CN112410304A

CN112410304A - Gene-modified exosome and preparation method and application thereof

Info

Publication number: CN112410304A
Application number: CN202011261034.8A
Authority: CN
Inventors: 邵文威; 明东
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-02-26

Abstract

The invention provides a gene modified exosome and a preparation method and application thereof, wherein the exosome expresses membrane protein with an immunoregulation function; the membrane protein with the immunoregulation function comprises any one or combination of at least two of PD-L1, PD-L1-ITGB1 fusion protein, HLA-G1 or HLA-G5. The invention adopts adeno-associated virus containing immunoregulation functional genes to infect the mesenchymal stem cells, and obtains exosomes expressing PD-L1, PD-L1-ITGB1, HLA-G1 or HLA-G5 by separating and purifying the culture supernatant of the mesenchymal stem cells, wherein the exosomes have immunoregulation function similar to the mesenchymal stem cells, have obvious inhibition effect on T cells activated by specific antigens, or induce immune cells to generate specific immune tolerance, thereby effectively overcoming the defect of mesenchymal stem cell treatment.

Description

Gene-modified exosome and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological engineering, relates to a genetically modified exosome and a preparation method and application thereof, and particularly relates to a mesenchymal stem cell-derived genetically modified exosome and a preparation method and application thereof in preparation of immune rejection disease prevention drugs and/or treatment drugs.

Background

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that can differentiate into various cells such as cardiac myocytes, adipocytes, osteoblasts, chondrocytes, and nerve cells. Clinically mesenchymal stem cells are mainly derived from bone marrow and infant umbilical cord. Under a normal physiological state, the mesenchymal stem cells reach the damaged part of the body through peripheral blood circulation, and are induced and differentiated into tissue cells through local environment, so that the purpose of repairing tissue damage is achieved. Mesenchymal stem cells also have a strong immunoregulatory function, and have been clinically applied to prevention and treatment of Graft Versus Host Disease (GVHD) in hematopoietic stem cell transplantation. The research shows that the mesenchymal stem cells regulate immune response mainly by secreting immunosuppressive cytokines, inhibiting immune cell contact and the like. In addition, research shows that the inhibition effect of glucocorticoid on immune response can be reduced by the common use of mesenchymal stem cells and glucocorticoid, so that the main function of mesenchymal stem cells is immune regulation rather than simple immune inhibition.

Graft Versus Host Disease (GVHD) is a common allogeneic stem cell transplantation complication that occurs by the following mechanisms: in donor stem cells

T cells recognize the recipient protein and recognize it as a foreign antigen, initiating a graft-versus-host response. The occurrence of GVHD has been thought to require the following three conditions: the transplant contains immunocompetent cells; the recipient's immune system is destroyed and does not respond to the graft; the recipient has an antigen that the donor does not express. However, according to current knowledge of GVHD, GVHD occurs even if the HLA of the donor and recipient are perfectly matched. When GVHD occurs, the donor immune cells recognize host antigens, liveThe tissue is damaged by a large amount of inflammatory factors generated by chemotherapy, the inflammatory environment is changed, and activated T cells, NK cells and macrophages attack host tissues. The major damaged organs of GVHD include liver, digestive tract and skin, and serious ones can endanger life. At present, the method of inputting mesenchymal stem cells into a transplanted patient is mainly adopted clinically to prevent and slow the symptoms of GVHD.

Mesenchymal stem cells may also be used to treat inflammation-related diseases such as Crohn's Disease, parkinsonism syndrome, liver transplant rejection, lung transplant rejection, diabetes, and the like. Although the pathogenesis of the above diseases is different, immune cells and immune reactions are involved, and mesenchymal stem cells are applied to the treatment of the above diseases by exerting an immunoregulatory function.

According to the current clinical results, mesenchymal stem cells show good inflammation inhibition, but still have the following defects: (1) mesenchymal stem cells are mainly derived from infant umbilical cords, and are cultured in vitro after being separated from the umbilical cords, so that the number of the obtained cells is limited, the large-scale clinical application is limited, and the quality and the effect of the mesenchymal stem cells in different batches are different, and the controllability is poor; (2) the mesenchymal stem cell therapy belongs to the category of cell therapy and has the problems of safety and ethical morality. Therefore, there is a need to provide new strategies to overcome the drawbacks of mesenchymal stem cell therapy.

Exosomes (exosomes) are spherical complexes with a biofilm structure produced by a variety of cells, about 50-150 nm in diameter and larger in size than low density lipoprotein complexes (LDL). Recent research shows that exosome has the functions of cell signal transduction, intercellular substance communication and the like. The breast cancer cells can generate a large amount of exosomes, inhibit the monitoring and eliminating capacity of immune cells in the local environment of the tumor, and specifically induce the immune cells to transform to the direction of immunosuppression. In addition, exosomes derived from tumor cells can enter peripheral blood, and the exosomes contain multiple microRNAs for signal transduction and expression regulation, so that clinical diagnosis and monitoring of tumors can be performed by detecting the expression level of the microRNAs related to the tumor cell exosomes in the peripheral blood. Antigen presenting cells, such as Dendritic Cells (DC), can also generate exosomes, some exosomes present carried antigens to immune cells, some exosomes activate T cells through T cell membrane surface receptors by using carried immune cell activation signals, and some exosomes transfer carried regulatory microRNA to target cells through fusion with the target cells, so that the aim of regulating and controlling the target cells is fulfilled. Mesenchymal stem cells are also capable of producing large amounts of exosomes, some of the functions of which have been reported but are still incomplete and unclear. Exosomes have weak immunoregulatory functions and cannot be directly used for the treatment of immune-related diseases.

Disclosure of Invention

Aiming at the defects and actual needs of the prior art, the invention provides a gene modified exosome, a preparation method and an application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a genetically modified exosome expressing a membrane protein having an immunomodulatory function;

the membrane protein with the immunoregulation function comprises any one or a combination of at least two of PD-L1, PD-L1-ITGB1, HLA-G1 or HLA-G5.

In the invention, exosomes derived from mesenchymal stem cells or exosomes expressing PD-L1, PD-L1-ITGB1, HLA-G1 or HLA-G5 have immunoregulation functions similar to the mesenchymal stem cells, have obvious inhibition effect on T cells activated by specific antigens or induce immune cells to generate specific immune tolerance.

Preferably, the PD-L1 comprises an amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence which has more than 80% of identity with the SEQ ID NO. 5 and has the same or similar biological functions;

SEQ ID NO:5：

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET。

preferably, the PD-L1-ITGB1 comprises an amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence which has more than 80% of identity with the SEQ ID NO. 6 and has the same or similar biological functions;

SEQ ID NO:6：

MNLQPIFWIGLISSVCCVFASFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERGGGGSGGGGSVDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRR。

in the invention, PD-L1-ITGB1 is a recombinant protein obtained by replacing the extramembranous region of ITGB1 with the extramembranous region of PD-L1 and adding 6 × his tag to the tail of the sequence.

