Protein combination, engineered cell expressing protein combination and application of protein combination in preparation of immune escape exosome

Abstract

The invention discloses a protein combination, an engineered cell for expressing the protein combination and application of the protein combination in preparation of an immune escape exosome. Wherein the protein combination comprises CD47 and CD63; the amino acid sequence of the CD47 is as shown in SEQ ID NO: 1, and the amino acid sequence of the CD63 is as shown in SEQ ID NO: 2. The engineered cell expressing the protein combination realizes in-vivo phagocytic system escape of the derived exosome and increase of the yield of the exosome, and the application effect of the exosome in regenerative medicine is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a protein combination, an engineered cell expressing the same and use thereof in preparation of immune escape exosomes. BACKGROUND

[0002] Extracellular vesicles (EVs) secreted by stem cells are small vesicles containing various bioactive molecules such as proteins, RNAs and lipids, which can transmit information between cells. Due to their important role in immune regulation, tissue repair and anti-inflammatory response, stem cell exosomes have multiple applications in the field of regenerative medicine, including cardiovascular repair, neural regeneration and skin wound healing. Exosomes can promote angiogenesis and myocardial repair by carrying bioactive molecules, thereby improving cardiac function. In the nervous system diseases, exosomes can promote neural regeneration by regulating inflammatory response and promoting neuronal repair. For skin damage, exosomes can accelerate wound healing and reduce scar formation. These characteristics make them a powerful tool for cell replacement therapy and show great application potential in regenerative medicine.

[0003] MSCs, as the main source cells for exosome production, have high heterogeneity, which leads to inconsistent therapeutic effects and becomes a major challenge. In contrast, iPSCs and MSCs derived therefrom as exosome sources not only provide better medical ethics advantages, but also better control cell consistency. At the same time, studies have shown that iPSC-derived exosomes have comparable or even better therapeutic effects than MSC-derived exosomes in some cases. However, the production of stem cell exosomes has low yield, complex production process and high cost, which limits large-scale application. In addition, the blood circulation half-life of exosomes in vivo is short and they are easily cleared by the mononuclear phagocyte system, resulting in low delivery efficiency and limited drug distribution at the treatment site. The above problems seriously limit the clinical translation and wide application of stem cell exosomes.

[0004] Therefore, there is an urgent need in the art for new cells for producing exosomes. SUMMARY

[0005] Therefore, there is an urgent need in the art for new cells for producing exosomes.

[0006] In a first aspect of the present application, an engineered cell is provided, which overexpresses a protein combination;

[0007] The protein combination comprises CD47 and CD63; the amino acid sequence of CD47 is shown in SEQ ID NO: 1, and the amino acid sequence of CD63 is shown in SEQ ID NO: 2;

[0008] The engineered cell is selected from the group consisting of Expi293F suspension cells, HEK293T cells, and pluripotent or totipotent stem cells; the pluripotent stem cell is optionally an induced pluripotent stem cell.

[0009] In some embodiments, the CD47 and the CD63 are present individually or operably linked as a recombinant protein.

[0010] In some embodiments, the CD47 and the CD63 are linked by a linking peptide in the recombinant protein.

[0011] In some embodiments, the linking peptide comprises a cleavable peptide.

[0012] In some embodiments, the recombinant protein comprises, in order from N-terminus to C-terminus, at least one CD47, a cleavable peptide, and at least one CD63.

[0013] In some embodiments, the recombinant protein has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In some embodiments, the engineered cell satisfies one or more of the following conditions i) to ii):

[0015] i) it comprises a nucleic acid encoding the protein combination as defined above; and,

[0016] ii) it is obtained after transduction of a cell to be engineered by a recombinant expression system comprising a nucleic acid encoding the protein combination as defined above.

[0017] In some embodiments, the nucleic acid has a kozak sequence at its 5' end in the recombinant expression system.

[0018] In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell.

[0019] In some embodiments, the nucleic acid comprises one or more of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0020] In some embodiments, the nucleic acid encodes the recombinant protein as defined above.

[0021] In some embodiments, the nucleic acid has a sequence comprising a sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0022] In some embodiments, the nucleic acid is a combination product and comprises one or more of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0023] In some embodiments, the recombinant expression system satisfies one or more of the following conditions 1) to 6):

[0024] 1) the system used is selected from the group consisting of a lentivirus vector, a gamma- retrovirus vector, a PiggyBac transposon system, a sleeping beauty vector, a PhiC31 recombinase system, an Epstein-Barr virus (EBV) vector, an adeno-associated virus vector, and an adenovirus vector; wherein the lentivirus vector is optionally pLenti, pLV, pCDH, pLVX, pWPI, or pSIN;

[0025] 2) the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6 are expressed separately by a dual promoter system;

[0026] 3) the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6 are expressed by an IRES element connection;

[0027] 4) the promoter in the recombinant expression system is selected from the group consisting of EF1a, CMV, CAG, CAGGS, SV40, TET-ON / OFF, PGK, and Ubc;

[0028] 5) the recombinant expression system is a combination product comprising a recombinant expression system 1 and a recombinant expression system 2, each of which comprises a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6; and,

[0029] 6) the 5' end of the nucleic acid in the recombinant expression system is provided with a kozak sequence.

[0030] In a second aspect of the present application, a method for preparing an exosome modified with CD47 and CD63 is provided, comprising:

[0031] culturing the engineered cell as defined in the first aspect to obtain a culture;

[0032] collecting exosomes from the culture.

[0033] In a third aspect of the present application, an exosome modified with CD47 and CD63 is provided, wherein the CD47 and the CD63 are as defined in the first aspect.

[0034] In some embodiments, it is secreted from the engineered cell as described in the first aspect.

[0035] In some embodiments, the exosome is prepared according to the method as described above.

[0036] In a fourth aspect of the present application, there is provided a protein combination comprising CD47 and CD63; wherein the amino acid sequence of the CD47 is as set forth in SEQ ID NO: 1, and the amino acid sequence of the CD63 is as set forth in SEQ ID NO: 2.

[0037] In some embodiments, the CD47 and the CD63 are present individually or operably linked as a recombinant protein, respectively.

[0038] In some embodiments, the CD47 and the CD63 in the recombinant protein are connected by a connecting peptide.

[0039] In some embodiments, the connecting peptide comprises a cleavable peptide.

[0040] In some embodiments, the recombinant protein comprises, in order from N-terminus to C-terminus, at least one CD47, a cleavable peptide, and at least one CD63.

[0041] In some embodiments, the recombinant protein has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0042] In a fifth aspect of the present application, there is provided a nucleic acid encoding the protein combination as described in the fourth aspect.

[0043] In some embodiments, the nucleic acid comprises one or more of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0044] In some embodiments, the nucleic acid encodes a recombinant protein as defined above.

