Exosomes derived from bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein and their application

Abstract

The present invention provides a bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein and its application, wherein the bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein can greatly improve its targeting ability to bone marrow mesenchymal stem cells, and the exosome containing both has the strongest ability to target bone marrow mesenchymal stem cells, proving the synergistic targeting characteristics of the two. Then, the Cas9 / sgRNA protein complex is loaded into the bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein by electroporation to prepare a dual-targeted engineered exosome with gene editing ability, which can effectively knock out the expression of ID1 protein in bone marrow mesenchymal stem cells, thereby affecting the regulatory effect of stem cells on hematological malignancies, thereby providing a new strategy for the treatment of hematological malignancies.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein and applications thereof. Background Art

[0002] Hematological malignancies are a common type of cancer, including leukemia, lymphoma, etc. The bone marrow microenvironment is one of the important factors in the occurrence and development of hematological malignancies, and mesenchymal stem cells are an important cell type in the bone marrow microenvironment. They can regulate the growth and differentiation of tumor cells by secreting various cytokines and growth factors, thereby promoting the development of tumors. By regulating the activity of bone marrow mesenchymal stem cells, the progression of hematological malignancies may be affected. At present, the treatment methods for hematological malignancies mainly include chemotherapy, radiotherapy, immunotherapy, etc., but these methods often cause certain damage to normal cells and have a high recurrence rate. Therefore, the development of a treatment method that can specifically target and regulate mesenchymal stem cells is of great significance for improving the treatment effect of hematological malignancies.

[0003] Exosomes are tiny vesicles secreted by cells. They can carry various biologically active substances in cells, such as proteins and RNA, to achieve information transmission and material exchange between cells. In recent years, the application of exosomes in disease diagnosis, treatment and prevention has received widespread attention. It is worth exploring whether exosomes can be used as carriers of gene editing systems, and whether the targeting of exosomes can be improved through genetic engineering and chemical modification to specifically regulate the activity of bone marrow mesenchymal stem cells. However, one of the difficulties of exosomes is their targeting. How to provide a substance that can change the targeting of exosomes, so that exosomes can specifically target and regulate exosomes in the bone marrow microenvironment of hematological malignancies, in order to provide a new strategy for the treatment of hematological malignancies. Summary of the invention

[0004] The purpose of the present invention is to provide a bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein and an application thereof. The loading of the Lamp2b-CXCR4 fusion protein of the present invention in the exosome can change the targeting of the exosome, and its loading amount determines the targeting intensity to inflammatory vascular endothelial cells. Furthermore, the bone marrow mesenchymal stem cell-derived exosome expressing the Lamp2b-CXCR4 fusion protein of the present invention can effectively knock out the expression of ID1 protein in bone marrow mesenchymal stem cells, thereby affecting the regulatory effect of stem cells on hematological malignancies, thereby providing a new strategy for the treatment of hematological malignancies.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a Lamp2b-CXCR4 fusion protein is provided, wherein the amino acid sequence of the Lamp2b-CXCR4 fusion protein is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:MCLSPVKGAKLILIFLFLGAVQSNALIVNLTDSKGTCLYAEWEMNFTITYE TTNQTNKTITIAVPDKATHDGSSCGDDRNSAKIMIQFGFAVSWAVNFTKEASHYSIHDIVLSYNTSDSTVFPGAVAKGVHTVKNPENFKVPLDVIFKCNSVLTYNLTPVVQKYWGIHLQAFVQNGTVSKNEQVCEEDQTPTTVAPIIHTTAPSTTTTLTPTSTTPTPTPTPTVGNYSIRNG NTTCLLATMGLQLNITEEKVPFIFNINPATTNFTGSCQPQSAQLRLNNSQIKYLDFIFAVKNEKRFYLKEVNVYMYLANGSAFNISNKNLSFWDAPLGSSYMCNKEQVLSVSRAFQINTFNLKVQPFNVTKGQYSTAEECAADSDLNFLIPVAVGVALGFLIIAVFISYMIGRRKSRTGYQS VGGSGGMEPISVSIYTSDNYSEEVGSGDYDSNKEPCFRDENVHFNRIFLPTIYFIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAMADWYFGKFLCKAVHIIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKAVYVGVWIPALLLTIPDFIF ADVSQGDISQGDDRYICDRLYPDSLWMVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILAFFACWLPYYVGISIDSFILLGVIKQGCDFESIVHKWISITEALAFFHCCLNPILYAFLGAKFKSSAQHALNSMSRGSSLKILSKGKRGGHSSVSTESESSSFHSS

[0008] The amino acid sequence of the p-selectin targeting polypeptide is DAEWVDVS.