Preferably, the HLA-G1 comprises an amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence which has more than 80% of identity with the SEQ ID NO. 7 and has the same or similar biological functions;

SEQ ID NO:7：

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPSRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEREGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETKPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD。

preferably, the HLA-G5 comprises an amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence which has more than 80% of identity with SEQ ID NO. 8 and has the same or similar biological functions;

SEQ ID NO:8：

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPSRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEREGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETKPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVLERPNKELRCIDQSVLVFCVTRRNP。

in a second aspect, the present invention provides an expression vector, which is an adeno-associated viral vector comprising a gene encoding a membrane protein having an immunoregulatory function.

Preferably, the expression vector comprises a nucleic acid molecule encoding PD-L1, a nucleic acid molecule encoding PD-L1-ITGB1, a nucleic acid molecule encoding HLA-G1 or a nucleic acid molecule encoding HLA-G5.

Preferably, the nucleic acid molecule encoding PD-L1 comprises the nucleic acid sequence shown in SEQ ID NO. 1, encodes PD-L1 shown in SEQ ID NO. 5;

SEQ ID NO:1：

atgaggatatttgctgtctttatattcatgacctactggcatttgctgaacgcatttactgtcacggttcccaaggacctatatgtggtagagtatggtagcaatatgacaattgaatgcaaattcccagtagaaaaacaattagacctggctgcactaattgtctattgggaaatggaggataagaacattattcaatttgtgcatggagaggaagacctgaaggttcagcatagtagctacagacagagggcccggctgttgaaggaccagctctccctgggaaatgctgcacttcagatcacagatgtgaaattgcaggatgcaggggtgtaccgctgcatgatcagctatggtggtgccgactacaagcgaattactgtgaaagtcaatgccccatacaacaaaatcaaccaaagaattttggttgtggatccagtcacctctgaacatgaactgacatgtcaggctgagggctaccccaaggccgaagtcatctggacaagcagtgaccatcaagtcctgagtggtaagaccaccaccaccaattccaagagagaggagaagcttttcaatgtgaccagcacactgagaatcaacacaacaactaatgagattttctactgcacttttaggagattagatcctgaggaaaaccatacagctgaattggtcatcccagaactacctctggcacatcctccaaatgaaaggactcacttggtaattctgggagccatcttattatgccttggtgtagcactgacattcatcttccgtttaagaaaagggagaatgatggatgtgaaaaaatgtggcatccaagatacaaactcaaagaagcaaagtgatacacatttggaggagacgtaa。

preferably, the nucleic acid molecule encoding PD-L1-ITGB1 comprises a nucleic acid sequence shown in SEQ ID NO. 2, encodes PD-L1-ITGB1 shown in SEQ ID NO. 6;

SEQ ID NO:2：

atgaatttacaaccaattttctggattggactgatcagttcagtttgctgtgtgtttgctagctttactgtcacggttcccaaggacctatatgtggtagagtatggtagcaatatgacaattgaatgcaaattcccagtagaaaaacaattagacctggctgcactaattgtctattgggaaatggaggataagaacattattcaatttgtgcatggagaggaagacctgaaggttcagcatagtagctacagacagagggcccggctgttgaaggaccagctctccctgggaaatgctgcacttcagatcacagatgtgaaattgcaggatgcaggggtgtaccgctgcatgatcagctatggtggtgccgactacaagcgaattactgtgaaagtcaatgccccatacaacaaaatcaaccaaagaattttggttgtggatccagtcacctctgaacatgaactgacatgtcaggctgagggctaccccaaggccgaagtcatctggacaagcagtgaccatcaagtcctgagtggtaagaccaccaccaccaattccaagagagaggagaagcttttcaatgtgaccagcacactgagaatcaacacaacaactaatgagattttctactgcacttttaggagattagatcctgaggaaaaccatacagctgaattggtcatcccagaactacctctggcacatcctccaaatgaaaggggaggcggtggctctggtggaggcggatctgtcgacatcattccaattgtagctggtgtggttgctggaattgttcttattggccttgcattactgctgatatggaagcttttaatgataattcatgacagaaggtga。

preferably, the nucleic acid molecule encoding HLA-G1 comprises the nucleic acid sequence shown in SEQ ID NO. 3, encodes HLA-G1 shown in SEQ ID NO. 7;

SEQ ID NO:3：

atggtcgtgatggctcctcgcacactgttcctgctgctgtctggggctctgacactgactgaaacttgggctggatcacactcaatgagatacttcagcgccgccgtgagcaggccatcccgcggcgagcccaggtttatcgctatgggctatgtggacgatacccagttcgtgcgctttgactccgattctgcctgccctaggatggagcctcgcgccccctgggtggagagggagggcccagagtactgggaggaggagacccgcaacacaaaggcccacgcccagaccgaccggatgaacctgcagacactgagaggctactataatcagtccgaggccagctcccacaccctgcagtggatgatcggctgtgacctgggctctgatggccggctgctgagaggctacgagcagtacgcctatgacggcaaggattatctggccctgaatgaggacctgcggtcttggaccgcagcagatacagcagcccagatcagcaagagaaagtgcgaggcagcaaacgtggcagagcagaggagagcatacctggagggaacctgcgtggagtggctgcaccggtatctggagaatggcaaggagatgctgcagagagccgacccccctaagacccacgtgacacaccacccagtgttcgattacgaggccacactgaggtgctgggcactgggcttttatcctgccgagatcatcctgacctggcagcgcgacggcgaggatcagacacaggacgtggagctggtggagaccaagccagcaggcgatggcacattccagaagtgggcagcagtggtggtgccttccggagaggagcagcggtatacctgtcacgtgcagcacgagggactgccagagccactgatgctgaggtggaagcagtctagcctgcccacaatccctatcatgggcatcgtggccggcctggtggtgctggccgccgtcgtcactggggcagccgtggcagccgtcctgtggcggaaaaagtcatctgattga。

preferably, the nucleic acid molecule encoding HLA-G5 comprises the nucleic acid sequence shown in SEQ ID NO. 4, encodes HLA-G5 shown in SEQ ID NO. 8;

SEQ ID NO:4：

atggtcgtgatggctcctcgcacactgttcctgctgctgtctggggctctgacactgactgaaacttgggctggatcacactcaatgagatacttcagcgccgccgtgagcaggccatcccgcggcgagcccaggtttatcgctatgggctatgtggacgatacccagttcgtgcgctttgactccgattctgcctgccctaggatggagcctcgcgccccctgggtggagagggagggcccagagtactgggaggaggagacccgcaacacaaaggcccacgcccagaccgaccggatgaacctgcagacactgagaggctactataatcagtccgaggccagctcccacaccctgcagtggatgatcggctgtgacctgggctctgatggccggctgctgagaggctacgagcagtacgcctatgacggcaaggattatctggccctgaatgaggacctgcggtcttggaccgcagcagatacagcagcccagatcagcaagagaaagtgcgaggcagcaaacgtggcagagcagaggagagcatacctggagggaacctgcgtggagtggctgcaccggtatctggagaatggcaaggagatgctgcagagagccgacccccctaagacccacgtgacacaccacccagtgttcgattacgaggccacactgaggtgctgggcactgggcttttatcctgccgagatcatcctgacctggcagcgcgacggcgaggatcagacacaggacgtggagctggtggagaccaagccagcaggcgatggcacattccagaagtgggcagcagtggtggtgccttccggagaggagcagcggtatacctgtcacgtgcagcacgagggactgccagagccactgatgctgaggtggaagcagtctagcctgcccacaatccctatcatgggcatcgtggccggcctggtggtgctggccgccgtcgtcctcgagaggcctaataaagagctcagatgcatcgatcagagtgtgttggttttttgtgtgacgcgtaggaacccctag。

preferably, the adeno-associated viral vector comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2 or AAV2/YF 3.

