimental results show that Cas9 / IL-10Exo@GelMA-TA can promote cartilage regeneration and achieve the treatment of osteoarthritis by inhibiting the expression of MMP-13 in cartilage tissue.

[0095] Among them, the preparation methods of G2: natural exosomes Nat ive Exo@GelMA-TA and G2: IL-10Exo@GelMA-TA are the same as Cas9 / IL-10Exo@GelMA-TA except that the types of exosomes obtained are different. Nat iveExo are exosomes secreted by natural 293T cells, and IL-10Exo are exosomes secreted by 293T cells transfected with plasmid pY4 only.

[0096] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0097] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A genetically engineered exosome for efficient delivery of Cas9 ribonucleoprotein complex, characterized in that: The genetically engineered exosomes include exosomes and Cas9 / sgRNA, the two ends of the characteristic protein CD63 of the exosomes are respectively connected to com nucleic acid aptamer binding proteins, the sgRNA scaffold ST2 loop of the Cas9 / sgRNA is modified with com nucleic acid aptamer, and the com nucleic acid aptamer binding protein binds to the com nucleic acid aptamer; The nucleotide sequence of the com nucleic acid aptamer is shown in SEQ ID NO.1, and the amino acid sequence of the com nucleic acid aptamer binding protein is shown in SEQ ID NO.

2.

2. A genetically engineered exosome for efficiently delivering Cas9 ribonucleoprotein complex according to claim 1, characterized in that: In the Cas9 / sgRNA, the nucleotide sequence of the sgRNA after modification by the com nucleic acid adaptor is shown in SEQ ID NO.

3.

3. A genetically engineered exosome for efficiently delivering Cas9 ribonucleoprotein complex according to claim 1 or 2, characterized in that: The exosomes are derived from 293T cells and are loaded with the anti-inflammatory factor IL-10.

4. A genetically engineered exosome for efficient delivery of Cas9 ribonucleoprotein complex, characterized in that: The genetically engineered exosomes are obtained by secretion from 293T cells transfected with plasmids pY4 and pY5; The pY4 is pY4-SB-EF1α-spCas9-Flag-IL-10-BleoR-PA, and the plasmid map is shown in FIG12 ; The pY5 is pY5-SB-U6-sgMMP-13-CMV-VSVG-com-CD63-com-PuroR-PA, and the plasmid map is shown in Figure 13.

5. Use of a genetically engineered exosome that efficiently delivers Cas9 ribonucleoprotein complex according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating osteoarthritis.

6. A genetically engineered exosome hydrogel complex, characterized in that: The complex comprises GelMA-TA hydrogel microspheres and genetically engineered exosomes encapsulated in the GelMA-TA hydrogel microspheres, wherein the genetically engineered exosomes are genetically engineered exosomes for efficiently delivering Cas9 ribonucleoprotein complexes as described in any one of claims 1 to 4; The GelMA-TA hydrogel microspheres are obtained by modifying tannic acid onto the surface of the GelMA hydrogel microspheres through Michael addition reaction.

7. Use of a genetically engineered exosome hydrogel complex as claimed in claim 6 as or in the preparation of a drug for preventing or treating osteoarthritis.

8. A method for preparing genetically engineered exosomes for efficiently delivering Cas9 ribonucleoprotein complexes according to any one of claims 1 to 4, characterized in that: The preparation method comprises: Construction of genetic engineering plasmids pY4 and pY5; Transfecting the pY4 and the pY5 into 293T cells to screen out stable monoclonal cell lines; Cultivating the stably transfected monoclonal cell line, and then extracting genetically engineered exosomes; Wherein, the pY4 is pY4-SB-EF1α-spCas9-Flag-IL-10-BleoR-PA, and the plasmid map is shown in FIG12 ; The pY5 is pY5-SB-U6-sgMMP-13-CMV-VSVG-com-CD63-com-PuroR-PA, and the plasmid map is shown in Figure 13.

9. The method for preparing genetically engineered exosomes for efficiently delivering Cas9 ribonucleoprotein complex according to claim 8, characterized in that: The transfection of the pY4 and the pY5 into 293T cells specifically comprises: The pY4 and the pY5 were transfected into 293T cells at a molar ratio of 1:

1.

10. The method for preparing genetically engineered exosomes for efficiently delivering Cas9 ribonucleoprotein complex according to claim 8, characterized in that: The step of culturing the stably transfected monoclonal cell line and then extracting genetically engineered exosomes specifically includes: The stably transformed monoclonal cell line is cultured, and then the cell supernatant is collected, and the genetically engineered exosomes in the cell supernatant are extracted by ultracentrifugation.

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