[0009] In the second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the Lamp2b-CXCR4 fusion protein.

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2.

[0011] In the third aspect of the present invention, a vector or an engineered bacterium or a cell line expressing the Lamp2b-CXCR4 fusion protein is provided.

[0012] In the fourth aspect of the present invention, a bone marrow mesenchymal stem cell expressing Lamp2b-CXCR4 fusion protein is provided. The bone marrow mesenchymal stem cell expressing Lamp2b-CXCR4 fusion protein is obtained by transfecting bone marrow mesenchymal stem cells with a lentivirus expressing the Lamp2b-CXCR4 fusion protein.

[0013] In the fifth aspect of the present invention, a bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein is provided, wherein the exosome is an extracellular vesicle obtained by centrifugation extraction of the cell culture supernatant collected after culturing the bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein.

[0014] Furthermore, the method for preparing bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein comprises:

[0015] Obtaining MSCs expressing Lamp2b-CXCR4 fusion protein: A nucleic acid molecule expressing Lamp2b-CXCR4 fusion protein is obtained by an in vitro de novo synthesis step, and sequences complementary to the bases of a part of the sequence of a lentiviral vector plasmid are added to both ends of the molecule sequence. The nucleic acid molecule sequence is constructed into a lentiviral vector plasmid by a DNA homologous recombinase, and then combined with packaging plasmids such as PMD2G and PSPAX2 to co-transfect HEK293 cell lines, and the exosomes produced after the lentivirus containing the target nucleic acid molecule (carrying a puromycin resistance gene) is transfected into MSCs can target CXCL12 (highly expressed in bone marrow mesenchymal stem cells);

[0016] The above technical solution specifically includes:

[0017] The bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein are taken, the cell culture supernatant is collected, and the extracellular vesicles are extracted by gradient centrifugation. After gradient centrifugation, exosomes derived from bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein are obtained.

[0018] Preferably, the gradient centrifugation method comprises: centrifuging the cell culture supernatant at 300G for 10 minutes, then centrifuging at 2000G for 20 minutes, then taking the supernatant and centrifuging at 10000G for 30 minutes, taking the supernatant, filtering it through a 0.22 μm filter membrane, and then continuing to centrifuge at 110000G for 70 minutes, and the precipitate is the exosomes secreted by the MSCs; preferably,

[0019] Preferably, exosomes are extracted from MSCs expressing Lamp2b-CXCR4 fusion protein after puromycin selection.

[0020] In a sixth aspect of the present invention, the present invention also provides a p-selectin targeted exosome, the preparation method of which comprises:

[0021] The peptide DAEWVDVS was co-incubated with DMPE-PEG2000 lipid to obtain

[0022] DMPE-PEG2000-DAEWVDVS

[0023] The bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein are co-incubated with the DMPE-PEG2000-DAEWVDVS to obtain p-selectin-targeted exosomes.

[0024] In the above technical scheme, DAEWVDVS is linked to DMPE-PEG2000 lipids through covalent bonds to obtain DMPE-PEG2000-DAEWVDVS, which is then incubated with exosomes derived from bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein at room temperature and passively mounted in exosomes to obtain targeted exosomes targeting p-selectin;

[0025] The DMPE-PEG2000 lipid may be DMPE-PEG2000-Mal (Cat. No. P007009, Shanghai Tuoyang Biotechnology Co., Ltd.);

[0026] The bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein and DMPE-PEG2000-DAEWVDVS were incubated in a PBS solution at room temperature overnight at a protein concentration mass ratio of 1:5 (i.e., the ratio of the total protein mass in the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein to the mass of the polypeptide DAEWVDVS is 1:4-1:6, preferably 1:5), and the supernatant was obtained by centrifugation at 110000G for 70 minutes. The resulting precipitate is the p-selectin targeted exosomes.

[0027] The bone marrow mesenchymal stem cells are derived from mouse bone marrow, and can also be replaced by embryonic mesenchymal stem cells or adipose mesenchymal stem cells.

[0028] In a seventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0029] Cas9 / sgRNA protein complex;

[0030] The bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein or the p-selectin-targeted exosomes.

[0031] Optionally, the Cas9 / sgRNA protein complex is loaded into the exosomes by electroporation; preferably, the setting parameters of electroporation are a voltage of 100 volts and a current stimulation time of 0.1 milliseconds.

[0032] As an optional factual mode, when the pharmaceutical composition is a Cas9 / sgRNA protein complex and the p-selectin targeted exosomes, the mass ratio of the protein in the p-selectin targeted exosomes, the Cas9 / sgRNA protein and the DMPE-PEG2000-DAEWVDVS polypeptide in the p-selectin targeted exosomes is 1:15~40:5~15.

