Use of arf6 as a scaffold protein in the preparation of engineered extracellular vesicles

Abstract

The application provides application of ARF6 as a scaffold protein in preparation of an engineered extracellular vesicle, and belongs to the technical field of biological medicine. The application provides application of ARF6 (SEQ ID NO. 1) as a scaffold protein in preparation of an engineered extracellular vesicle (EV). The application uses natural full-length ARF6 as a scaffold protein to prepare an engineered EV, can fully exert the comprehensive advantages of short gene sequence, high editing and synthesis efficiency, strong loading capacity and good universality, effectively breaks through the technical bottlenecks of low loading efficiency and complex construction process of the existing EV drug loading system, and successfully constructs a kind of efficient, universal and safe protein and polypeptide drug delivery platform. The technical scheme of the application shows good anti-inflammatory effect in a cell inflammation model and a sepsis mouse model, indicating that the application has important clinical application value and industrialization prospect in the fields of drug delivery and disease treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ARF6 as a scaffold protein in the preparation of engineered extracellular vesicles. Background Technology

[0002] Extracellular vesicles (EVs) are lipid bilayer vesicles secreted by various cells, including exosomes, microvesicles, and apoptotic bodies. Their surfaces are rich in various specific membrane proteins, and they carry bioactive molecules such as proteins, nucleic acids, and lipids, playing important roles in various physiological and pathological processes. In recent years, due to their natural nanocarrier properties, EVs have been considered an ideal platform for delivering proteins, nucleic acids, and small molecule drugs, enabling the efficient delivery of various therapeutic drugs to target cells to mediate disease treatment. Compared with synthetic nanocarriers such as liposomes, EVs possess unique advantages such as low immunogenicity, no cytotoxicity, excellent biocompatibility, and the ability to overcome biological barriers. Therefore, they are considered a new generation of drug delivery carriers with great clinical translational potential, showing broad application prospects in the field of disease treatment.

[0003] Despite the significant advantages of endothelial cells (EVs) in drug delivery, their natural cargo loading efficiency is often insufficient to meet clinical treatment needs. In particular, the efficient loading of protein or peptide therapeutic drugs remains a core technological bottleneck that EV drug delivery systems urgently need to overcome. One common strategy involves genetically engineering parental cells to fuse the target protein (POI) with membrane proteins highly enriched in EVs. Utilizing the sorting signals of these membrane proteins, the POI accumulates on the EV membrane surface or within the lumen, thereby achieving efficient loading and delivery of therapeutic cargo. These membrane proteins, highly enriched in EVs and capable of acting as a loading framework, are commonly referred to as scaffold proteins.

[0004] Currently reported commonly used scaffold proteins include four-transmembrane proteins (such as CD9, CD63, and CD81), single-transmembrane proteins (such as LAMP2B), and lipid-anchored proteins. However, EV drug delivery systems developed based on these scaffold proteins still suffer from problems such as low loading efficiency and unstable expression, resulting in insufficient biological activity and limited therapeutic effects. In addition, some scaffold proteins have large molecular weights, often requiring truncation or modification to optimize expression. This not only increases the difficulty of construction but may also affect host cell activity and EV generation and function. Therefore, there is an urgent need to develop a novel scaffold protein with stable expression, no need for truncation or modification, and high loading capacity to overcome the technical limitations of existing scaffold proteins, maximize the therapeutic potential of EV drug delivery platforms, and promote their clinical translation. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of ARF6 as a scaffold protein in the preparation of engineered extracellular vesicles, wherein ARF6 as a scaffold protein can efficiently mediate the enrichment of target proteins in extracellular vesicles without truncation or additional modification, effectively overcoming the problems of low drug loading efficiency and complex modification of traditional scaffold proteins.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of ARF6 as a scaffold protein in the preparation of engineered extracellular vesicles, wherein the amino acid sequence of ARF6 is shown in SEQ ID NO.1.

[0007] The present invention also provides a fusion protein composed of an ARF6 scaffold protein and a target protein, wherein the amino acid sequence of the ARF6 is shown in SEQ ID NO.1.

[0008] Preferably, the target protein includes a reporter protein, a therapeutic protein, a diagnostic protein, or a functional fragment of the aforementioned proteins; the reporter protein includes green fluorescent protein EGFP; and the therapeutic protein includes IL1R2, TNFR1, and / or IL6ST.