In the invention, the AAV of different subtypes has certain infection efficiency on the mesenchymal stem cells, wherein the infection efficiency of the AAV2 and the AAV2/YF3 on the mesenchymal stem cells is the highest.

In a third aspect, the present invention provides a recombinant adeno-associated virus, which is a mammalian cell transfected with the expression vector and helper plasmid of the second aspect.

In a fourth aspect, the present invention provides a method for preparing the recombinant adeno-associated virus according to the third aspect, wherein the method comprises the following steps:

(1) co-transfecting the expression vector of the second aspect with a helper plasmid and a transfection reagent into mammalian cells, and culturing for a period of time prior to cell lysis;

(2) adding the cell lysate into a cesium chloride solution for ultracentrifugation, and collecting the cesium chloride solution containing the adeno-associated virus;

(3) and (3) dialyzing the cesium chloride solution containing the adeno-associated virus to obtain the adeno-associated virus.

As a preferred embodiment, the present invention provides a method for packaging AAV, comprising the steps of:

co-transfecting HEK293 cells with the master plasmid, the packaging plasmid and a transfection reagent (PEI), and collecting the cells after 48 hours; repeatedly freezing and thawing cells and ultrasonically cracking the cells; adding the lysate into a cesium chloride solution with a certain density for ultracentrifugation overnight; collecting cesium chloride solutions in different layers in a centrifugal tube, detecting and picking out the cesium chloride solution containing AAV; and placing the AAV-containing cesium chloride solution into a dialysis bag, placing the dialysis bag into PBS for dialysis, and finally obtaining the AAV-containing PBS solution. The concentration of AAV is detected by real-time fluorescent quantitative PCR, and the infection efficiency of AAV is determined by infecting Hela cells and observing the relationship between the gene expression level and the AAV amount.

In a fifth aspect, the present invention provides a method for preparing the exosome of the first aspect, the method comprising the steps of:

infecting mesenchymal stem cells with the recombinant adeno-associated virus of the third aspect, culturing for a period of time, and collecting a culture supernatant of the mesenchymal stem cells;

and separating and purifying the exosome from the culture supernatant of the mesenchymal stem cells.

Preferably, the MOI of the recombinant adeno-associated virus infected mesenchymal stem cell is 5000-10000.

Preferably, the culture medium of the mesenchymal stem cells is serum-free and exosome-free.

Preferably, the culture time of the mesenchymal stem cells is not shorter than 3 days, and the culture supernatant of the mesenchymal stem cells is preferably collected every 3 days.

Preferably, the method for separating and purifying the exosome comprises ultracentrifugation and/or polyethylene glycol precipitation.

In the invention, the ultracentrifugation method is suitable for separating and purifying exosomes rapidly with small quantity and high concentration, and is used for basic research and mouse experiments; the polyethylene glycol (PEG) precipitation method is suitable for separating and purifying a large amount of exosomes with high efficiency, and is used for large animal experiments and clinical application.

As a preferred technical scheme, the invention provides a method for specifically expressing a target protein on the surface of an exosome derived from a mesenchymal stem cell, which comprises the following steps:

(1) designing a recombinant protein gene specifically expressed on the surface of an exosome, wherein the recombinant protein gene comprises a signal peptide, an extracellular region, a transmembrane region and an intracellular region;

(2) integrating the recombinant protein gene into a pTR-selfcompletementary-CMV (pTR-sc-CMV) plasmid;

(3) packaging AAV2-sc virus;

(4) infecting the mesenchymal stem cells by AAV virus according to the proportion that the MOI is 10000, and adopting a culture medium without serum or exosome component to culture the mesenchymal stem cells;

(5) collecting culture supernatant every 3 days, and separating and purifying exosome by using an ultracentrifugation method and/or a polyethylene glycol precipitation method;

(6) and detecting the expression condition of the recombinant protein on the surface of the exosome by using a flow cytometer or Western blot.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising an exosome according to the first aspect.

Preferably, the pharmaceutical composition further comprises mesenchymal stem cells.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a seventh aspect, the present invention provides an exosome of the first aspect, an expression vector of the second aspect, a recombinant adeno-associated virus of the third aspect, or a pharmaceutical composition of the sixth aspect, for use in preparing a medicament for preventing and/or treating an immune rejection disease.

Preferably, the immune rejection disease comprises graft versus host disease.

In an eighth aspect, the present invention provides a method for specifically inducing immune cell activation using exosomes, comprising the steps of:

(1) integrating the coding gene of the OVA protein into pTR-sc-CMV and packaging AAV2-sc adeno-associated virus;

(2) culturing mouse mesenchymal stem cells (mouse MSCs) using a serum-free medium;

(3) infecting mouse mesenchymal stem cells by adopting the adeno-associated virus in the step (1) according to the MOI (molar equivalent of identity) of 1:10000, and collecting cell culture supernatant after 3 days;

(4) isolating purified exosomes from the cell culture supernatant of step (3);

(5) taking the spleen of an OT-1 mouse, and separating spleen T cells;

(6) mixing and culturing the exosome obtained in the step (4) and OT-1 mouse splenocytes;

(7) after 12 hours, flow cytometry detected the expression of the T cell early activation signal CD 69.