[0033] Furthermore, the mass ratio of the Cas9 / sgRNA complex to the total protein of the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein is 1:15-40.

[0034] Preferably, the mass ratio of the Cas9 protein to the total protein of the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein is 0.01 to 0.1:1.

[0035] Furthermore, the sgRNA targeting sequence in the Cas9 / sgRNA complex is shown in SEQ ID NO.3, and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.4. The sequence of the sgRNA is shown in SEQ ID NO.5.

[0036] Optionally, the Cas9 / sgRNA protein complex is provided by co-incubating the Cas9 protein and sgRNA in an aqueous solution. Preferably, the Cas9 protein is expressed and purified from Escherichia coli, and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.4.

[0037] Preferably, the pharmaceutical composition can also be prepared into a pharmaceutical preparation, comprising the above-mentioned pharmaceutical composition and further comprising pharmaceutically acceptable excipients.

[0038] Preferably, the pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

[0039] Preferably, the pharmaceutical preparation is selected from an injection, an oral preparation or an external preparation.

[0040] More preferably, the injection is selected from injection solution or powder injection; the oral preparation is selected from tablets, solutions, capsules, powders, pills, granules, syrups, suspensions or oral sustained-release preparations; the external preparation is selected from ointments, sprays or patches.

[0041] In an eighth aspect of the present invention, a dual-targeting engineered exosome with gene editing capability is provided, wherein the exosome comprises the pharmaceutical composition.

[0042] That is, the dual-targeted engineered exosomes with gene editing capability may be composed of a Cas9 / sgRNA protein complex and bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein;

[0043] Alternatively, the dual-targeting engineered exosomes with gene editing capabilities may be composed of a Cas9 / sgRNA protein complex and the p-selectin targeted exosomes.

[0044] In the above technical solution, the Cas9 / sgRNA protein complex is encapsulated in exosomes by electroporation.

[0045] Furthermore, the protein in the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein, the protein of the Cas9 / sgRNA, and the targeted exosomes of the p-selectin

[0046] The mass ratio of the polypeptide of DMPE-PEG2000-DAEWVDVS is 1:15~40:5~15.

[0047] Furthermore, the dual-targeting engineered exosomes with gene editing capability also include an aqueous solution, and the aqueous solution is selected from water, physiological saline, glucose aqueous solution or cell culture medium.

[0048] In the ninth aspect of the present invention, provided are the bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein, the exosomes derived from bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein, the pharmaceutical composition, and the use of the engineered exosomes in the preparation of drugs for treating hematological malignancies.

[0049] The above-mentioned anti-hematological malignancies include myelodysplastic syndrome, myeloproliferative neoplasms, acute myeloid leukemia, multiple myeloma and lymphoma.

[0050] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0051] The present invention provides bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein and its application. First, the present application finds that bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein can greatly improve its targeting ability to bone marrow mesenchymal stem cells, and exosomes containing both have the strongest ability to target bone marrow mesenchymal stem cells, proving the synergistic targeting characteristics of the two. Then, the Cas9 / sgRNA protein complex is loaded into the bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein by electroporation to prepare dual-targeted engineered exosomes with gene editing ability, which can effectively knock out the expression of ID1 protein in bone marrow mesenchymal stem cells, thereby affecting the regulatory effect of stem cells on hematological malignancies, thereby providing a new strategy for the treatment of hematological malignancies.

[0052] The preparation method of dual-targeted exosomes with gene editing function provided by the present invention has the advantages of simple operation, low cost, etc., and has important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] 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.

[0054] Figure 1 This is an image of bone marrow mesenchymal stem cells (MSCs) expressing Lamp2b-CXCR4 screened in Experimental Example 1 of the present invention;

[0055] Figure 2 This is a schematic diagram of the research on separation and preparation of dual-targeted exosomes in Experimental Example 2 of the present invention;

[0056] Figure 3 It is a western blotting image verifying the high expression of CXCR4 in MSCs-derived exosomes in Experimental Example 3 of the present invention; wherein A is a transmission electron microscopy schematic diagram of exosomes, and B is the expression of exosome-related protein markers and CXCR4;

[0057] Figure 4 This is a confocal image verifying the efficient targeting of CXCR4-expressing exosomes C-EX to bone marrow mesenchymal stem cells in Experimental Example 4 of the present invention;

[0058] Figure 5 It is a flow cytometry image verifying that DMPE-PEG2000-DAEWVDVS can be efficiently inserted into C-EX in Experimental Example 5 of the present invention; wherein A is a typical fluorescence intensity distribution diagram of FITC of exosomes after adding different proportions of DMPE-PEG2000-DAEWVDVS-FITC to exosomes by flow cytometry; B is the average fluorescence intensity of FITC of exosomes after adding different proportions of DMPE-PEG2000-DAEWVDVS-FITC to exosomes by flow cytometry;