[0045] In some embodiments, the nucleic acid has a sequence comprising a sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0046] In some embodiments, it is a combination product and comprises one or more of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0047] In a sixth aspect of the present application, there is provided a recombinant expression system comprising the nucleic acid as described in the fifth aspect.

[0048] In some embodiments, the recombinant expression system satisfies one or more of the following conditions 1) to 6):

[0049] 1) the system used is selected from the group consisting of a lentivirus vector, a gamma- retrovirus vector, a PiggyBac transposon system, a sleeping beauty vector, a PhiC31 recombinase system, an Epstein-Barr virus (EBV) vector, an adeno-associated virus vector, and an adenovirus vector;

[0050] 2) the nucleic acid fragments of SEQ ID NO: 5 and SEQ ID NO: 6 are expressed by a dual promoter system, respectively;

[0051] 3) the nucleic acid fragments of SEQ ID NO: 5 and SEQ ID NO: 6 are expressed by an IRES element;

[0052] 4) the promoter in the recombinant expression system is selected from the group consisting of EF1a, CMV, CAG, CAGGS, SV40, TET-ON / OFF, PGK, and Ubc;

[0053] 5) the recombinant expression system is a combination product comprising a recombinant expression system 1 and a recombinant expression system 2, each of which comprises the nucleic acid fragments of SEQ ID NO: 5 and SEQ ID NO: 6, respectively; and

[0054] 6) the nucleic acid in the recombinant expression system has a kozak sequence at the 5' end.

[0055] In some embodiments, the lentivirus vector is pLenti, pLV, pCDH, pLVX, pWPI, or pSIN.

[0056] In a seventh aspect of the present application, a pharmaceutical composition of cells is provided, comprising at least one of the engineered cells of the first aspect and the exosomes of the third aspect, and a pharmaceutically acceptable carrier.

[0057] In an eighth aspect of the present application, the use of the engineered cells of the first aspect or the exosomes of the third aspect in the preparation of an anti-aging or regenerative medicine product is provided.

[0058] Applicants have achieved the escape of the derived exosomes from the phagocytic system in vivo and the improvement of the yield of exosomes by molecular modification of CD47 and CD63 and co-expression thereof by cells, thereby improving the application effect of the exosomes in regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, and more completely understand the present application and its beneficial effects, the accompanying drawings required to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings. It should be noted that the drawings are drawn in a simplified form and are only used to conveniently and clearly assist in the description of the present application.

[0060] Figures 1A-1C For the target gene (GOI) of the plasmid used to construct the cell stable expression strain, wherein Figure 1A and Figure 1B are the natural sequence and the sequence after codon optimization, respectively, and the huCD47 and huCD63 expression sequences (CDS) are connected by a P2A element. Figure 1C Two huCD63 expression sequences are connected in series to further improve the expression amount of CD63.

[0061] Figure 2A and Figure 2B are the transcription levels of the target gene under different multiplicity of infection for the wild type and the modified type cells detected by RT-qPCR. Among them Figure 2A are the results of the wild type and the modified type of Expi293F, Figure 2B are the results of the wild type and the modified type of iPSC, which show that the corresponding cell stable expression strain is successfully constructed.

[0062] Figures 3A-3D are the cell morphologies before and after the modification of the corresponding cells. Figure 3A and Figure 3B are the cell morphologies before and after the modification of Expi293F, which show consistent characteristics of suspension cells. Figure 3C and Figure 3D are the cell morphologies before and after the modification of iPSC, which show consistent characteristics of colony formation.

[0063] Figures 4A-4F are the derived exosome particle size distribution and membrane protein CD63 and CD47 load of the wild type and the modified type of Expi293F. Figure 3A and Figure 4B show that the wild type and the modified type of derived exosome particle size distribution are basically consistent. Figure 4D (Compared with Figure 4C ) and Figure 4F (Compared with Figure 4E ) show that the modified type of Expi293F derived exosome has higher CD63 and CD47 membrane protein load, respectively.

[0064] Figures 5A-5FParticle size distribution and membrane protein CD63 and CD47 loading of iPSC wild type and engineered derived exosomes. Figure 5A and Figure 5B The wild type and engineered derived exosomes show similar particle size distribution. Figure 5D The engineered iPSC derived exosomes show higher CD63 and CD47 membrane protein loading compared to Figure 5C and Figure 5F Figure 5E The engineered iPSC derived exosomes show higher CD63 and CD47 membrane protein loading compared to

[0065] Figures 6A-6C Particle morphology of corresponding cell derived exosomes under transmission electron microscope. Figure 6A and Figure 6B The engineered / wild type Expi293F derived exosomes show typical cup shape morphology. Figure 6C The engineered iPSC derived exosomes show typical cup shape morphology, while the wild type iPSC under the same harvest condition, no clear exosome particles under electron microscope can be seen. Figure 6D

[0066] Figure 7 Western blot band comparison of engineered and wild type cells and derived exosomes. The overexpression of target proteins (CD63 / CD47) is shown to be successful, while the positive and negative markers of derived exosomes are effectively retained.

[0067] Figures 8A-8C The engineered cell lines show higher exosome yield compared to wild type. Figure 8A The Expi239F-MUT derived exosomes show about 300% higher exosome yield compared to wild type. Figure 8B The iPSC-MUT derived exosomes show about 20% higher exosome yield compared to wild type. Figure 8C The iPSC-MUT derived exosomes show about 600 times higher exosome yield compared to wild type after optimization of purification process.

[0068] Figures 9A-9C The Raw264.7 cell uptake results of fluorescently labeled exosomes in an embodiment of the present application are shown. Figure 9A and Figure 9B The fluorescence signal results of Raw264.7 cell uptake of Expi293F-WT and Expi293F-MUT derived exosomes after fluorescent labeling. Figure 9C The fluorescence signal of FITC is counted, and it is shown that the engineered derived exosomes are less taken up by Raw264.7 cells, with about 65% reduction of fluorescence signal.

[0069] Figure 10 ​​Expi293F cells using other fusion proteins and the yield of exosomes secreted by the obtained cell lines.

[0070] Figure 11 Expi293F cells using other fusion proteins and the yield of exosomes secreted by the obtained cell lines. DETAILED DESCRIPTION

[0071] For the purposes of this application, reference will be made to the accompanying drawings in which preferred embodiments of the application are shown. The application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0073] In this application, "one or more" means any one of the listed items or any combination of the listed items, unless otherwise specified. Similarly, "one or more" and the like, where used in other contexts, also mean "one or more" unless otherwise specified.

[0074] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0075] In this application, "suitable combination manner", "suitable manner", "any suitable manner" and the like, "suitable" means that the technical solutions of the application can be implemented, the technical problems of the application can be solved, and the expected technical effects of the application can be achieved.