[0009] The present invention also provides an engineered extracellular vesicle comprising the above-described fusion protein.

[0010] Preferably, the extracellular vesicles include one or more of exosomes, microvesicles, and apoptotic bodies.

[0011] The present invention also provides an engineered cell for producing the above-described engineered extracellular vesicles.

[0012] Preferably, the engineered cells include mammalian cells, insect cells, bacterial or yeast cells; the mammalian cells include human embryonic kidney HEK293 cells, and the human embryonic kidney HEK293 cells include HEK293F cells.

[0013] The present invention also provides a method for preparing the above-mentioned engineered extracellular vesicles, comprising the following steps: separating and purifying the culture supernatant of the above-mentioned engineered cells to obtain engineered extracellular vesicles; the method for preparing the engineered cells comprises the following steps: constructing an expression vector encoding the above-mentioned fusion protein, transfecting the expression vector into host cells and culturing them to obtain engineered cells.

[0014] Preferably, the separation and purification employs differential ultracentrifugation combined with size exclusion chromatography, comprising the following steps: centrifugation at 300g for 5 minutes, centrifugation of the supernatant at 2000g for 20 minutes, centrifugation of the supernatant at 15000g for 30 minutes, and then ultracentrifugation of the supernatant at 100000g for 2 hours to obtain extracellular vesicle precipitate; after resuspending the extracellular vesicle precipitate, purification is performed using size exclusion chromatography, and the enriched extracellular vesicle components are collected to obtain engineered extracellular vesicles.

[0015] The present invention also provides the use of any one of the following in the preparation of a drug for treating macrophage inflammation and / or sepsis: (1) the above-mentioned fusion protein; (2) the above-mentioned engineered extracellular vesicles; (3) the above-mentioned engineered cells.

[0016] The present invention also provides the application of any of the following in the preparation of a drug delivery system: (1) the above-mentioned fusion protein; (2) the above-mentioned engineered extracellular vesicles; (3) the above-mentioned engineered cells.

[0017] The present invention also provides a pharmaceutical composition comprising any one of the following and a pharmaceutically acceptable carrier or excipient: (1) the above-described fusion protein; (2) the above-described engineered extracellular vesicles; (3) the above-described engineered cells.

[0018] The beneficial effects of this invention are: This invention is the first to propose that ARF6 can be used as a scaffold protein for the preparation of engineered extracellular vesicles. Using ARF6 as a scaffold protein to prepare engineered extracellular vesicles offers the following advantages: low engineering difficulty, high loading efficiency, which helps reduce the dosage of extracellular vesicles while ensuring efficacy; no need for truncation or modification, resulting in high biosafety; good versatility, without affecting the biological activity of the target protein; and the short sequence characteristics of ARF6 allow it to load proteins, peptides, and other bioactive molecules with larger molecular weights or more complex structures, facilitating the flexible construction of diverse drug delivery platforms for different diseases and possessing broad clinical translational potential; and it is suitable for large-scale production.

[0019] In summary, this invention uses natural full-length ARF6 as a scaffold protein to prepare engineered extracellular vesicles, which can fully leverage its advantages of short gene sequence, high editing and synthesis efficiency, strong loading capacity and good versatility. It effectively overcomes the technical bottlenecks of existing extracellular vesicle drug delivery systems, such as low loading efficiency and complex construction process, and successfully constructs a highly efficient, universal and safe protein and peptide drug delivery platform with important clinical application value. Attached Figure Description

[0020] Figure 1Figure 1 shows the screening and expression validation results of candidate scaffold proteins. A is the flowchart of candidate scaffold protein screening; B is the Venn diagram of the co-expression of the top 300 high-abundance proteins in HEK293F-EV samples; C is the heatmap of the abundance distribution of 19 candidate scaffold proteins in each sample; D is the membrane protein type composition diagram of candidate scaffold proteins; and E is the expression results of EV marker proteins and candidate scaffold proteins detected by Western blot.

[0021] Figure 2 This is a diagram of the GV712 carrier skeleton.

[0022] Figure 3 The image shows the fluorescence expression results of different EGFP candidate scaffold proteins in HEK293T cells.