[0059] Figure 6 This is a confocal image of Experimental Example 6 of the present invention verifying that the dual-targeted exosomes CP-EX can efficiently target inflammatory vascular endothelial cells;

[0060] Figure 7 This is a picture of the in vivo imaging results of small animals in experimental example 7 of the present invention verifying the in vivo targeting of exosomes of different groups such as simple exsomes, C-EX, P-EX and CP-EX in leukemia model mice;

[0061] Figure 8 This is a diagram showing the results of verifying the targeting of different groups such as exsome, C-EX, P-EX and CP-EX to bone marrow mesenchymal stem cells in leukemia model mice by flow cytometry in Experimental Example 8 of the present invention;

[0062] Fig. 9 This is a western blotting image of Experimental Example 9 of the present invention verifying that CP-EX carries Cas9 / sgRNA through electroporation;

[0063] Fig.10 This is an image of the results of validating the efficiency of Cas9@CP-EX in knocking out the ID1 gene in bone marrow mesenchymal stem cells in Experimental Example 10 of the present invention;

[0064] Fig.11 This is a result graph of the experimental example 11 of the present invention verifying that CP-EX and Cas9@CP-EX prolong the survival curve of leukemia model mice through tail vein injection. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0068] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0069] How to induce the secretion of target proteins into exosomes, change the targeting of exosomes, and improve the targeting intensity to inflammatory vascular endothelial cells has become a difficulty in the preparation of exosomes.

[0070] The inventors of this application discovered that:

[0071] (1) The role of the exosome sorting protein Lamp2b in inducing the secretion of target proteins into exosomes.

[0072] (2) Exosomes containing CXCR4 target MSCs more efficiently;

[0073] (3) The loading of DAEWVDVS peptide (p-selectin targeting peptide) in exosomes can change the targeting of exosomes, and its loading amount determines the targeting intensity to inflammatory vascular endothelial cells;

[0074] Next, the inventors of the present application formed a fusion protein with CXCR4 and Lamp2b (exosome sorting protein), and the exosomes produced after transfection of MSCs with a three-plasmid system lentivirus (carrying a puromycin resistance gene) could target CXCL12 (highly expressed in bone marrow mesenchymal stem cells);

[0075] In the above technical scheme, DAEWVDVS is connected to DMPE-PEG2000 lipid through a covalent bond, and after incubation with exosomes at room temperature, it is passively mounted in the exosomes to obtain targeting to p-selectin; the bone marrow mesenchymal stem cells are derived from mouse bone marrow and can also be replaced by embryonic mesenchymal stem cells or adipose mesenchymal stem cells.

[0076] Furthermore, the Cas9 / sgRNA protein complex for knocking out the ID1 protein is loaded on the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein to prepare dual-targeted engineered exosomes with gene editing capabilities, which can effectively knock out the expression of ID1 protein in bone marrow mesenchymal stem cells, thereby affecting the regulatory effect of stem cells on hematological malignancies.

[0077] In other embodiments, the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein can be loaded with other target proteins.

[0078] As a specific embodiment, the method for preparing the dual-targeting exosomes with gene editing function may specifically include the following steps:

[0079] (1) Obtain mouse bone marrow-derived mesenchymal stem cells, construct a lentiviral plasmid containing Lamp2b-CXCR4 protein (with a puromycin resistance gene), use a three-plasmid lentiviral system and HEK293T cells to form a lentiviral system that can transfect cells, and use the virus combined with puromycin to select MSCs cell lines that highly express Lamp2b-CXCR4 protein, collect cell culture supernatants, and obtain exosomes by density gradient centrifugation. The gradient centrifugation method is as follows: the cell culture supernatant is centrifuged at 300G for 10 minutes, then at 2000G for 20 minutes, and then the supernatant is centrifuged at 10000G for 30 minutes, the supernatant is taken, filtered through a 0.22μm filter membrane, and then centrifuged at 110000G for 70 minutes. The precipitate is the exosomes secreted by the MSCs (C-EX); the exosomes are incubated with DMPE-PEG2000-DAEWVDVS in a PBS solution at room temperature overnight at a protein concentration mass ratio of 1:5, the supernatant is obtained, and centrifuged at 110000G for 70 minutes to obtain dual-targeted exosomes (CP-EX) expressing CXCR4 and targeting p-selectin; thereafter, the precipitate is washed twice with sterile saline, and finally resuspended in 1mL of sterile saline and placed at 4°C for use.