In a ninth aspect, the present invention provides a method for specifically inducing tolerance of immune cells to antigens by using genetically modified exosomes, comprising the following steps (immune regulatory molecule is exemplified by PD-L1, and antigens are exemplified by OVA protein):

(1) preparing pTR-sc-CMV-OVA and pTR-sc-CMV-PD-L1-ITGB1 plasmids, wherein the coding genes of PD-L1-ITGB1 are shown in SEQ ID NO:2, and packaging AAV2-sc adeno-associated virus;

(4) isolating purified exosomes from the cell culture supernatant of step (3);

(5) taking the spleen of an OT-1 mouse, and separating spleen T cells;

(7) exosomes were supplemented every 2 days for a total of 7 days:

(8) adding OVA polypeptide SIINFEKL into HEK293/H2kb cells, culturing overnight, and preparing antigen presenting cells;

(9) mixing and culturing the treated HEK293/H2kb cells and OT-1 splenocytes;

(10) after 12 hours, flow cytometry detected the expression of the T cell early activation signal CD 69.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention adopts adeno-associated virus to infect the mesenchymal stem cells, and adeno-associated viruses of different subtypes have higher infection efficiency and biosafety to the mesenchymal stem cells, wherein the infection efficiency of wild type AAV2 and mutant AAV2YF3 to the mesenchymal stem cells is highest;

(2) the invention adopts adeno-associated virus containing immunoregulation functional gene to infect mesenchymal stem cells, and the exosome expressing membrane protein with immunoregulation function is obtained by separation and purification from the culture supernatant of the mesenchymal stem cells, has immunoregulation function similar to that of the mesenchymal stem cells, has obvious inhibition effect on T cells activated by specific antigen, or induces immune cells to generate specific immune tolerance;

(3) the mesenchymal stem cells and/or the genetically modified exosomes of the present invention are of great significance in the prevention and/or treatment of immune rejection diseases.

Drawings

FIG. 1A shows the infection efficiency of AAV1/luc, AAV2/Iuc, AAV3/Iuc, AAV6/Iuc, AAV8/luc, AAV9/luc and AAV10/luc on mesenchymal stem cells, and FIG. 1B shows the infection efficiency of AAV2/Iuc and AAV2YF3/luc on mesenchymal stem cells;

FIG. 2 is a graph showing the comparison of the infection efficiency of AAV2-self complementary-GFP and Lentivirus-GFP on mesenchymal stem cells;

FIG. 3A shows the expression of markers CD9 and CD63 of exosomes derived from mesenchymal stem cells separated and purified by western blot detection ultracentrifugation, and FIG. 3B shows the corresponding flow cytometer detection results;

FIG. 4A is the expression of markers CD9 and CD63 of exosomes derived from mesenchymal stem cells separated and purified by western blot detection polyethylene glycol precipitation method, and FIG. 4B is the corresponding flow cytometer detection result;

FIG. 5 is a comparison of the inhibitory effect of human mesenchymal stem cells and exosomes on proliferation of anti-CD3 activated T cells in vitro;

FIG. 6 is a histogram of T cell activation proliferation rates of the different experimental groups of FIG. 5;

FIG. 7 is a comparison of the effect of mouse mesenchymal stem cells and exosomes in inhibiting proliferation of OT-1 splenocytes after activation in C57 mice;

FIG. 8 is a histogram of T cell activation proliferation rates of the different experimental groups of FIG. 7;

FIG. 9 shows the body weight changes of GVHD mice in different experimental groups;

FIG. 10 shows the change in the peripheral blood leukocyte ratios of GVHD mice of different experimental groups;

FIG. 11 shows HE staining results of liver tissue sections of GVHD mice of different experimental groups, at microscope magnification of 100 ×;

FIG. 12 shows the results of HE staining of colon tissue sections of GVHD mice of different experimental groups, at a microscope magnification of 100 ×;

FIG. 13 shows the expression of PD-L1 molecule in the cell membrane and cells of exosomes;

FIG. 14 shows the PD-L1 positive rate of exosomes obtained by infecting human mesenchymal stem cells with AAV2sc-PD-L1 and AAV2sc-PD-L1-ITGB 1;

FIG. 15 is a comparison of the effect of different genetically modified exosomes in inhibiting the early activation signal CD69 in T cells in vitro;

figure 16 is a comparison of the effect of different genetically modified exosomes in inducing Treg cells in vitro;

FIG. 17 is a graph of the inhibitory effect of the T cell early activation signal of antigen loaded exosomes.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 design and construction of recombinant expression vectors

In this example, the full-length sequences of OVA protein, PD-L1, ITGB1, HLA-G1 and HLA-G5 genes are firstly retrieved from NCBI gene database, the signal peptide region, the membrane-outer region, the transmembrane region and the membrane-inner region of PD-L1 and ITGB1 are determined by using protein analysis in Lasergene and combining the retrieval result of Uniprot database, the membrane-outer region of ITGB1 is replaced by the membrane-outer region of PD-L1, and 6 × his tag is added at the tail of the sequence to form the coding gene SEQ ID NO. 2 of the recombinant protein PD-L1-ITGB1, and the corresponding amino acid sequence is shown in SEQ ID NO. 6.

The coding gene SEQ ID NO 2 of PD-L1-ITGB1 recombinant protein is synthesized by whole genes and is connected into pTR-sc-CMV plasmid to construct a recombinant expression vector pTR-sc-CMV-PD-L1-ITGB 1.

In this example, an OVA protein-encoding gene, a PD-L1-encoding gene SEQ ID NO:1 (amino acid sequence shown in SEQ ID NO: 5), an HLA-G1-encoding gene SEQ ID NO:3 (amino acid sequence shown in SEQ ID NO: 7), and an HLA-G5-encoding gene SEQ ID NO:4 (amino acid sequence shown in SEQ ID NO: 8) were ligated to a pTR-sc-CMV plasmid, respectively, to construct recombinant expression vectors pTR-sc-CMV-OVA, pTR-sc-CMV-PD-L1, pTR-sc-CMV-HLA-G1, and pTR-sc-CMV-HLA-G5.

Example 2 adeno-associated virus packaging and purification

(1) The plasmid is greatly extracted to obtain pTR-sc-CMV-OVA, pTR-sc-CMV-PD-L1, pTR-sc-CMV-PD-L1-ITGB1, pTR-sc-CMV-HLA-G1, pTR-sc-CMV-HLA-G5 and pXR2 and pXX6-80 plasmids, wherein the integrity of ITR is ensured by the restriction enzyme detection of Sma1 on the pTR-sc-CMV plasmid, and the single strip and the supercoiled structure are ensured by the agarose gel electrophoresis detection of all the plasmids;

(2) the cultured HEK293 is subcultured to a new culture dish in advance for 16 hours, the density is about 90% of the saturation density, the HEK293 is required to be free of bacteria, viruses and pathogen pollution, the culture medium does not obviously turn yellow after normal subculture for 3 days, and the culture medium of the HEK293 is 10% FBS + DMEM;

(3) 9 mu g of pTR-sc-CMV plasmid, 12 mu g of pXR2 plasmid and 15 mu g of pXX6-80 plasmid are required for each cell of a 15cm culture dish, the three plasmids are added into 500 mu L of opti-MEM according to the proportion and mixed evenly, 150 mu L of 1 mu g/mu L PEI is added dropwise while shaking, the mixture is kept stand at room temperature for 10min, and the mixture is added into the cell culture supernatant;

(4) blowing HEK293 to be in a suspension state after 48 hours, centrifuging at 2000rpm for 5min to collect cell precipitates, resuspending with 8.7mL of ultrapure water, repeatedly freezing and thawing for three times between dry ice and warm water, ultrasonically cracking the cells for 2min, adding 5g of cesium chloride, ultrasonically treating for 2min, and placing on ice;

(5) centrifuging at 12000rpm for 20min at 4 deg.C, suspending insoluble cell debris on the surface of cesium chloride solution, carefully transferring the transparent cesium chloride solution below to an Ultracentrifuge tube, centrifuging at 65000rpm for 18 h at 15 deg.C by using Sorvall WX 80+ Ultracentrifuge, and reducing the brake to 5;

(6) taking out the centrifuged cesium chloride solution layer by layer from the bottom, wherein the volume of each layer is about 1mL, measuring the refractive index by a refractometer, and obtaining the cesium chloride solution with the refractive index of about 1.37, namely the cesium chloride solution containing AAV;

(7) transferring the AAV-containing cesium chloride solution into a dialysis bag, dialyzing in pre-precooled PBS three times, and finally collecting the AAV-containing PBS solution.