[0023] Figure 4 Figure 1 shows the expression and loading efficiency analysis results of different EGFP candidate scaffold proteins in HEK293F cells and their derived EVs. A shows the EGFP expression in cells detected by fluorescence microscopy; B shows the statistical results of the mean fluorescence intensity (MFI) of EGFP in cells; C shows the EGFP positivity rate in cells detected by flow cytometry; D shows a representative graph of the EGFP positivity rate in each engineered EV detected by nanoflow cytometry; E shows the statistical results of the EGFP positivity rate in each engineered EV; F shows the statistical results of the mean fluorescence intensity (MFI) of EGFP in each engineered EV; G shows the expression of EGFP fusion proteins in cells and EVs detected by Western blot. This means p < 0.001.

[0024] Figure 5 Figure 1 shows the results of ARF6-engineered EV-mediated loading and localization validation of different functional protein cargoes. A shows the particle size distribution of the control-EV and each ARF6-engineered EV as detected by NTA; B shows the morphology of the control-EV and each ARF6-engineered EV as observed by transmission electron microscopy (scale bar: 100 nm); C shows the mRNA expression level of the target POI in cells transfected by each fusion plasmid as detected by qRT-PCR; D shows the protein expression level of the target POI in cells transfected by each fusion plasmid and its derived EV as detected by Western blot; E shows the localization of the IL6ST fusion protein on the membrane surface of IL6ST-ARF6-EV (IL6ST-EV) as observed by exosome immunogold electron microscopy. This means p < 0.001.

[0025] Figure 6This diagram illustrates the anti-inflammatory effect of engineered IL6ST-EV in an LPS-induced RAW 264.7 macrophage inflammation model. A represents the mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α, and CCL2 in cells; B represents the protein secretion levels of IL-6 and TNF-α in the cell culture supernatant. This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.001.

[0026] Figure 7 This diagram illustrates the therapeutic effect of engineered IL6ST-EV in a mouse model of sepsis, where A is the mouse survival curve; B is the serum biochemical index detection results; and C is a heatmap of serum inflammatory factors and chemokine expression levels. This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.001. Detailed Implementation

[0027] This invention provides the application of ARF6 as a scaffold protein in the preparation of engineered extracellular vesicles, wherein the amino acid sequence of ARF6 is shown in SEQ ID NO.1.

[0028] In this invention, ARF6 can efficiently mediate the enrichment of target proteins in EVs without truncation or modification, effectively overcoming the technical difficulties of low drug loading efficiency or the need for truncation modification of traditional scaffold proteins. In this invention, the nucleotide sequence encoding ARF6 is shown in SEQ ID NO.2. This invention uses full-length ARF6 as the scaffold protein, which is only 175 amino acids (aa), significantly shorter than commonly used existing scaffold proteins (such as PTGFRN, which is 879 aa). The shorter coding sequence used in this invention is more conducive to efficient gene editing and stable transfection, reduces the difficulty of cell engineering, and improves the efficiency of protein synthesis and stable expression, thereby significantly increasing the loading level of the target protein in EVs and helping to reduce the dosage of EVs while ensuring efficacy. In this invention, the naturally short ARF6 sequence enables the efficient enrichment of target proteins in EVs without any truncation or modification, effectively simplifying vector construction and engineering processes and reducing technical difficulty; at the same time, it avoids the potential adverse effects of scaffold protein modification on cell activity, EV generation, and function, improving the biosafety of engineered EVs. Furthermore, ARF6 can efficiently mediate the loading of functional proteins of different molecular weights (such as IL1R2, TNFR1, and IL6ST) into EVs, demonstrating excellent platform versatility. The fusion expression of ARF6 with POIs does not affect the biological activity of the POIs; for example, engineered IL6ST-ARF6-EVs loaded with IL6ST exhibited significant anti-inflammatory efficacy in both cellular and animal inflammation models. In addition, the short sequence of ARF6 allows it to load proteins, peptides, and other bioactive molecules with larger molecular weights or more complex structures, facilitating the flexible construction of diverse drug delivery platforms for different diseases and possessing broad clinical translational potential.

[0029] The present invention also provides a fusion protein composed of an ARF6 scaffold protein and a target protein, wherein the amino acid sequence of the ARF6 is shown in SEQ ID NO.1.

[0030] In the fusion protein of the present invention, the target protein and the ARF6 scaffold protein can be directly fused end-to-end, or the target protein can be linked to the N-terminus or C-terminus of ARF6 via a linker. In the present invention, the linker preferably includes a flexible peptide, and the nucleotide sequence of the flexible peptide preferably includes GGAGGTGGAGGATCA (SEQ ID NO.3) or GGGGGCGGGTCC (SEQ ID NO.4).