[0080] (2) The present invention uses a homemade Cas9 / sgRNA protein complex that can specifically knock out the ID1 gene, and its specific preparation method is as follows:

[0081] The Cas9 protein sequence with the amino acid sequence of nuclear entry function was constructed in a prokaryotic plasmid (with kanamycin resistance), and BL21 Escherichia coli was transfected by heat shock method to screen the BL21 strain expressing Cas9 protein, and then the BL21 strain was expanded using conventional culture medium; the broken strain was collected, and the supernatant was taken, and the Cas9 protein with a purity of up to 95% WT was obtained based on histidine tag affinity purification, anion and cation column purification and desalting column purification. The Cas9 protein was combined with a synthetic backbone sequence containing

[0082] sgRNA (GAACCGCAAAGTGAGCAAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC, SEQ ID NO.5) was incubated in an aqueous solution at a molar concentration ratio of 1:5 overnight, and the Cas9 / sgRNA protein complex containing the sgRNA sequence was obtained using fast liquid chromatography, which was concentrated and configured and placed at 4°C for use.

[0083] (3) CP-EX was prepared into 1 mg / mL protein concentration and Cas9 / sgRNA protein complex was prepared into 1 mg / mL solution. The two were mixed and electroporated in a biorad electroporator with the operating parameters of 100 volts, 0.1 millisecond current pulse stimulation, and 20 consecutive electric pulses. The supernatant was obtained and centrifuged at 110000G for 70 minutes. The precipitate was the dual-targeted exosomes (Cas9@CP-EX) with gene editing ability. The precipitate was washed twice with sterile saline and finally resuspended in 1 mL of sterile saline and placed at 4°C for use.

[0084] The engineered exosomes can be used in the preparation of drugs for treating hematological malignancies.

[0085] The above-mentioned anti-hematological malignancies include myelodysplastic syndrome, myeloproliferative neoplasms, acute myeloid leukemia, multiple myeloma and lymphoma.

[0086] The relevant terms of the present invention are explained as follows:

[0087] (1) Exosomes without Cas9 / sgRNA: exosomes obtained from primary bone marrow stem cells;

[0088] (2) Exosomes containing Cas9 / sgRNA: Exosomes obtained from primary bone marrow stem cells carry the Cas9 / sgRNA gene editing system through electroporation;

[0089] (3) C-EX containing Cas9 / sgRNA: Exosomes obtained from bone marrow stem cells expressing Lamp2b-CXCR4 (i.e., bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein of the present invention) carry the Cas9 / sgRNA gene editing system by electroporation, i.e., dual-targeted engineered exosomes with gene editing capability composed of the Cas9 / sgRNA protein complex and the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein.

[0090] (4) P-EX containing Cas9 / sgRNA: Exosomes obtained from primary bone marrow stem cells carry the p-selectin polypeptide through a membrane fusion strategy in vitro, and then carry the Cas9 / sgRNA gene editing system through electroporation.

[0091] (5) CP-EX containing Cas9 / sgRNA: Exosomes obtained from bone marrow stem cells expressing Lamp2b-CXCR4 carry p-selectin polypeptide in vitro through a membrane fusion strategy to obtain p-selectin targeted exosomes, which are then electroporated to carry the Cas9 / sgRNA gene editing system, i.e., dual-targeted engineered exosomes with gene editing capability composed of the Cas9 / sgRNA protein complex and the p-selectin targeted exosomes.

[0092] The following will describe in detail the use of the fibroblast reticular cells from mesenteric tissue or the extracellular vesicles derived from the fibroblast reticular cells in the preparation of a drug for treating and / or preventing sepsis-induced acute kidney injury in combination with examples and experimental data.

[0093] Example 1. Bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein

[0094] This embodiment provides a method for screening bone marrow mesenchymal stem cells that highly express Lamp2b-CXCR4 fusion protein. The specific scheme is as follows:

[0095] Lamp2b was covalently linked to CXCR4 via a flexible linker (GGSGG) to form a fusion protein, the corresponding base sequence (SEQ ID NO.2:

[0096]

[0097] The lentiviral plasmid containing Lamp2b-CXCR4 and the packaging plasmids PSPAX2 and PMD2G were co-transfected into HEK293T cells using PEI reagent to obtain a lentiviral vector loaded with the target sequence plasmid;

[0098] The lentivirus was transfected into mouse bone marrow-derived mesenchymal stem cells (MSCs). After 48 hours, stem cell complete culture medium containing 0.8 μg / mL puromycin was added for screening. The screening period was 3 weeks.

[0099] The bone marrow mesenchymal stem cells prepared above were taken and combined with flow cytometry antibodies to observe the proportion of stem cells expressing CXCR4 by flow cytometry. The obtained image is as follows: Figure 1 shown.