Measuring the concentration of the obtained AAV, taking 90 μ L of AAV-containing PBS solution, adding 10 μ L of DNase1 working solution, digesting for 1 hour at 37 ℃, and then adding 6 μ L of 0.5M EDTA to terminate the reaction; adding 100 μ L protease digestion solution, digesting at 55 deg.C for 2 hr, and incubating at 95 deg.C for 5min to inactivate protease; diluting AAV solution 1000 times with ultrapure water, using as template of real-time quantitative PCR, performing quantitative PCR analysis, and calculating AAV concentration.

As a result, it was found that the concentration of AAV was more than 1X 10 in each group⁹vg/μL。

Example 3 isolation and culture of human umbilical cord-derived mesenchymal Stem cells

This example collects mesenchymal stem cells from neonatal umbilical cord by the following steps:

(1) flushing the umbilical cord with physiological saline, cutting the umbilical cord into small sections of about 2cm by using scissors, longitudinally planing open, and continuously flushing;

(2) peeling off the inner skin of the inner surface of the opened umbilical cord, and cutting the umbilical cord into pieces with a size of about 1mm in PBS³Small pieces of (2);

(3) adding trypsin digestion solution with one volume, and digesting at 37 deg.C for 20 min;

(4) adding 10 times of DMEM and 10% FBS to neutralize trypsin, placing the trypsin in an incubator for culture, and replacing fresh culture solution every 3 days;

(5) the cell morphology was observed and when the degree of cell fusion reached around 90%, trypsinization was used and subcultured.

The isolated and cultured human mesenchymal stem cells of the embodiment gradually disperse and grow into single cells, have good growth state and are used for subsequent experiments.

Example 4 infection efficiency of different subtypes of AAV on human mesenchymal stem cells

Based on the human mesenchymal stem cells isolated and cultured in example 3, the human mesenchymal stem cells were scraped from the culture dish and resuspended in fresh medium at a cell density of 10⁵Per mL, the cell suspension was aliquoted into 48-well plates, 400 μ L per well and multiple wells were set up;

adding AAV1, AAV2, AAV2YF3, AAV3, AAV6, AAV8, AAV9, AAV10 and PBS (carrying luciferase gene in AAV) according to the proportion that AAV is 10000:1, after 48 hours, using 200 muL of passive lysate to lyse human mesenchymal stem cells for 12min, transferring 50 muL of lysate of each well to luciferase measuring wells, adding 100 muL of reaction substrate to each well, and using a measuring instrument to read the value;

meanwhile, human mesenchymal stem cells (AAV: cell: 10000:1, lentivirus-GFP: cell: 10000:1) were infected with AAV2-sc-GFP and lentivirus-GFP, and after 48 hours, the expression of GFP was observed with a fluorescence microscope.

As shown in fig. 1A, AAV2 has the highest infection efficiency on human mesenchymal stem cells, and as shown in fig. 1B, the mutant AAV2YF3 of AAV2 has the same infection efficiency on human mesenchymal stem cells as wild-type AAV2, so both wild-type AAV2 and AAV2YF3 can be used as vectors for infecting human mesenchymal stem cells.

In addition, in this example, human mesenchymal stem cells were infected with AAV2-sc-GFP and Lentivirus-GFP at the same MOI, and as a result, as shown in fig. 2, AAV2-sc has higher efficiency of infection of human mesenchymal stem cells than Lentivirus, and it was found that AAV has higher biosafety because its insertion rate into host cell genome is much lower than Lentivirus (Lentivirus).

Example 5 ultracentrifugation method for isolation and purification of exosomes from human mesenchymal stem cell supernatant

In this example, the small amount of exosomes were isolated and purified from human mesenchymal stem cells by ultracentrifugation, the steps of which are as follows:

(1) culturing human mesenchymal stem cells with serum-free medium (X-VIVO10) for 3 days, collecting culture supernatant, transferring into 50mL centrifuge tube, centrifuging at 4 deg.C for 10min at 500g, and removing cell debris;

(2) transferring the centrifuged supernatant into a new 50mL centrifuge tube, centrifuging at 4 ℃ and 12000rpm for 20min, and further removing cell fragments and organelles;

(3) transferring the centrifuged supernatant into an ultracentrifuge tube (35mL), centrifuging by adopting a horizontal rotor, and centrifuging for 2 hours at 100000g at 4 ℃;

(4) carefully taking out the ultracentrifuge tube, discarding the supernatant, and placing the ultracentrifuge tube on absorbent paper upside down for 5 min;

(5) using 500 mu L PBS to resuspend the sediment at the bottom of the tube, lightly blowing and beating the sediment for several times, then completely resuspending the sediment as much as possible, and standing the sediment for 1 hour at 4 ℃;

(6) taking 10 mu L of exosome solution for exosome identification, and freezing and storing the rest part of exosome solution at-80 ℃ after subpackaging;

(7) the quality and quantity of exosomes were examined using western blot and flow cytometry (microsphere adsorption, 1atex beads), while the morphology of exosomes was observed using scanning electron microscopy.

The expression of CD9 and CD63 in the exosome sample was detected using western blot, beta-actin was a negative control, and the results are shown in FIG. 3A, where no beta-actin was present in the sample, indicating that the sample was not contaminated with cytoskeletal elements.

All proteins in a sample are coated on the surface of the microsphere in a coupling adsorption mode, and then flow cytometry detection is carried out by adopting fluorescence-labeled anti-CD 9, CD63 and beta-actin antibodies, and the result is shown in figure 3B, wherein both CD9 and CD63 are highly expressed, and beta-actin is not detected on the surface of the microsphere.

Scanning electron microscope results show that the purified exosome of the present example is complete in morphology and free of adhesion.