[0031] In this invention, the target protein preferably includes a reporter protein, a therapeutic protein, a diagnostic protein, or a functional fragment of the aforementioned proteins; the reporter protein preferably includes green fluorescent protein (EGFP), the amino acid sequence of which is shown in SEQ ID NO. 5; the therapeutic protein preferably includes IL1R2, TNFR1, and / or IL6ST, the amino acid sequence of which is shown in SEQ ID NO. 6, the amino acid sequence of which is shown in SEQ ID NO. 7, and the amino acid sequence of which is shown in SEQ ID NO. 8.

[0032] This invention also provides an engineered extracellular vesicle comprising the aforementioned fusion protein. In this invention, the extracellular vesicle preferably comprises one or more of exosomes, microvesicles, and apoptotic bodies. This invention demonstrates that ARF6, as a scaffold protein, can stably and efficiently mediate the loading of functional proteins of different molecular weights (such as IL1R2, TNFR1, and IL6ST) into extracellular vesicles, indicating that ARF6-mediated cargo loading is robust and universal, rather than specific protein-dependent, thereby enabling the construction of engineered extracellular vesicles for different therapeutic purposes. In some specific embodiments, the engineered extracellular vesicle is IL6ST-ARF6-EV loaded with IL6ST. Experimental results show that IL6ST-ARF6-EV can correctly anchor IL6ST to the outer surface of the EV membrane and effectively maintain its biological activity. As in vitro validation, in an LPS-induced RAW 264.7 macrophage inflammation model, IL6ST-ARF6-EV can significantly reduce the expression of inflammatory factors, exhibiting good anti-inflammatory activity. In vivo validation, in a mouse model of sepsis, the IL6ST-ARF6-EV significantly improved mouse survival, mitigated liver and kidney function damage, and effectively inhibited systemic inflammatory responses, demonstrating significant anti-inflammatory activity and therapeutic potential. Therefore, engineered EVs based on the ARF6 scaffold protein have significant clinical application value and industrialization prospects in drug delivery and disease treatment.

[0033] This invention also provides an engineered cell for producing the aforementioned engineered extracellular vesicles. In this invention, the engineered cells preferably include mammalian cells, insect cells, bacterial cells, or yeast cells; the mammalian cells preferably include human embryonic kidney HEK293 cells, and the human embryonic kidney HEK293 cells preferably include HEK293F cells. HEK293F cells can be cultured in high-density suspension, offering advantages such as simple culture, high transfection efficiency, high EV yield, suitability for serum-free chemically limited culture media (avoiding animal-derived component contamination), and ease of engineering modification, making them highly suitable for the large-scale production of engineered EV drug delivery systems. This invention does not specifically limit the source of each cell; conventional commercially available products in the art can be used.

[0034] The present invention also provides a method for preparing the above-mentioned engineered extracellular vesicles, comprising the following steps: separating and purifying the culture supernatant of the above-mentioned engineered cells to obtain engineered extracellular vesicles; the method for preparing the engineered cells comprises the following steps: constructing an expression vector encoding the above-mentioned fusion protein, transfecting the expression vector into host cells and culturing them to obtain engineered cells.

[0035] In this invention, the expression vector preferably includes a eukaryotic expression plasmid vector or a viral plasmid vector. The eukaryotic expression plasmid vector preferably includes a CMV promoter, a multiple cloning site, and an resistance gene initiated by the SV40 promoter; more preferably, it is a GV712 vector with the element composition CMV enhancer-MCS-SV40-puromycin. In this invention, when the fusion protein is composed of EGFP and ARF6, the nucleotide sequence encoding the fusion protein EGFP-ARF6 is shown in SEQ ID NO. 9; when the fusion protein is composed of IL1R2 and ARF6, the nucleotide sequence encoding the fusion protein IL1R2-ARF6 is shown in SEQ ID NO. 10; when the fusion protein is composed of TNFR1 and ARF6, the nucleotide sequence encoding the fusion protein TNFR1-ARF6 is shown in SEQ ID NO. 11; and when the fusion protein is composed of IL6ST and ARF6, the nucleotide sequence encoding the fusion protein IL6ST-ARF6 is shown in SEQ ID NO. 12. When constructing the expression vector encoding the above fusion protein, the nucleotide sequence encoding the above fusion protein is inserted into the multiple cloning site XbaI / XbaI.