[0076] In this application, "further", "furthermore", "in particular", "for example", "such as", "example", "for instance" and the like are used for description purposes and mean that the preceding and following technical solutions have a correlation in terms of coverage, but should not be understood as limiting the previous technical solution, nor as limiting the protection scope of the application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0077] In the present application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, if there is no special description, and there is no contradiction or mutual restriction relationship, each "optional" is independent. If there is no other description, the present application is described as "optionally includes", "optionally contains" and the like. For example, "optionally includes" means "may include or not include".

[0078] The terms "contain", "include" and "comprise" used in the present application are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; Non-limiting examples of members or features also include actions, conditions, timing, states, etc.

[0079] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0080] In the present application, the exemplary description involving "in some embodiments (or examples)", "in one embodiment (or example)" and the like can cover but is not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical solutions.

[0081] In the present application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth" and the like are only for description purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and it should be understood that they do not constitute a closed limitation on the quantity.

[0082] In this application, when a numerical interval (i.e., a numerical range) is involved, the distribution of the selectable numbers in the numerical interval is considered to be continuous and includes both numerical endpoints (i.e., the minimum and maximum values) of the numerical interval and every number between the two numerical endpoints, unless otherwise specified. When a numerical interval refers only to integers within the numerical interval, including both endpoint integers and every integer between the two endpoint integers, it is equivalent to directly listing each integer, unless otherwise specified. When multiple numerical ranges are provided to describe a feature or characteristic, the numerical ranges can be combined. In other words, unless otherwise indicated, numerical ranges disclosed herein are to be understood to include any and all sub-ranges of the numbers within the range. A "number" in a numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. A "numerical interval" is intended to broadly include numerical interval types such as percentage intervals, ratio intervals, and the like.

[0083] In this application, when a method flow involves multiple steps, unless there is an explicit different description herein, the execution of these steps does not have strict order restrictions, and they can be executed in other orders described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0084] In a first aspect of the application, an engineered cell is provided, which overexpresses a protein combination;

[0085] The protein combination comprises CD47 and CD63; the amino acid sequence of CD47 is shown in SEQ ID NO: 1, and the amino acid sequence of CD63 is shown in SEQ ID NO: 2;

[0086] The engineered cell is selected from the group consisting of Expi293F suspension cells, HEK293T cells, and pluripotent or totipotent stem cells; the pluripotent stem cell is optionally an induced pluripotent stem cell.

[0087] In some embodiments, the CD47 and the CD63 are present individually or operably linked as a recombinant protein.

[0088] In some embodiments, the CD47 and the CD63 in the recombinant protein are connected by a connecting peptide.

[0089] In some embodiments, the connecting peptide comprises a cleavable peptide.

[0090] In some embodiments, the cleavable peptide comprises a self-cleaving peptide.

[0091] The term "self-cleaving peptide" in the present application, if not otherwise specified, is a short peptide, usually containing 18-25 amino acids, which was first discovered in foot-and-mouth disease virus, and can make a transcript produce multiple proteins. The self-cleaving peptide is not a true "self-cleaving", but works by causing the ribosome to skip the synthesis of glycine and proline peptide bond at the C-terminal of the 2A element. This process causes the 2A sequence to separate from the downstream product, while the C-terminal of the upstream protein will add some extra 2A residues, and the N-terminal of the downstream protein will have an extra proline. Common self-cleaving peptides include but are not limited to P2A, T2A, E2A and F2A, etc., which are derived from different viruses.

[0092] The cleavable peptide in the present application can comprise a self-cleaving peptide, for example, can be P2A, T2A, E2A or F2A. The number of self-cleaving peptides in the cleavable peptide can be one or more.

[0093] In some embodiments, the recombinant protein comprises, in order from N-terminus to C-terminus: at least one CD47, a P2A, and at least one CD63.

[0094] It should be understood that different CD47s or different CD63s can be connected by a flexible linker. The flexible linker can be a flexible linker commonly used in the art, for example, a linker comprising G (glycine) and S (serine). In some embodiments, the flexible linker is (GGGGS)n, wherein n is an integer of 1 or greater than 1. In some embodiments, the flexible linker is (GGGGS)3.

[0095] In some embodiments, the recombinant protein has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0096] In some embodiments, the engineered cell satisfies one or more of the following conditions i) to ii):

[0097] i) it comprises a nucleic acid encoding the combination of proteins as defined above; and,

[0098] ii) it is obtained after transduction of a cell to be engineered with a recombinant expression system comprising a nucleic acid encoding the combination of proteins as defined above.

[0099] In some embodiments, the pluripotent stem cell is optionally an induced pluripotent stem cell.

[0100] In some embodiments, the nucleic acid comprises a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and / or a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0101] In some embodiments, the nucleic acid encodes a recombinant protein as defined above.

[0102] In some embodiments, the nucleic acid has a sequence comprising a sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0103] In some embodiments, the nucleic acid is a combination product and comprises a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and / or a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0104] In some embodiments, the recombinant expression system satisfies one or more of the following conditions 1) to 6):

[0105] 1) the system used is selected from the group consisting of a lentivirus vector, a gamma- retrovirus vector, a PiggyBac transposon system, a sleeping beauty vector, a PhiC31 recombinase system, an Epstein-Barr virus (EBV) vector, an adeno-associated virus vector, and an adenovirus vector; wherein the lentivirus vector is optionally pLenti, pLV, pCDH, pLVX, pWPI, or pSIN;

[0106] 2) the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6 are expressed separately by a dual promoter system;

[0107] 3) the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and the nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6 are expressed by an IRES element connection;

[0108] 4) the promoter in the recombinant expression system is selected from the group consisting of EF1a, CMV, CAG, CAGGS, SV40, TET-ON / OFF, PGK, and Ubc;

[0109] 5) the recombinant expression system is a combination product comprising a recombinant expression system 1 and a recombinant expression system 2, each of which comprises a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6; and,

[0110] 6) the 5' end of the nucleic acid in the recombinant expression system is provided with a kozak sequence.

[0111] In a second aspect of the present application, a method for preparing an exosome modified with CD47 and CD63 is provided, comprising:

[0112] culturing the engineered cell as defined in the first aspect to obtain a culture;

[0113] collecting exosomes from the culture.

[0114] In a third aspect of the present application, an exosome modified with CD47 and CD63 is provided, wherein the CD47 and the CD63 are as defined in the first aspect.

[0115] In some embodiments, the exosome is secreted from the engineered cell as defined in the first aspect.

[0116] In some embodiments, the exosome is prepared according to the method as defined above.

[0117] In a fourth aspect of the present application, a protein combination comprising CD47 and CD63 is provided; wherein the amino acid sequence of the CD47 is as shown in SEQ ID NO: 1, and the amino acid sequence of the CD63 is as shown in SEQ ID NO: 2.

[0118] In some embodiments, the CD47 and the CD63 are present individually or operably linked as a recombinant protein.