Example 6 isolation and purification of exosomes from human mesenchymal stem cell supernatant by polyethylene glycol precipitation

In this example, a polyethylene glycol (PEG) precipitation method is used to separate and purify a large amount of exosomes from human mesenchymal stem cells, and the steps are as follows:

(1) expanding and culturing the human mesenchymal stem cells to 10 culture dishes of 15cm, washing the cells twice by PBS when the cell fusion degree reaches 90%, and replacing the culture medium with a serum-free culture medium X-VIVO 10;

(2) collecting culture supernatant on day 3, adding new culture medium into the culture dish, centrifuging the collected culture supernatant at 4 deg.C for 5min to obtain supernatant, filtering with 0.22 μm sterile filter, removing residual cell debris and organelles by negative pressure suction, adding 10% (W/V) PEG8k into 300mL culture supernatant, mixing, and standing at 4 deg.C overnight;

(3) transferring the culture supernatant containing the protein precipitate into a 500mL centrifuge bottle, and centrifuging at 4 ℃ and 8000g for 15 min;

(4) resuspend the pellet with 10mL of precooled PBS, and stand overnight at 4 ℃;

(5) dialyzing the solution containing exosome in a large amount of PBS 3 times by using a dialysis bag (100KDa), taking 10 mu L of the solution for exosome identification and analysis, and freezing and storing the rest part of the solution at-80 ℃ after subpackaging;

(6) collecting cell culture supernatant containing exosomes on 6 th, 9 th and 12 th days, and carrying out exosome separation and purification according to the steps (2) to (5);

Theoretically, before the mesenchymal stem cell state is deteriorated, cell culture supernatant containing exosomes can be continuously collected for clinical experiments, the quality consistency of the exosomes can be kept in the process, and meanwhile, the cost is reduced.

The expression of CD9 and CD63 in the exosome sample was detected using western blot, beta-actin was a negative control, and the results are shown in FIG. 4A, where no beta-actin was present in the sample, indicating that the sample was not contaminated with cytoskeletal elements.

All proteins in a sample are coated on the surface of the microsphere in a coupling adsorption mode, and then flow cytometry detection is carried out by adopting fluorescence-labeled anti-CD 9, CD63 and beta-actin antibodies, and the result is shown in FIG. 4B, wherein both CD9 and CD63 are highly expressed, and beta-actin is not detected on the surface of the microsphere.

Example 7 comparison of the Effect of human mesenchymal Stem cells and exosomes in inhibiting inflammatory response in vitro

This example uses the isolated and purified exosomes of example 5 and example 6 for the experiments, as follows:

(1) peripheral Blood Mononuclear Cells (PBMC) were isolated using lymphocyte separation media, followed by isolation of T lymphocytes from PBMC using beads conjugated with anti-CD3 antibody, stained with CFSE (carboxyfluorescein diacetate succinimidyl ester) and then added with 100U/mL IL-2 and 100U/mL anti-CD3 antibody (OKT-3):

(2) evenly dividing a culture medium containing a stimulating factor and T lymphocytes into 24-well plates, wherein each well contains 500 mu L of the culture medium;

(3) one group of 3 wells were filled with PBS and mesenchymal stem cells (1X 10 per well)⁵One) or 10 μ L, 25 μ L, 50 μ L exosome solution;

(4) after 3 days of culture, CFSE was detected by flow cytometry, and the proliferation level of activated T cells was estimated.

As shown in FIG. 5, the anti-CD3 antibody and IL-2 synergistically stimulated splenic T cells in mice in the positive control group, the ratio of proliferated T cells was 86.01% after 3 days, and under the precondition of synergistic stimulation, mesenchymal stem cells, 10. mu.L, 25. mu.L or 50. mu.L of exosome solution were added, and the ratio of proliferated T cells was 41.01%, 40%, 39.61% and 42.84%, respectively.

As shown in fig. 6, statistics of the proliferation rate of T cells in each experimental group shows that both mesenchymal stem cells and exosomes have inhibitory effects on activation and proliferation of T cells, suggesting that exosomes derived from mesenchymal stem cells also have inhibitory effects on immune responses of mesenchymal stem cells.

Example 8 comparison of Effect of mouse mesenchymal Stem cells and exosomes in inhibiting inflammatory response in vivo

(1) day 0, AAV8/OVA was administered at 1X 10¹⁰Mu.g of each mouse dose was injected intravenously into C57 mice;

(2) on day 2, T cells were isolated from splenocytes from OT-1 mice and labeled CFSE;

(3) OT-1 cells were mixed with PBS and mesenchymal stem cells (1X 10)⁶Each mouse), 50 μ L, 100 μ L or 200 μ L of exosomes were mixed and injected intravenously into C57 mice;

(4) on day 7, the spleens of the mice were removed for lysis, and the level of CFSE was measured by flow cytometry to calculate the level of proliferation of activated T cells.

As shown in FIG. 7, after AAV8/OVA infection of C57 mice, the immune system of the mice recognized OVA antigen, followed by intravenous injection through PBS, mesenchymal stem cells (1X 10)⁶Each mouse), 50 mu L, 100 mu L and 200 mu L of exosomes, OVA antigen is presented to OT-1 cells, the OT-1 cells recognize the antigen and activate proliferation, and the proportion of the proliferated T cells is 42.6%, 14.6%, 33.1%, 14.2% and 10.6% respectively.

As shown in fig. 8, it is found that, after statistics of the T cell proliferation rate of each experimental group, both the mesenchymal stem cell and the exosome can generate an inhibitory effect on T cell activation stimulated by a specific antigen in vivo, suggesting that the exosome derived from the mesenchymal stem cell also has an immune reaction inhibitory effect on the mesenchymal stem cell in vivo.

Example 9 comparison of the Effect of mouse mesenchymal Stem cells and exosomes on the treatment of mouse GVHD

This example uses the isolated and purified exosomes of example 6 for the experiments, as follows:

(1) transplanting allogeneic bone marrow after Co60 ray irradiation is carried out on the mouse, constructing a GVHD mouse model, and simulating GVHD after human allogeneic stem cell transplantation;

(2) on day 1, recipient mice were transplanted with bone marrow cells, and injected intravenously with PBS (1X 10) through the inner canthus⁶Mesenchymal stem cells, 50 μ g, 100 μ g or 200 μ g exosomes;

(3) monitoring the weight change and the change of the peripheral blood leukocyte ratio of each group of mice within 2 weeks after transplantation;

(4) after two weeks, organs such as liver and colon of the mouse are taken, tissue sections are made, and HE staining is carried out to evaluate the tissue damage degree.

As shown in FIG. 9, in two weeks of induction of GVHD in mice, the body weight of each group of mice changed with time, the mesenchymal stem cells had significant alleviation on the body weight loss of the mice, and the exosomes of 50 ug, 100 ug and 200 ug had significant alleviation on the body weight loss of the mice.

As shown in FIG. 10, in two weeks of induction of GVHD in mice, the peripheral blood leukocyte ratios of the mice in each group varied with time, and the increase in the leukocyte ratios of the mesenchymal stem cells, 50. mu.g, 100. mu.g, and 200. mu.g of exosomes was inhibited.