[0036] This invention does not specifically limit the transfection method for transfecting the expression vector into host cells; conventional transfection methods in the art can be used. In this invention, it is preferred to separate and purify the cell culture supernatant 72 hours after transfection. In this invention, the separation and purification preferably employs differential ultracentrifugation combined with size exclusion chromatography, including the following steps: centrifugation at 300g for 5 minutes, centrifugation of the supernatant at 2000g for 20 minutes, centrifugation of the supernatant at 15000g for 30 minutes, and then ultracentrifugation of the supernatant at 100000g for 2 hours to obtain extracellular vesicle precipitate; after resuspending the extracellular vesicle precipitate, purification is performed using size exclusion chromatography, and the enriched extracellular vesicle components are collected to obtain engineered extracellular vesicles. In this invention, the size exclusion chromatography purification is preferably performed using an exosome purification kit (ES933) purchased from Beijing Enzekangtai Biotechnology Co., Ltd.

[0037] The present invention also provides the use of any one of the following in the preparation of a disease treatment drug or in the preparation of a drug delivery system: (1) the above-mentioned fusion protein; (2) the above-mentioned engineered extracellular vesicles; (3) the above-mentioned engineered cells.

[0038] In this invention, the drug delivery system is particularly suitable for delivering therapeutic protein or polypeptide molecules; The diseases preferably include macrophage inflammation and / or sepsis.

[0039] The present invention also provides a pharmaceutical composition comprising any one of the following and a pharmaceutically acceptable carrier or excipient: (1) the above-described fusion protein; (2) the above-described engineered extracellular vesicles; and (3) the above-described engineered cells. In the present invention, the pharmaceutical composition can be prepared into various dosage forms, including but not limited to injections, lyophilized powders, inhalers, capsules, granules, tablets, oral preparations, gels, ointments, or patches, to suit different routes of administration.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] In the following examples, statistical data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. p < 0.05 was considered statistically significant. All experimental results were repeated at least three times.

[0044] Example 1 Screening and expression validation of candidate high-abundance scaffold proteins 1. Preparation of HEK293F-EV (HEK293F cell-derived extracellular vesicles) Human embryonic kidney Expi293F suspension cells (HEK293F, A14527CN, ThermoFisher) were cultured at a concentration of 0.3–0.5 × 10⁻⁶ cells / mL. 6 Cells / mL were inoculated into serum-free, chemically defined medium (prepared by adding 500 mL of SMM293-TⅡ (M293TⅡ, Beijing Yiqiao Shenzhou Technology Co., Ltd.), 500 mL of OPM-293 CD05Medium (81075-001, Shanghai Aopuma Biotechnology Co., Ltd.), and 20 mL of GlutaMAX). TM Mix the supplement (35050-061, Gibco) thoroughly to prepare a complete culture medium for HEK293F cells. Store at 4°C (preheat in a 37°C water bath for 30 min before use). Incubate at 37°C, 8% CO2, 80-90% humidity, and 100 rpm with shaking until the cell density reaches 3-5 × 10⁶ cells / year. 6 Cells / mL were collected, and the cell culture supernatant was extracted and purified by differential ultracentrifugation combined with size exclusion chromatography: first, the cell pellet was removed by centrifugation at 300g for 5 minutes; the supernatant was then centrifuged at 2000g for 20 minutes to remove cell debris; the supernatant was then centrifuged at 15000g for 30 minutes to remove large particulate impurities; finally, the supernatant was centrifuged at 100000g for 2 hours to obtain the EV pellet; the obtained pellet was resuspended in sterile PBS, and then further purified using an exosome purification kit based on size exclusion chromatography (ES933, Beijing Enzekangtai Biotechnology Co., Ltd.), and the EV enriched fraction (fractions 6-8) was collected.

[0045] 2. Proteomics analysis of HEK293F-EV Five different batches of HEK293F-EV samples (n=5) prepared in step 1 were subjected to proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The main procedure is as follows: total protein of EVs was extracted and quantified, followed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining for pre-mass spectrometry quality control; after passing quality control, the samples were enzymatically digested and then analyzed by LC-MS / MS. Based on the acquired raw mass spectrometry data, the Proteome Discoverer database search software was used for spectral matching and database retrieval according to the Uniprot protein database to complete protein identification and analysis, and finally obtain the proteomic information of HEK293F-EV.