[0100] Depend on Figure 1 It was found that more than 95% of the bone marrow mesenchymal stem cells obtained in Example 1 expressed the Lamp2b-CXCR4 fusion protein.

[0101] Example 2: Exosomes from bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein

[0102] The bone marrow mesenchymal stem cells obtained in Example 1 were lysed with RIPA lysis buffer, and a loading buffer containing a reducing agent was added. A western blotting experiment was performed with primary antibodies against CXCR4 and Lamp2b from mice and a secondary antibody against rabbit mouse containing horseradish peroxidase to verify whether the exosomes contained high levels of CXCR4. The control groups were exosomes derived from stem cells expressing only CXCR4 and exosomes derived from conventional MSCs. The experimental results are as follows: Figure 3 shown.

[0103] from Figure 3 It can be seen from the results that the content of CXCR4 in the exosomes secreted by stem cells that only overexpress CXCR4 is very low, while making CXCR4 and Lamp2b into fusion proteins can greatly enhance the effective enrichment of CXCR4 in exosomes, proving the role of the exosome sorting protein Lamp2b in inducing the secretion of target proteins into exosomes.

[0104] Example 3: Exosomes with CXCR4 are more efficient in targeting MSCs

[0105] The exosomes obtained in Example 2 were taken, and the exosomes of the control group did not contain CXCR4 molecules; two groups of exosomes (exosomes and CXCR4-exsomes) containing a protein concentration of 0.5 mg / mL were labeled with a membrane dye PKH26 at a concentration of 1 μM in a physiological saline solution, centrifuged at 110000G for 60 minutes, and the dye-labeled exosome precipitate was obtained. The exosome protein was prepared into a working solution with an exosome protein concentration of 0.1 mg / mL by using a stem cell culture medium, and incubated with bone marrow mesenchymal stem cells grown in a confocal dish for 24 hours. The uptake of exosomes by stem cells was observed by confocal microscopy. The relevant experimental results are shown in the figure Figure 4 shown.

[0106] from Figure 4 It can be seen from the results that exosomes without CXCR4 can target bone marrow mesenchymal stem cells to a certain extent, mainly due to the homing characteristics of mesenchymal stem cell exosomes; at the same time, exosomes containing CXCR4 are more efficient in targeting MSCs, proving the important role of targeting CXCR4 / CXCL12 in exosome targeting stem cells.

[0107] Example 4: Targeted exosomes of p-selectin

[0108] DMPE-PEG2000-Mal (Cat. No. P007009, Shanghai Tuoyang Biotechnology Co., Ltd.) was covalently linked to the amino acid sequence DAEWVDVS targeting p-selectin to obtain a DMPE-PEG2000-DAEWVDVS component that can be inserted into the cell membrane;

[0109] The bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein obtained in Example 2 were co-incubated with DMPE-PEG2000-DAEWVDVS in physiological saline, the incubation ratio was 1:1-40 calculated according to the protein concentration, and the incubation time was 24 hours; the exosomes incubated at different concentrations were centrifuged and washed, and the highest concentration dose of the polypeptide inserted in the exosomes was identified by flow cytometry, and the relevant results are shown in Figure 5.

[0110] from Figure 5 It can be seen from the results that saturation was reached when the ratio of exosomes and DMPE-PEG2000-DAEWVDVS was 1:15, and incubation with a higher concentration of DMPE-PEG2000-DAEWVDVS could not promote the exosomes to carry a higher concentration of p-selectin targeting peptide.

[0111] Example 5: Loading of DAEWVDVS polypeptide in exosomes can change the targeting of exosomes

[0112] Exosomes containing different concentrations of the p-selectin targeting polypeptide prepared in Example 4 (5 concentration gradient ratios, exosome protein concentration: polypeptide concentration ratios were no p-selectin polypeptide, 1:2, 1:5, 1:10, and 1:15, respectively) were incubated with LPS or TNF-α activated vascular endothelial cells b End.3 (mimicking the inflammatory condition of vascular endothelial cells in the bone marrow when hematological malignancies occur) for 24 hours, and flow cytometry was used to observe the effects of exosomes of different concentrations on activated vascular endothelial cells. The relevant experimental results are shown in Figure 2. Figure 6 shown.

[0113] from Figure 6 The results show that the more p-selectin targeting polypeptides are loaded into exosomes, the stronger their targeting to inflammatory vascular endothelial cells, while their targeting to non-activated vascular endothelial cells is weaker, which proves that the loading of DAEWVDVS polypeptide in exosomes can change the targeting of exosomes, and its loading amount determines the targeting intensity to inflammatory vascular endothelial cells.