As shown in FIG. 11, after the mice are induced with GVHD successfully for two weeks, the results of HE staining of liver tissue sections of the mice in each group show that the liver injury of the mice in the PBS treatment group is obvious, and the mesenchymal stem cells and the exosomes of 50 ug, 100 ug and 200 ug have obvious repairing effect on the liver injury of the mice in the GVHD.

As shown in FIG. 12, after two weeks of successful mouse GVHD induction, the HE staining results of colon tissue sections of mice in each group show that the damage of colon mucosal tissue of mice in the PBS treatment group is obvious, and the mesenchymal stem cells and exosomes 50 mug, 100 mug and 200 mug have obvious repairing effect on the damage of colon mucosal tissue of GVHD mice.

Example 10 exosome-directed modification based on adeno-associated virus and engineered expression plasmids

This example is based on adeno-associated virus AAV2-PD-L1, AAV2-PD-L1-ITGB1, AAV2-HLA-G1, and AAV2-HLA-G5 constructed in example 2, and the targeted modification of exosomes is carried out by the following steps:

(1) infecting a HeLa cell line (10% FBS + DMEM) and human mesenchymal stem cells (X-VIVO10) with packaged AAV2-PD-L1, AAV2-PD-L1-ITGB1, AAV2-HLA-G1 or AAV2-HLA-G5 according to MOI & gt 10000, collecting HeIa cells after 48 hours, and detecting the distribution condition of PD-L1 in the cells by a flow cytometer;

(2) after 3 days of adeno-associated virus infection, collecting culture supernatant of mesenchymal stem cells, separating and purifying exosomes, and detecting the expression condition of PD-L1 on the surface of the exosomes by a flow cytometer.

As shown in fig. 13, when HeLa cells were not perforated, the proportion of PD-L1 positive cells in the group of HeLa cells infected with AAV2-PD-L1 was 48.18%, and the proportion of PD-L1 positive cells in the group of HeLa cells infected with AAV2-PD-L1-ITGB1 was 29.01%; when HeLa cells were punctured, the proportion of PD-L1 positive cells in the group of HeLa cells infected with AAV2-PD-L1 was 48.93%, and the proportion of PD-L1 positive cells in the group of HeLa cells infected with AAV2-PD-L1-ITGB1 was 44.68%. The results show that PD-L1 is a membrane protein molecule, and flow cytometry detection shows that PD-L1 is mostly expressed on the surface of a cell membrane, and PD-L1-ITGB1 is mostly expressed in cells, because ITGB1 is mainly distributed in lysosomes in the cells.

As shown in FIG. 14, the positive rate of PD-L1 in exosomes generated after AAV2-PD-L1 infected with mesenchymal stem cells was about 40%, while the positive rate of PD-L1 in exosomes generated after AAV2-PD-L1-ITGB1 infected with mesenchymal stem cells was about 44%. The results show that ITGB1 can effectively improve the expression level of PD-L1 on the surface of an exosome membrane.

Example 11 Effect of modified exosomes on inhibiting inflammation in vitro

In this example, experiments were carried out using the exosome PD-L1-exosome expressing PD-L1, the exosome PD-L1-ITGB1-exosome expressing PD-L1-ITGB1, the exosome HLA-G1-exosome expressing HLA-G1, and the exosome HLA-G5-exosome expressing HLA-G5 obtained in example 10, according to the following steps:

(1) taking OT-1 mouse spleen cells to be cracked into single cells;

(2) one day in advance, 1. mu.g of OVA polypeptide (SEQ ID NO: 9: SIINFEKL) was mixed with HEK293/H2kb in 24-well plates at 500. mu.L per well;

(3) mixing OT-1 spleen cells and HEK293 cells at a ratio of 10:1, adding PBS and mesenchymal stem cells into 3 wells (1 × 10 per well)⁵50 μ L of exosome, 50 μ L of PD-L1-exosome, 50 μ L of PD-L1-ITGB1-exosome, 50 μ L of HLA-G1-exosome, 50 μ L of HLA-G5-exosome solution;

(4) culturing for 1 day, and detecting the proportion of CD69+ (T cell early activation signal) T cells by a flow cytometer;

(5) after 5 days of culture, the proportion of Treg cells is detected by a flow cytometer.

As shown in FIG. 15, the anti-CD3 antibody and IL-2 in the positive control group synergistically stimulated splenic T cells of mice, the proportion of activated T cells after 3 days was 87%, and PBS, 50. mu.L exosome, 50. mu.L PD-L1-exosome, 50. mu.L PD-L1-ITGB1-exosome, 50. mu.L HLA-G1-exosome and 50. mu.L HLA-G5-exosome were added under the premise of synergistic stimulation, and the proportion of activated T cells was 35.5%, 25%, 20.8%, 21%, 18.3% and 15.6%, respectively, wherein the inhibition effect of HLA-G1 and HLA-G5 on the specific activation of T cells was stronger than that of PD-L1, especially HLA-G5 on the specific antigen-activated T cells.

As shown in FIG. 16, the ratios of FOXP3+ cells of PBS, exosome, PD-L1-exosome, PD-L1-ITGB1-exosome, HLA-G1-exosome and HLA-G5-exosome groups in CD4+ CD25+ cells are respectively 5.45%, 43.5%, 41.9%, 41.2%, 39.2% and 36.8%, which indicates that the exosome can obviously induce the increase of the proportion of Treg cells, and the effect of inducing the Treg cells cannot be obviously improved after the exosome is modified.

Example 12 antigen-loaded exosomes induce activation of immune cells

This example was based on an in vitro induced T cell activation experiment, and the following steps were performed:

(1) infecting mouse mesenchymal stem cells by combination of AAV2-OVA, AAV2-PD-L1-ITGB1, AAV2-OVA and AAV2-PD-L1-ITGB1 according to MOI & gt 10000, culturing for 3 days, collecting cell supernatant, separating and purifying exosome to obtain OVA-exosome, PD-L1-ITGB1-exosome, OVA-PD-L1-ITGB 1-exosome;

(2) taking OT-1 mouse splenocytes, adding 1 × 10 per well of 12-well plate⁶PermL mouse splenocytes, cultured in serum-free medium (X-VIVO10) with 50. mu.L exosome, OVA-exosome, PD-L1-ITGB1-exosome, OVA-PD-L1-ITGB1-exosome added per well for mixed culture for seven days;

(3) on day 6, 1 μ g SIINFEKL polypeptide was mixed with HEK293/H2kb cells and cultured for one day to prepare antigen presenting cells;

(4) on the 7 th day, after replacing the culture medium of the mouse spleen cells and the HEK293 cells, mixing and culturing the mouse spleen cells and the HEK293 cells according to the proportion of 10:1 for 12 hours;

(5) flow cytometry detected the expression of the T cell early activation signal C69.