[0046] 3. Screening of candidate scaffold proteins Based on the proteomics analysis results of the five HEK293F-EV samples in step 2, a total of 2633 proteins were identified. Figure 1 The screening process shown in A follows the following criteria for systematically screening candidate scaffold proteins: (1) High abundance: Select the top 300 proteins by abundance in each sample, and then screen out proteins that are stably co-expressed in ≥3 samples; the results are as follows. Figure 1 As shown in B, a total of 292 high-abundance proteins were stably co-expressed in ≥3 samples; (2) Membrane proteins: based on the subcellular localization annotation of the UniProtKB database and related literature reports, proteins with membrane localization were screened; (3) based on a systematic literature review, some membrane proteins that were too large in molecular weight, too complex in structural domain, might interfere with EV formation and release, or had potential biosafety risks were excluded as unsuitable as scaffold proteins. After the above screening, 19 membrane proteins were finally obtained as candidate scaffold proteins: MFGE8, PTGFRN, ATP1A1, PTPRK, ITGB1, CPD, SDCBP, BSG, SLC1A5, PLXNB2, IGSF8, ARF6, BASP1, CNTN1, NCSTN, MARCKSL1, CD47, ARRDC1, and MARCKS, for subsequent verification. The omics detection abundance distribution of the 19 candidate scaffold proteins in each sample is shown in the figure below. Figure 1 As shown in C, the membrane protein type composition of the 19 candidate scaffold proteins is as follows: Figure 1 As shown in D, it mainly includes multiple transmembrane proteins, single transmembrane proteins, lipid anchor proteins, and peripheral membrane proteins.

[0047] 4. Validation of candidate scaffold protein expression Following the method described in step 1, three different batches of HEK293F-EV samples (293F-EV-1, 293F-EV-2, and 293F-EV-3) were prepared again to verify the expression of the aforementioned 19 candidate scaffold proteins in HEK293F-EV. First, the marker proteins of EVs were detected by Western blot. The results are as follows... Figure 1 As shown in E, the positive marker proteins ALIX, TSG101, CD9 and CD81 were highly expressed in the prepared EV samples, while the negative marker proteins Calnexin (endoplasmic reticulum marker) and GM130 (Golgi marker) were not expressed, indicating that the obtained EV samples had high purity and met the requirements for subsequent experiments.

[0048] Subsequently, Western blot was used to verify the expression of the candidate scaffold protein in EVs. The results are as follows: Figure 1As shown in E, except for PTPRK, IGSF8, and BASP1, which may not have been detected due to insufficient antibody sensitivity (results not shown), the remaining 16 candidate scaffold proteins all showed clear specific bands, indicating that these proteins are stably expressed in HEK293F-EV, thus further verifying the reliability of the proteomics identification results.

[0049] Example 2 Construction of expression carrier To further evaluate the ability of the 19 candidate scaffold proteins selected in Example 1 to load the target protein (POI) into the EV, this invention uses enhanced green fluorescent protein (EGFP) as an exemplary POI to construct expression vectors for 19 EGFP-candidate scaffold protein fusion proteins.

[0050] Preparation of EGFP-candidate scaffold protein fusion protein: The nucleotide sequence encoding EGFP and the nucleotide sequence encoding the candidate scaffold protein were linked by a flexible peptide (SEQ ID NO.3: GGAGGTGGAGGATCA) to obtain the nucleotide sequences encoding each fusion protein, wherein the nucleotide sequence encoding the ARF6 protein is shown in SEQ ID NO.2 (the nucleotide sequence encoding the EGFP-ARF6 fusion protein is shown in SEQ ID NO.9).

[0051] The nucleotide sequences encoding each fusion protein were synthesized by Shanghai Jikai Gene Co., Ltd., using the GV712 vector backbone. The GV712 vector backbone consists of CMV enhancer-MCS-SV40-puromycin (the GV712 vector backbone was purchased from Shanghai Jikai Gene Co., Ltd., and the diagram is shown below). Figure 2 As shown in the figure, the coding sequences of EGFP-candidate scaffold protein fusion proteins were inserted into the multiple cloning sites (XbaI / XbaI) to obtain 19 recombinant plasmids of EGFP-candidate scaffold protein fusion proteins (the above process was carried out by Shanghai Jikai Gene Co., Ltd., and the order number of the recombinant plasmid for EGFP-ARF6 fusion protein is GOSE0386887), which were used for subsequent cell transfection and screening verification.