[0119] As used herein, the term “operably linked” refers to the functional relationship between the two regions of the recombinant protein, i.e., the CD47 and the CD63, unless otherwise specified; wherein the two regions are connected to produce a recombinant protein or a fusion protein.

[0120] In some embodiments, the CD47 and the CD63 in the recombinant protein are connected by a connecting peptide.

[0121] In some embodiments, the connecting peptide comprises a cleavable peptide.

[0122] As described above, the cleavable peptide can include a self-cleavable peptide, and exemplary self-cleavable peptides include P2A, T2A, E2A, F2A, and the like.

[0123] In some embodiments, the recombinant protein comprises, in order from N-terminus to C-terminus, at least one CD47, a P2A, and at least one CD63.

[0124] In some embodiments, the recombinant protein comprises, in order from N-terminus to C-terminus, a CD47, a P2A, and at least one CD63. Exemplarily, the at least one is 2 or more.

[0125] Further included within the scope of the present application are functional variants of the recombinant proteins of the present application described herein. The term "functional variant" as used herein refers to a recombinant protein, polypeptide or protein having substantial or significant sequence identity or similarity to a parent recombinant protein that retains the biological activity of the recombinant protein variant. Functional variants encompass, for example, those variants of the recombinant proteins described herein (parent recombinant proteins) that retain similar functionality to the parent recombinant protein. The amino acid sequence of the functional variant can, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99% or more identity to the amino acid sequence of the parent recombinant protein.

[0126] The functional variant may, for example, comprise the amino acid sequence of the parent recombinant protein with at least one conservative amino acid substitution. Alternatively or additionally, the functional variant can comprise the amino acid sequence of the parent recombinant protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent recombinant protein.

[0127] The amino acid substitution of the recombinant proteins of the present application is preferably a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain for another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain for another amino acid having a beta-branched side chain (e.g., He, Thr, and Val), an amino acid having an aromatic side chain for another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0128] The recombinant proteins of the embodiments of the present application, including the functional parts and functional variants of the present application, can comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino normal decanoic acid, homoserine, S-acetylamino methyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl- lysine, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2- norbornane)-carboxylic acid, a, y-diaminobutyric acid, a, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0129] Mutants having a certain degree of amino acid homology with the amino acid sequence of the recombinant proteins as described above, for example between 70% and 99% homology, further between 80% and 99% homology, further between 90% and 99% homology, and 99% homology, also belong to the scope of protection of the present application.

[0130] “Homology” (percent sequence identity (%)) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by determining the number of amino acid residues (or bases) in the candidate sequence that are identical to the reference sequence to which it is being compared, and dividing the number of identical amino acid residues (or bases) by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not counted as differences. Alignment can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J. Mol. Biol. 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al. Bioinformatics (Cambridge, England), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. The alignment can be achieved by the default parameters provided by the tools or can be appropriately customized according to the needs of the alignment by the person skilled in the art, for example by choosing a suitable algorithm.

[0131] As used herein, the term “amino acid” refers to an organic compound that includes an amino (-NH2) and carboxyl (-COOH) functional group and a side chain specific to each amino acid. The amino acid names are also denoted in the standard one-letter or three-letter code in the present disclosure, which is summarized as follows.

[0132]

[0133]

[0134] In some embodiments, the amino acid sequence of the recombinant protein is as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0135] In a fifth aspect of the present application, there is provided a nucleic acid encoding the protein combination of the fourth aspect.

[0136] In the present application, unless otherwise specified, "nucleic acid" has the meaning commonly understood in the art, also referred to as "polynucleotide", is a molecule formed by a plurality of nucleotide monomers.

[0137] In some embodiments, the nucleic acid comprises one or more of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0138] In some embodiments, the nucleic acid comprises both of the above-mentioned nucleic acid fragments as a single molecule. Although the two nucleic acid fragments are contained in the same nucleic acid molecule, they can be expressed separately.

[0139] In the present application, the manner of separately expressing the two nucleic acid fragments can be conventional in the art, illustratively, the two can be linked by an IRES element, or expressed by a dual promoter system.

[0140] The terms "IRES" and "IRES element" in the present application are used interchangeably herein. IRES element, the full name of which is internal ribosome entry site, is a nucleic acid sequence found in eukaryotes and viruses capable of infecting eukaryotes, and its presence enables protein translation initiation to be independent of 5' cap structure, thus enabling direct initiation of translation from the middle of messenger RNA (mRNA).

[0141] In some embodiments, the nucleic acid encodes a recombinant protein as defined above.

[0142] In some embodiments, the sequence of the nucleic acid comprises a sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0143] In some embodiments, it is a combination product and comprises at least one of a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 5 and a nucleic acid fragment having a sequence as set forth in SEQ ID NO: 6.

[0144] In a sixth aspect of the present application, there is provided a recombinant expression system comprising the nucleic acid of the fifth aspect.

[0145] In some embodiments, the recombinant expression system satisfies one or more of the following conditions 1) to 6):

[0146] 1) the system used is selected from the group consisting of lentivirus vector, gamma-retrovirus vector, PiggyBac transposon system, sleeping beauty virus vector, PhiC31 recombinase system, centromere virus (EBV) vector, adeno-associated virus vector and adenovirus vector;

[0147] 2) the nucleic acid fragment with sequence as shown in SEQ ID NO: 5 and the nucleic acid fragment with sequence as shown in SEQ ID NO: 6 are respectively expressed by a double promoter system;

[0148] 3) the nucleic acid fragment with sequence as shown in SEQ ID NO: 5 and the nucleic acid fragment with sequence as shown in SEQ ID NO: 6 are expressed by IRES element connection;

[0149] 4) the promoter in the recombinant expression system is selected from the group consisting of EF1a, CMV (Cytomegalovirus Promoter), CAG (Chicken beta-Actin Promoter with CMV Enhancer), CAGGS, SV40 (Simian Virus 40), TET-ON / OFF, PGK (Phosphoglycerate Kinase Promoter) and Ubc (Ubiquitin C promoter);

[0150] 5) the recombinant expression system is a combination product, which comprises recombinant expression system 1 and recombinant expression system 2 respectively comprising the nucleic acid fragment with sequence as shown in SEQ ID NO: 5 and the nucleic acid fragment with sequence as shown in SEQ ID NO: 6; and,

[0151] 6) in the recombinant expression system, the 5' end of the nucleic acid is provided with a kozak sequence.

[0152] The CMV promoter in the present application also relates to its related modified promoters, such as CMV-beta, CMV-EF1a, etc.