The grouping of this embodiment is as follows:

exosome+HEK293/H2kb；

exosome+HEK293/H2kb-SIINFEKL；

exosome-PD-L1+HEK293/H2kb-SIINFEKL；

exosome-PD-L1-OVA+HEK293/H2kb-SIINFEKL；

exosome-PD-L1-ITGB1+HEK293/H2kb-SIINFEKL；

exosome-PD-L1-ITGB1-OVA+HEK293/H2kb-SIINFEKL。

the results are shown in fig. 17, after each experimental group is stimulated by siifngl for the second time, the proportion of CD69 positive cells in CD8 positive cells is 10.2%, 21.1%, 18.4%, 10.3%, 16.3%, 13.25%, which indicates that OT-1 spleen cells after primary induction by exosome-PD-L1-OVA or exosome-PD-L1-ITGB1-OVA produce specific immune tolerance, and after antigen presenting cells of SIINFEKL polypeptide are encountered again, the OT-1T cells have significantly reduced response to OVA antigen SIINFEKL.

In conclusion, the invention adopts adeno-associated virus containing immunoregulation functional genes to infect the mesenchymal stem cells, and the exosome expressing the membrane protein with the immunoregulation function is obtained by separating and purifying the culture supernatant of the mesenchymal stem cells, has the immunoregulation function similar to that of the mesenchymal stem cells, and effectively overcomes the defect of mesenchymal stem cell treatment.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Tianjin university

<120> gene modified exosome and preparation method and application thereof

<130> 20201109

<160> 9

<170> PatentIn version 3.3

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ggcgatggca cattccagaa gtgggcagca gtggtggtgc cttccggaga ggagcagcgg 840

tatacctgtc acgtgcagca cgagggactg ccagagccac tgatgctgag gtggaagcag 900

tctagcctgc ccacaatccc tatcatgggc atcgtggccg gcctggtggt gctggccgcc 960

gtcgtcctcg agaggcctaa taaagagctc agatgcatcg atcagagtgt gttggttttt 1020

tgtgtgacgc gtaggaaccc ctag 1044

<210> 5

<211> 290

<212> PRT

<213> Artificial sequence

<400> 5

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 6

<211> 286

<212> PRT

<213> Artificial sequence

<400> 6

Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys

1 5 10 15

Cys Val Phe Ala Ser Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val

20 25 30

Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu

35 40 45

Lys Gln Leu Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp

50 55 60

Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln

65 70 75 80

His Ser Ser Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser

85 90 95

Leu Gly Asn Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala

100 105 110

Gly Val Tyr Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg

115 120 125

Ile Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile

130 135 140

Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala

145 150 155 160

Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln

165 170 175

Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys

180 185 190

Leu Phe Asn Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu

195 200 205

Ile Phe Tyr Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr

210 215 220

Ala Glu Leu Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu

225 230 235 240

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Asp Ile Ile Pro

245 250 255

Ile Val Ala Gly Val Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu

260 265 270

Leu Leu Ile Trp Lys Leu Leu Met Ile Ile His Asp Arg Arg

275 280 285

<210> 7

<211> 338

<212> PRT

<213> Artificial sequence

<400> 7

Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Ser Ala Ala Val Ser Arg Pro Ser Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser

50 55 60

Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Arg Glu Gly

65 70 75 80

Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln

85 90 95

Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly

115 120 125

Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu

245 250 255

Thr Lys Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro

290 295 300

Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala

305 310 315 320

Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg Lys Lys Ser

325 330 335

Ser Asp

<210> 8

<211> 347

<212> PRT

<213> Artificial sequence

<400> 8

Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Ser Ala Ala Val Ser Arg Pro Ser Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser

50 55 60

Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Arg Glu Gly

65 70 75 80

Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln

85 90 95

Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly

115 120 125

Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu

245 250 255

Thr Lys Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro

290 295 300

Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala

305 310 315 320

Val Val Leu Glu Arg Pro Asn Lys Glu Leu Arg Cys Ile Asp Gln Ser

325 330 335

Val Leu Val Phe Cys Val Thr Arg Arg Asn Pro

340 345

<210> 9

<211> 8

<212> PRT

<213> Artificial sequence

<400> 9

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

Claims

1. A genetically modified exosome, wherein the exosome expresses a membrane protein with immunomodulatory function;

2. Exosome according to claim 1, characterized in that PD-L1 comprises the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence with more than 80% identity to SEQ ID No. 5 and with the same or similar biological function;

preferably, the HLA-G5 comprises an amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence which has more than 80% of identity with SEQ ID NO. 8 and has the same or similar biological functions.

3. An expression vector, characterized in that the expression vector is an adeno-associated virus vector containing a coding gene of a membrane protein with an immunoregulatory function;

preferably, the expression vector comprises a nucleic acid molecule encoding PD-L1, a nucleic acid molecule encoding PD-L1-ITGB1, a nucleic acid molecule encoding HLA-G1, or a nucleic acid molecule encoding HLA-G5;

preferably, the nucleic acid molecule encoding PD-L1 comprises the nucleic acid sequence shown in SEQ ID NO 1;

preferably, the nucleic acid molecule encoding PD-L1-ITGB1 comprises the nucleic acid sequence shown in SEQ ID NO. 2;

preferably, the nucleic acid molecule encoding HLA-G1 comprises the nucleic acid sequence shown in SEQ ID NO. 3;

preferably, the nucleic acid molecule encoding HLA-G5 comprises the nucleic acid sequence shown in SEQ ID NO. 4.

4. The expression vector of claim 3, wherein the adeno-associated viral vector comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2 or AAV2/YF 3.

5. A recombinant adeno-associated virus, which is a mammalian cell transfected with the expression vector of claim 3 or 4 and a helper plasmid.

6. A method for producing the recombinant adeno-associated virus according to claim 5, wherein the method comprises the steps of:

(1) co-transfecting the expression vector of claim 3 or 4 with a helper plasmid and a transfection reagent into mammalian cells, and culturing for a period of time followed by cell lysis;

7. A method for the preparation of exosomes according to claim 1 or 2, comprising the steps of:

infecting mesenchymal stem cells with the recombinant adeno-associated virus according to claim 5, culturing for a period of time, and collecting a culture supernatant of the mesenchymal stem cells;

8. The method of claim 7, wherein the MOI of the recombinant adeno-associated virus infected mesenchymal stem cells is 5000-10000;

preferably, the culture medium of the mesenchymal stem cells is a serum-free, exosome-free culture medium;

preferably, the culture time of the mesenchymal stem cells is not shorter than 3 days;

9. A pharmaceutical composition comprising an exosome according to claim 1 or 2;

preferably, the pharmaceutical composition further comprises mesenchymal stem cells;

10. Use of the exosome according to claim 1 or 2, the expression vector according to claim 3 or 4, the recombinant adeno-associated virus according to claim 5 or the pharmaceutical composition according to claim 9 for the preparation of a prophylactic and/or therapeutic drug for immune rejection diseases;

preferably, the immune rejection disease comprises graft versus host disease.