[0114] Example 6: Exosomes containing both CXCR4 and p-selectin targeting peptides can more efficiently target the bone marrow of diseased mice

[0115] 1. Use 0.1 mg / mL PKH26 cell membrane dye to incubate unmodified exosomes (the preparation method is to incubate 0.1 mg of PKH26 dye with 1 mg of exosomes with a total protein content in PBS at room temperature for 1 hour, and then use the above-mentioned strategy for preparing exosomes to obtain exosomes containing the fluorescent dye PKH26), exosomes containing CXCR4 (the preparation method is to incubate 0.1 mg of PKH26 dye with 1 mg of exosomes containing CXCR4 with a total protein content in PBS at room temperature for 1 hour, and then use the above-mentioned strategy for preparing exosomes to obtain exosomes containing the fluorescent dye PKH26), exosomes containing p-selectin polypeptide (the preparation method is to incubate 0.1 mg of PKH26 dye with 1 mg of p-selectin polypeptide with a total protein content of 1 mg) at room temperature for 1 hour. The exosomes of the peptide were co-incubated in PBS at room temperature for 1 hour, and then the exosomes containing the fluorescent dye PKH26 were obtained using the aforementioned strategy for preparing exosomes) and the dual-targeted exosomes prepared in Example 2 were stained, and then the dye-labeled exosomes were harvested by centrifugation at 110000G, and the exosomes were washed twice with physiological saline to finally obtain different groups of exosomes with a protein concentration of 1 mg / mL; an AML-related C57 leukemia model was constructed using an acute myeloid leukemia C57 mouse transplantation model driven by AE9a / MLL-AF9, and different groups of exosomes with a total protein concentration of 0.2 mg were injected into the tail vein. The mice were imaged in vivo 24 hours after the injection, and the ex vivo organs were taken out for imaging to evaluate the targeting effect of different groups of exosomes on the bone marrow. The results are shown in Figure 2. Figure 7 shown.

[0116] from Figure 7 The results show that the exosomes derived from bone marrow mesenchymal stem cells have certain characteristics of targeting the bone marrow of diseased mice. Among them, the exosomes containing CXCR4 alone or p-selectin targeting peptide alone can improve their targeting characteristics in the bone marrow of diseased mice, while the exosomes containing both CXCR4 and p-selectin targeting peptides can target the bone marrow of diseased mice more efficiently, proving the synergistic targeting effect of CXCR4 and p-selectin targeting peptides.

[0117] 2. Obtain the mouse bone marrow in Experimental Example 4, collect the single cell suspension in the mouse bone marrow, use flow cytometry antibodies to label the bone marrow mesenchymal stem cell population, whose molecular marker is CD45-CD31-Sca-1+CD44+, and count the proportion of bone marrow mesenchymal stem cells containing fluorescence in the PBS group, exosome group (exosome), exosome group containing CXCR4 (C-EX), exosome group containing p-selectin targeting polypeptide (P-EX), and dual-targeting exosome group (CP-EX) by flow cytometry. The relevant results are as follows: Figure 8 shown.

[0118] from Figure 8 The results show that simple exosomes derived from bone marrow mesenchymal stem cells have a very small ability to target stem cells, while exosomes containing CXCR4 or p-selectin targeting peptides can greatly enhance their targeting ability to bone marrow mesenchymal stem cells. Exosomes containing both of them have the strongest ability to target bone marrow mesenchymal stem cells, proving the synergistic targeting characteristics of the two.

[0119] Example 7: Dual-targeted engineered exosomes with gene editing capabilities

[0120] The Cas9 / sgRNA protein complex was loaded into the dual-targeting exosomes (i.e., the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein in Example 2) by electroporation, and the protein concentration ratio was 0.01-0.1:1, the latter being the exosome protein concentration. The parameters selected for electroporation were a voltage of 100 volts and a current stimulation time of 0.1 milliseconds; centrifugation at 110000G for 60 minutes, and then the exosomes containing the Cas9 / sgRNA protein complex were washed twice with physiological saline, the exosomes were collected, and the maximum loading Cas9 / sgRNA content in the exosomes was identified by western blotting. The relevant results are shown in the figure Fig. 9 shown.

[0121] from Fig. 9 It can be seen from the results that the amount of Cas9 loaded in exosomes can be regulated by adjusting the concentration ratio of Cas9 protein to exosome protein. When the ratio of Cas9 protein to exosome protein is 0.05:1, the amount of Cas9 protein loaded in exosomes is basically saturated.