[0153] The term "promoter" in the present application is used to refer to a set of transcription control modules clustered around the start site for RNA polymerase II, unless otherwise specified. Many ideas about how promoters are organized have been derived from analysis of several viral promoters, including the promoters for the HSV thymidine kinase (tk) and the SV40 early transcription unit. These studies, reinforced by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of about 7-20 bp of DNA and containing one or more recognition sites for transcriptional activation proteins. At least one module in each promoter acts to position the start site for RNA synthesis. The best known example of this is the TATA box, but in certain promoters that lack a TATA box, such as the promoters for the mammalian terminal deoxynucleotidyl transferase gene and the SV 40 late genes, discrete elements overlapping the start site itself help fix the initiation position.

[0154] Additional promoter elements modulate the frequency of transcription initiation. Generally, these are located in the region 30-110 bp upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between elements is flexible, such that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between elements can be increased to 50 bp apart before activity begins to drop. Depending on the promoter, individual elements can appear to act together or independently to activate transcription. Any promoter that can direct the initiation of transcription of a sequence encoded by a nucleic acid construct can be used in the present application.

[0155] In some embodiments, the lentiviral vector is pLenti, pLV, pCDH, pLVX, pWPI, or pSIN.

[0156] The term "vector" in the present application refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and expressed, unless otherwise specified. A vector can be, for example, a plasmid, a cosmid, a virus (e.g., a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus), an RNA vector, or a linear or circular DNA or RNA molecule, which can include a chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecule. The term includes both vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of host cells into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0157] In some embodiments, the backbone of the recombinant vector is a viral vector.

[0158] As used herein, the term "viral vector" refers to a vector based on a virus that has been modified to carry a foreign gene and associated genetic elements, which can be packaged into a viral particle and can mediate the transfer and expression of the foreign gene. The viral vector can be a recombinant viral vector or a viral vector without viral genes. Non-limiting examples of viral vectors include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and the like.

[0159] In a seventh aspect of the present application, there is provided a pharmaceutical composition comprising at least one of the engineered cell of the first aspect and the exosome of the third aspect, and a pharmaceutically acceptable carrier.

[0160] As used herein, the term "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for administration to patients and are commensurate with a reasonable benefit / risk ratio.

[0161] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0162] In an eighth aspect of the present application, there is provided use of the engineered cell of the first aspect or the exosome of the third aspect in the manufacture of an anti-aging or regenerative medicine product.

[0163] In a ninth aspect of the present application, there is provided a method of treating an aging-related disease using the engineered cell of the first aspect or the exosome of the third aspect.

[0164] As used herein, the term "treatment" refers to therapeutic treatment, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treatment" embraces reduction or alleviation of at least one symptom or parameter associated with a disease or disorder. Treatment is generally "effective" if there is a reduction in one or more symptoms or clinical markers. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted, that is, "treatment" includes not just the improvement of symptoms but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or remission of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0165] Some embodiments are provided below.

[0166] The embodiments of the present application will be described in detail with reference to the following examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental methods in the following examples, for which no conditions are specified, are preferably performed according to the indications given in the present application, but also according to the protocols known in the art or according to the conditions recommended by the manufacturer or according to the experimental methods known in the art.

[0167] Example 1

[0168] 1. Engineering of stable cell lines

[0169] a) Engineering of plasmid construction and optimization

[0170] Exosomes production cells for engineering stable cell lines (subject cells can be engineered cell lines such as Expi293F suspension cells, HEK293T cells, etc.; or stem cells with pluripotency or totipotency such as induced pluripotent stem cells iPSC or mesenchymal stem cells MSC, etc.) are constructed into corresponding stable expression cell lines (stable cell lines) by lentivirus infection method or transposon method or Crispr-Cas9 gene editing technology, etc. The cell lines can be polyclonal cell populations or further screened as monoclonal cell lines. The key materials for constructing stable expression cell lines are vector plasmids loaded with functional elements for stable expression. The GOI of the corresponding sequence is inserted into the expression frame of the vector plasmid (pLent-EF1a-MF-pgk-Puro-KanR, sequence as shown in SEQ ID NO: 10) by molecular cloning, etc. to obtain GOI-loaded vector plasmids such as construct-1, construct-2 or construct-3. Among them, construct 1 involves the connection of the natural expression sequences (CDS) of huCD47 and huCD63 through P2A element; construct 2 involves the connection of the codon-optimized expression sequences (CDS; as shown in SEQ ID NO: 5 and 6, respectively) of huCD47 and huCD63 through P2A element; and construct 3 involves the construction of the tandem expression sequences of huCD47 and two huCD63 to further improve the expression amount of CD63 (see Figure 1A 、 Figure 1B and Figure 1C ). The amino acid sequences of CD47 and CD63 encoded by construct-1, construct-2 and construct-3 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and the nucleic acid sequences of the three constructs are shown in SEQ ID NO: 7-9, respectively.

[0171] The success of plasmid construction is confirmed by sanger sequencing, and the confirmed plasmid is used for subsequent construction of stable expression cell lines.

[0172] b) Stable cell line construction

[0173] The engineering of target exosome producing cells (both stable cell line construction, such as in Expi293F suspension cells (Thermo Fischer) or the engineering of induced pluripotent stem cells (iPSC), and the packaging of corresponding lentivirus) were performed by GenScript. Specifically, using lentivirus vector plasmid construct 1 (or construct 2, construct 3), after extraction and endotoxin removal, and the third generation lentivirus packaging plasmid system, lentivirus preparation was performed in HEK293T cells. Then, using the lentivirus prepared as described above, the target cells were infected, drug screened, and clonogenicity tested. 1-3 virus infection multiplicity of infection (or MOI) was set respectively, and positive cell stable strains were obtained by resistance screening. The mRNA level of the transcription of the target gene of the positive stable strain cell sample and the control cell (unmodified wild type cell) was detected by RT-qPCR to identify and confirm whether the stable strain was successfully constructed. The successfully constructed stable strain can be used for subsequent exosome production and harvesting.

[0174] The transcription product mRNA abundance of the target gene (GOI) in the engineered cells (MUT) and the control wild type cells (WT) was detected by RT-qPCR. The exosome producing cell surface gene engineered Expi293F-MUT and iPSC-MUT stable expression cell strains were successfully constructed. The relative abundance of the transcription product mRNA of the target gene on Expi293F-MUT and iPSC-MUT was presented as Figure 2A and Figure 2B respectively. And after genetic engineering, the cell morphology did not change. As shown in Figure 3A and Figure 3B , Expi293F-MUT and Expi293F-WT had similar suspension cell morphology and state, as shown in Figure 3C and Figure 3D , iPSC-WT and iPSC-MUT had similar colony cell morphology.

[0175] 2. Exosome collection and physicochemical characterization

[0176] c) Exosome producing cell culture

[0177] Exosome producing cells were cultured according to the requirements of the corresponding cells. Specifically, Expi293F wild type (Expi293F-WT) or modified type (Expi293F-MUT) were cultured in Expire293 expression medium (Gibco), and the cell culture conditions were 95% air, 5% carbon dioxide, and 37°C suspension culture. Induced pluripotent stem cells wild type (iPSC-WT) or modified type (iPSC-MUT) were cultured in mTeSR1 culture system (STEM CELL Technologies), and the cell culture conditions were 95% air, 5% carbon dioxide, and 37°C culture.

[0178] d) Purification and collection of exosomes from cell culture supernatant

[0179] Exosome samples were obtained by purification and collection of cell culture supernatant. Specifically, the cell supernatant was first centrifuged at 300xg for 10 min to remove cells, and then centrifuged at 2,000xg for 10 min to remove cell debris. The obtained cell supernatant was further filtered through a 0.22μm or 0.45μm filter to obtain pretreated cell culture supernatant. Then the sample was transferred to a new centrifuge tube, and the ultracentrifuge was selected, 4°C, 100,000xg centrifugation for 70 min. After removing the supernatant and resuspending with an appropriate volume of pre-cooled 1xPBS, the ultracentrifuge was selected again, filtered through a 0.22μm filter, and ultracentrifuged at 4°C, 100,000xg for 70 min. The supernatant was removed again, and the sample was resuspended with an appropriate volume of 1xPBS to obtain purified exosomes for subsequent detection and experiments. For smaller volume of culture supernatant samples (such as <3-10mL), ultrafiltration tubes (Millipore) can also be used for ultrafiltration concentration and purification of pretreated culture supernatant. The specific method is described in the ultrafiltration tube instruction manual, and the obtained purified exosome sample is used for subsequent detection and experiments.

[0180] e) Nano flow cytometry and nanoparticle tracking analyzer detection to determine exosome particle concentration and particle size distribution

[0181] Nano flow cytometry detection (Nano FCM, N30E), specifically, 10μL of exosomes was diluted to an appropriate multiple. After passing the instrument performance test with standard samples, exosome samples can be loaded. Gradient dilution is required to avoid sample clogging the sample needle. After the sample is detected, the particle size and particle concentration information of the exosome detected by the instrument can be obtained. Alternatively, the exosome sample is diluted to an appropriate multiple, and the nanoparticle tracking analyzer (NanoSight300, Yikeshi) is used to sample the sample with an automatic sample needle to detect the particle concentration and particle size distribution of the sample, and the particle size and particle concentration information of the exosome sample is obtained.

[0182] Figure 4A and Figure 4B The particle size distribution of engineered and wild type Expi293F derived exosomes was not significantly changed.

[0183] As Figure 6A and Figure 6B The particle size distribution of engineered and wild type Expi293F derived exosomes was not significantly changed. As Figure 6C The TEM morphology of iPSC-MUT derived exosomes showed a cup-shaped morphology characteristic of exosomes, while iPSC-WT collected in the same process did not show typical exosome particle morphology in the entire field of view due to the lack of sufficient derived exosomes (see Figure 6D ).

[0184] f) Nano flow cytometer assay of exosome membrane surface proteins

[0185] Specifically, an appropriate amount of exosome sample was diluted, and the diluted exosomes were added to the fluorescently labeled antibody to be tested and mixed uniformly, avoiding light, and incubated at 37°C for 30 min. Add 1 mL of pre-cooled PBS, select the ultracentrifuge, 4°C, 110,000 x g, ultracentrifuge for 70 min. Carefully remove the supernatant, add 1 mL of pre-cooled PBS, select the ultracentrifuge, 4°C, 110,000 x g, ultracentrifuge for 70 min. Carefully remove the supernatant, resuspend with 50 μL of pre-cooled 1 x PBS. First, use the standard to test the performance of the instrument (NanoFCM, N30E) to pass the test before loading the exosome sample. Note that gradient dilution is required to avoid clogging the sample needle. After the sample is detected, the protein index results of the instrument detection can be obtained.

[0186] Referring to Figure 4C and Figure 4D , which respectively show wild type Expi293F derived exosomes and engineered Expi293F derived exosomes, the results show that the engineered CD63 protein is more loaded on the membrane of the exosomes. As Figure 4E and Figure 4F , which respectively show wild type Expi293F derived exosomes and engineered Expi293F derived exosomes, the results show that the engineered CD47 protein is more loaded on the membrane of the exosomes.

[0187] Referring to Figure 5C and Figure 5D , which respectively show wild type iPSC derived exosomes and engineered iPSC derived exosomes, the results show that the engineered CD63 protein is more loaded on the membrane of the exosomes. As Figure 5E and Figure 5FWild type iPSC-derived exosomes and engineered iPSC-derived exosomes were displayed, respectively, and the results showed that the engineered CD47 protein was more loaded on the membrane of the exosomes. Figure 5A and Figure 5B Wild type and engineered derived exosomes showed basically consistent particle size distribution.

[0188] g) Transmission electron microscopy detection of exosome morphology

[0189] Specifically, 10 μL of exosome sample was taken. The sample was dropped on a copper mesh and precipitated for 1 min, and the supernatant was absorbed by filter paper. 10 μL of uranyl acetate (Henan Xinrui, GZ02625-5) was dropped on the copper mesh and precipitated for 1 min, and the supernatant was absorbed by filter paper. Dry for a few minutes at room temperature. Use an acceleration voltage of 80 KV to detect imaging by electron microscope (Hitachi, HT-7700) and obtain transmission electron microscopy imaging photos.

[0190] See Figure 6A and Figure 6B , the transmission electron microscopy morphology of Expi293F-WT and Expi293F-MUT derived exosomes showed a tea tray-like morphology characteristic of exosomes.

[0191] h) Western blot detection of target protein expression in cells and derived exosomes

[0192] Specifically:

[0193] 1. Prepare SDS PAGE electrophoresis gel with a concentration of 12% according to the protein size of the sample to be detected; after the electrophoresis device is assembled, add appropriate amount of Running Buffer.

[0194] 2. Take out the protein sample, boil in a constant temperature metal bath for 3-5 min, and centrifuge; add the protein sample and protein Maker to the hole on the electrophoresis gel in the required order using a pipette or a loading needle.

[0195] 3. Cover the tank cover, turn on the power, run the gel at 80V until the sample runs out of the concentrated gel, and then run the gel at 100V until the bromophenol blue reaches the bottom of the gel.

[0196] 4. After electrophoresis, take out the electrophoresis gel, trim the size of the gel, cut off the edges and excess part, and cut the corner mark at the lower left corner; according to the size of the trimmed gel, cut a PVDF membrane of the same size, cut the corner at the lower left corner as a mark, activate in appropriate amount of methanol for 20 s, and then soak in pre-cooled transfer buffer.

[0197] 5. Take 8 pieces of filter paper and cut them into 8 cm x 10 cm size, and also soak them in pre-cooled transfer buffer; make a transfer "sandwich" in the order of sponge-filter paper-electrophoresis gel-membrane-filter paper-sponge.