[0122] Example 8. Dual-targeted engineered exosomes with gene editing capabilities

[0123] The exosomes containing Cas9 / sgRNA were configured into a 0.1 mg / mL solution, wherein the exosome groups provided were an exosome group (exosome), an exosome group containing CXCR4 (C-EX), an exosome group containing p-selectin targeting polypeptide prepared in Example 4 (P-EX), and a dual-targeting exosome group (CP-EX, i.e., bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein prepared in Example 2);

[0124] The exosomes of the different groups were co-incubated with Cas9 / sgRNA; after the exosomes of the same concentration in different groups were co-incubated with bone marrow-derived mesenchymal stem cells of diseased mice for 24 hours, the stem cells of different groups were collected, lysed with RIPA lysis buffer and heat-inactivated, and western blotting experiments were performed with anti-ID1 and anti-GAPDH mouse primary antibodies and rabbit anti-mouse secondary antibodies containing horseradish peroxidase. The relevant results are shown in Fig.10 shown.

[0125] from Fig.10 The results show that dual-targeting exosomes carrying Cas9 / sgRNA protein complexes have the best silencing effect on bone marrow-derived mesenchymal stem cells in diseased mice. The main reason is that the exosomes can be most efficiently taken up by mesenchymal stem cells.

[0126] Experimental Example 11: Therapeutic effect of dual-targeted engineered exosomes with gene editing capabilities

[0127] The AML-related C57 leukemia model was constructed using the AE9a / MLL-AF9-driven acute myeloid leukemia C57 mouse transplant model. Starting from the second day after the successful construction of the model, exosomes without Cas9 / sgRNA, exosomes containing Cas9 / sgRNA, C-EX containing Cas9 / sgRNA, P-EX containing Cas9 / sgRNA, and CP-EX containing Cas9 / sgRNA were injected respectively. The total injection dose was 0.2 mg protein concentration, and the injection was once every 3 days for a total of 6 injections. The survival rate statistics of mice in different groups are shown in the figure below. Fig.11 shown.

[0128] from Fig.11 The results show that dual-targeted exosomes carrying Cas9 / sgRNA gene editors have the best therapeutic effect on diseased mice, proving the superiority of this system in treating leukemia models. Finally, it should be noted that the terms "include", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or device.

[0129] 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.

[0130] 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 Lamp2b-CXCR4 fusion protein, characterized in that: The amino acid sequence of the Lamp2b-CXCR4 fusion protein is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Lamp2b-CXCR4 fusion protein of claim 1.

3. A vector, engineered bacteria or cell line expressing the Lamp2b-CXCR4 fusion protein according to claim 1.

4. A bone marrow mesenchymal stem cell expressing Lamp2b-CXCR4 fusion protein, characterized in that: The bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein are obtained by transfecting the bone marrow mesenchymal stem cells with a lentivirus expressing the Lamp2b-CXCR4 fusion protein according to claim 1.

5. A bone marrow mesenchymal stem cell-derived exosome expressing Lamp2b-CXCR4 fusion protein, characterized in that: The exosomes are extracellular vesicles obtained by centrifugation and extraction of the cell culture supernatant collected after culturing the bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein as claimed in claim 4.

6. A p-selectin-targeted exosome, characterized in that: The method for preparing the p-selectin targeted exosomes comprises: The peptide DAEWVDVS was co-incubated with DMPE-PEG2000 lipid to obtain DMPE-PEG2000-DAEWVDVS; The exosomes derived from bone marrow mesenchymal stem cells expressing the Lamp2b-CXCR4 fusion protein according to claim 5 are co-incubated with the DMPE-PEG2000-DAEWVDVS to obtain p-selectin targeted exosomes.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: Cas9 / sgRNA protein complex; The bone marrow mesenchymal stem cell-derived exosomes expressing Lamp2b-CXCR4 fusion protein according to claim 5 or the p-selectin-targeted exosomes according to claim 6.

8. A pharmaceutical composition according to claim 7, characterized in that: The mass ratio of the Cas9 / sgRNA complex to the total protein of the bone marrow mesenchymal stem cell-derived exosomes expressing the Lamp2b-CXCR4 fusion protein is 1:15-40; the targeting sequence of the sgRNA in the Cas9 / sgRNA complex is shown in SEQ ID NO.3, and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.4; The mass ratio of the protein in the p-selectin targeting exosomes, the protein of the Cas9 / sgRNA, and the polypeptide of the DMPE-PEG2000-DAEWVDVS in the p-selectin targeting exosomes is 1:15-40:5-15.

9. Use of the bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein according to claim 4, the exosomes derived from bone marrow mesenchymal stem cells expressing Lamp2b-CXCR4 fusion protein according to claim 5, the p-selectin targeted exosomes according to claim 6, and the pharmaceutical composition according to any one of claims 7-8 in the preparation of anti-leukemia drugs.

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