[0198] 6. Add transfer buffer to the tank, assemble the transfer device, add ice, bury the device in ice bath or put in the refrigerator, turn on the power, 300mA transfer, the transfer time is determined according to the size of the protein to be detected, the general rule is 1 kd = 1 min.

[0199] 7. After the transfer is completed, take out the PVDF membrane, immerse the membrane in 5% skim milk TBST with the protein facing up, and seal for 1 h.

[0200] 8. Primary antibody blocking: the membrane after milk blocking is cut according to the need, the membrane is immersed in the prepared primary antibody solution, and incubated at 4°C overnight.

[0201] 9. Recover the primary antibody, wash the membrane with 20 mL TBST at room temperature for 10 min, repeat three times.

[0202] 10. According to the selection of secondary antibody, the secondary antibody is prepared in 5% skim milk TBST solution at 1:5000, the membrane is immersed in the secondary antibody solution, and incubated at room temperature for about 1 h.

[0203] 11. Recover the secondary antibody, wash the membrane with 20 mL TBST at room temperature for 10 min, repeat three times.

[0204] 12. Take out the PVDF membrane, drain the water, lay the protein facing up on the plastic wrap, drop the mixed ECL A / B liquid mixture of equal volume on the membrane, avoid light reaction for 5 min; take the membrane and move it into the plastic sealed film, the protein always faces up.

[0205] 13. Place the membrane in the imager, set the parameters, start exposure; adjust the brightness and contrast, save the picture.

[0206] See Figure 7 , compared with the cell samples and the derived exosome samples of Expi293F-WT, the corresponding Expi293F-MUT cells and the derived exosomes show the target protein (overexpression of CD47 and CD63).

[0207] i) Calculation of the yield of cell-derived exosomes

[0208] Calculation of the yield of exosomes from cell supernatant, calculated according to the following formula,

[0209] Exosome yield per unit volume of cell supernatant (exosome particle number corresponding to each unit volume of supernatant, such as Calculate and compare the yield of harvested exosomes under different conditions or compare the relative changes in yield.

[0210] See Figure 8AAfter the modification of Expi293F, Expi293F-MUT showed higher number of derived exosome particles per volume compared to wild type, showing about 300% improvement. See Figure 8B After the modification of iPSC, iPSC-MUT showed higher number of derived exosome particles per volume compared to wild type, showing about 20% improvement. By further optimizing the purification condition (from ultracentrifugation of cell culture supernatant to ultrafiltration purification), reference Figure 8C iPSC-MUT derived exosomes showed 600-fold improvement of exosome yield per volume compared to iPSC-WT wild type derived.

[0211] 2. Fluorescently labeled exosomes and Raw264.7 cell uptake experiment

[0212] j) Fluorescent dye labeling of exosomes

[0213] Prepare dye according to the instruction (PKH67, Sigma). Add PKH67 dye to the appropriate amount of exosomes, mix well, and incubate at room temperature for 10 min in the dark. Add 1 mL of PBS, Optima MAX-XP + rotor TLA120.2, 100000g, centrifuge for 17 min, discard the supernatant, and resuspend in sterile PBS. Repeat the above steps three times with sterile PBS. Resuspend in 100 μL of sterile PBS (store in the dark), take 10 μL to detect particle size and concentration, and store the rest at -80°C for subsequent cell uptake experiments.

[0214] k) Fluorescently labeled exosomes and Raw264.7 cell uptake experiment

[0215] Specifically, Raw264.7 cells were cultured in culture medium containing 1% BSA, 10% fetal bovine serum, at 37°C, 5% CO2, saturated humidity, and subcultured every 2 days. In the exosome uptake experiment, the target amount of fluorescently labeled exosomes was plated in a 48-well plate, and Raw264.7 cells were plated and labeled exosomes were added, respectively. Cell slides were prepared, and after the cells adhered, the labeled exosomes were added to the cells, and the cells were incubated in the cell incubator for 8 h. Nuclear staining and microscopy were performed, and first the cell slides were removed and washed with PBS three times. Then 4% paraformaldehyde was added for room temperature fixation for 30 min, and washed with PBS three times. Then the cells were stained for 10 min, and the slides were mounted. Confocal microscopy was performed and photos of the exosomes taken by Raw264.7 cells were obtained. Figure 9A Expi293F-WT derived exosomes were fluorescently labeled and taken up by Raw264.7 cells, and the fluorescence signal was Figure 9BExpi293F-MUT derived exosomes were shown to be taken up by Raw264.7 cells after being labeled with the same fluorescent marker. The results showed that the engineered cell line-derived exosomes showed lower macrophage uptake, and the Raw264.7 cells took up less engineered exosomes than wild-type exosomes, with a decrease of about 65% of the fluorescent signal. Figure 9C ) It can be seen that the engineered cell line shows an escape effect on macrophages compared to the wild type.

[0216] Comparative Example

[0217] The engineered stable cell line construction, stable cell line construction, and exosome collection and physicochemical characterization were carried out according to the method shown in Example 1 above. The main difference is that other proteins (PDGFRbeta transmembrane region, lactadherin protein C1C2 transmembrane region, and Arc protein, respectively) other than CD63 were expressed using amino acid sequences shown in SEQ ID NO: 11, 13, or 15 (nucleic acid sequences shown in SEQ ID NO: 12, 14, or 16, respectively). At the same time, the CD63 with the amino acid sequence shown in SEQ ID NO: 17 (nucleic acid sequence shown in SEQ ID NO: 18, containing a linker element) was also expressed.

[0218] The results are shown in Figure 10 and Figure 11 , which show that unlike the modification in the example, other modifications do not show a significant increase in exosome yield compared to the wild type (WT group), showing the superiority of the modification method described in this application.

[0219] Summary:

[0220] 1. Through exosome particle concentration and particle size distribution experiments, exosome membrane surface protein detection, exosome morphology detection, and Western blot detection, we successfully verified the feasibility of the application. The prepared exosomes not only have regular particle morphology and sufficient membrane surface loading, but also have excellent target protein expression levels.

[0221] 2. By calculating its yield, we can learn that the exosome yield of the application has been improved by hundreds of times compared to the wild type, which suggests that through the recombination method of the application, especially by connecting the target gene through the linker element, the independent expression of the target protein is one of the important guarantees for achieving this improvement.

[0222] In addition, through the cell uptake experiment, we confirmed that the exosomes of the application have a strong escape effect on macrophages.

[0223] 3. Through the horizontal comparison of the comparative example and the example, it is further confirmed that other proteins do not contribute much to the yield of exosomes.

[0224] Each of the technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, but it should be understood that any combination of the technical features is within the scope of the present disclosure as long as the combination does not result in a contradiction.

[0225] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these are within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An engineered cell, characterized in that, Its overexpressed protein composition; The protein combination includes CD47 and CD63; the amino acid sequence of CD47 is shown in SEQ ID NO:1, and the amino acid sequence of CD63 is shown in SEQ ID NO:

2. The engineered cells are selected from a group consisting of Expi293F suspension cells, HEK293T cells, and pluripotent or all-purpose stem cells; the pluripotent stem cells may optionally be induced pluripotent stem cells.

2. The engineered cell as described in claim 1, characterized in that, The CD47 and CD63 may exist individually or be operatively linked as a recombinant protein. Optionally, CD47 and CD63 in the recombinant protein are linked by a linker peptide; the linker peptide may optionally include a cleavable peptide. Further optionally, the recombinant protein comprises, from the N-terminus to the C-terminus, at least one CD47, a cleavable peptide, and at least one CD63; Alternatively, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:3 or SEQ ID NO:

4.

3. The engineered cell as described in claim 1 or 2, characterized in that, The engineered cells satisfy one or more of the following conditions i) to ii): i) It comprises a nucleic acid encoding a combination of proteins as defined in claim 1 or 2; and, ii) It is obtained by transducing cells to be engineered using a recombinant expression system comprising a nucleic acid encoding a combination of proteins as defined in claim 1 or 2; optionally, in the recombinant expression vector, the nucleic acid has a kozak sequence at its 5' end.

4. The engineered cell as described in claim 3, characterized in that, The nucleic acid comprises one or more of the nucleic acid fragments with sequences as shown in SEQ ID NO:5 and nucleic acid fragments with sequences as shown in SEQ ID NO:6; Optionally, the nucleic acid encodes a recombinant protein as defined in claim 2; further optionally, the sequence of the nucleic acid comprises a sequence as shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; Optionally, the nucleic acid is a combination product and comprises one or more of the nucleic acid fragments with sequences as shown in SEQ ID NO:5 and nucleic acid fragments with sequences as shown in SEQ ID NO:

6.

5. The engineered cell as described in claim 3 or 4, characterized in that, The recombinant expression system satisfies one or more of the following conditions 1) to 6): 1) The system used is selected from the group consisting of lentiviral vectors, γ-retroviral vectors, PiggyBac transposon systems, virus-specific sleeping beauty vectors, PhiC31 recombinase systems, centromere virus (EBV) vectors, adeno-associated virus vectors, and adenovirus vectors; wherein, the lentiviral vector may be pLenti, pLV, pCDH, pLVX, pWPI, or pSIN; 2) The nucleic acid fragments shown in SEQ ID NO:5 and SEQ ID NO:6 are expressed separately using a dual-initiator subsystem; 3) Nucleic acid fragments with sequences as shown in SEQ ID NO:5 and SEQ ID NO:6 are expressed by linking them together using an IRES element; 4) The promoters in the recombinant expression system are selected from the group consisting of EF1a, CMV, CAG, CAGGS, SV40, TET-ON / OFF, PGK and Ubc; 5) The recombinant expression system is a combinatorial product comprising recombinant expression system 1 and recombinant expression system 2, each containing a nucleic acid fragment with the sequence shown in SEQ ID NO:5 and a nucleic acid fragment with the sequence shown in SEQ ID NO:6, respectively; and, 6) In the recombinant expression system, the 5' end of the nucleic acid carries a kozak sequence.

6. A method for preparing exosomes modified with CD47 and CD63, characterized in that, It includes: Culture the engineered cells as described in any one of claims 1 to 5 to obtain a culture; Exosomes were collected from the culture.

7. An exosome modified with CD47 and CD63, characterized in that, The CD47 and the CD63 are as defined in claim 1 or 2.

8. The exosomes as described in claim 7, characterized in that, It is secreted from engineered cells as described in any one of claims 1 to 5; Optionally, the exosomes are prepared according to the method of claim 6.

9. A protein composition, characterized in that, It contains CD47 and CD63; The amino acid sequence of CD47 is shown in SEQ ID NO:1, and the amino acid sequence of CD63 is shown in SEQ ID NO:

2.

10. The protein combination as described in claim 9, characterized in that, The CD47 and CD63 may exist individually or be operatively linked as a recombinant protein. Optionally, CD47 and CD63 in the recombinant protein are linked by a linker peptide; the linker peptide may optionally include a cleavable peptide. Further optionally, the recombinant protein comprises, from the N-terminus to the C-terminus, at least one CD47, a cleavable peptide, and at least one CD63; Alternatively, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:3 or SEQ ID NO:

4.

11. A nucleic acid encoding a protein combination as described in claim 9 or 10; Optionally, the nucleic acid comprises a nucleic acid fragment with a sequence as shown in SEQ ID NO:5 and / or a nucleic acid fragment with a sequence as shown in SEQ ID NO:6; Further, optionally, the nucleic acid encodes a recombinant protein as defined in claim 10; Alternatively, the nucleic acid sequence may include sequences as shown in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

12. The nucleic acid as described in claim 11, characterized in that, It is a combination product and contains one or more of the nucleic acid fragments with sequences as shown in SEQ ID NO:5 and SEQ ID NO:

6.

13. A recombinant expression system comprising the nucleic acid as described in claim 11 or 12; Optionally, the recombinant expression system satisfies one or more of the conditions shown in 1) to 6) below: 1) The systems used were selected from a group consisting of lentiviral vectors, gamma-retroviral vectors, PiggyBac transposon systems, viral sleeping beauty vectors, PhiC31 recombinase systems, centromere virus (EBV) vectors, adeno-associated virus vectors, and adenovirus vectors; among them, The lentiviral vector may optionally be pLenti, pLV, pCDH, pLVX, pWPI, or pSIN; 2) The nucleic acid fragments shown in SEQ ID NO:5 and SEQ ID NO:6 are expressed separately using a dual-initiator subsystem; 3) Nucleic acid fragments with sequences as shown in SEQ ID NO:5 and SEQ ID NO:6 are expressed by linking them together using an IRES element; 4) The promoters in the recombinant expression system are selected from the group consisting of EF1a, CMV, CAG, CAGGS, SV40, TET-ON / OFF, PGK and Ubc; 5) The recombinant expression system is a combinatorial product comprising recombinant expression system 1 and recombinant expression system 2, each containing a nucleic acid fragment with the sequence shown in SEQ ID NO:5 and a nucleic acid fragment with the sequence shown in SEQ ID NO:6, respectively; and, 6) In the recombinant expression system, the 5' end of the nucleic acid carries a kozak sequence.

14. A cell-based drug composition comprising at least one of engineered cells as described in any one of claims 1 to 5 and exosomes as described in claim 7 or 8, and a pharmaceutically acceptable carrier.

15. Use of engineered cells as described in any one of claims 1 to 5 or exosomes as described in claim 7 or 8 in the preparation of products for anti-aging or regenerative medicine.

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