[0052] Example 3 First-round screening and validation of candidate scaffold proteins in HEK293T cells 1. HEK293T cell transfection The recombinant plasmids of 19 EGFP-candidate scaffold protein fusion proteins constructed in Example 2 were transiently transfected into HEK293T cells, with HEK293T cells transfected with empty plasmids serving as a control group. The specific procedures were as follows: Cells were seeded in 12-well culture plates, and when the confluence reached 70-80%, transfection was performed according to the Lipofectamine 3000 (Invitrogen, USA) reagent instructions. For a 12-well plate, the transfection system for each well was prepared as follows: Solution A was prepared by dissolving 1 μg of the recombinant plasmid of the fusion protein in 50 μL of Opti-MEM medium (Gibco, USA), and Solution B was prepared by dissolving 2.25 μL of Lipofectamine 3000 in 50 μL of Opti-MEM medium. Solution A and Solution B were then mixed thoroughly and allowed to stand at room temperature for 10-15 minutes before adding the cells. After transfection, the cells were cultured at 37°C in a 5% CO2 incubator to achieve fusion protein expression.

[0053] 2. Detection of EGFP expression using fluorescence microscopy Seventy-two hours after transfection, the EGFP fluorescence signal in the cells was observed using a fluorescence microscope. The results are as follows: Figure 3 As shown, ARF6, MARCKS, MARCKSL1, BASP1, SDCBP, and ARRDC1, when expressed as scaffold proteins fused with EGFP, produced significantly higher green fluorescence signals in cells than other EGFP-scaffold protein fusion constructs.

[0054] The results of this embodiment indicate that ARF6, MARCKS, MARCKSL1, BASP1, SDCBP, and ARRDC1 can mediate higher GFP expression in HEK293T cells, suggesting that these scaffold proteins may have stronger EV sorting potential. Given that this round of screening required simultaneous validation of 19 candidate scaffold proteins, and the supernatant obtained from the 12-well plate culture system was insufficient for EV extraction, only initial screening at the cellular level was performed, and EV-level validation was not conducted. Based on the initial screening results, and considering that MARCKS and MARCKSL1 belong to the same protein family and have similar functions, only MARCKS was retained as a representative in the second round of screening. Therefore, ARF6, MARCKS, BASP1, SDCBP, and ARRDC1 were ultimately selected for the second round of screening using a HEK293F suspension cell system to collect sufficient supernatant and further evaluate the efficiency of each scaffold protein in mediating POI loading into EVs.

[0055] Example 4 The second round of screening and validation of the five candidate high-abundance scaffold proteins obtained in Example 3 in HEK293F cells 1. HEK293F cell transfection To further screen for scaffold proteins with optimal EV loading efficiency, recombinant plasmids of the five EGFP-candidate scaffold protein fusion proteins screened in Example 3 (EGFP-ARF6, EGFP-MARCKS, EGFP-BASP1, EGFP-SDCBP, and EGFP-ARRDC1) were transiently transfected into HEK293F cells with polyethyleneimine (PEI-40K, Polysciences) (HEK293F cells transfected with empty plasmids served as a control group) to prepare engineered EVs. The steps are as follows: (1) Cell preparation: Detect cell density and viability 1 hour before transfection and adjust to 3×10⁻⁶ cells / year. 6 Cells / mL with a viability ≥95% were inoculated into 500mL conical shake flasks (783011, NEST) with a culture medium volume of 110mL and cultured in an incubator at 37℃, 8% CO2, 80~90% humidity, and 100rpm for later use.

[0056] (2) Preparation of plasmid / PEI-40K complex: Calculate the mixed liquid volume as 5% of the total culture system volume (e.g., 5.5 mL for a 110 mL culture system, including 2.75 mL each of plasmid diluent and PEI-40K diluent). The plasmid diluent is prepared at 1 μg / 10... 6 Cells were prepared in Opti-MEM medium and incubated at room temperature for 5 minutes. PEI-40K dilution was prepared in Opti-MEM medium at a DNA:PEI mass ratio of 1:2 and incubated at room temperature for 5 minutes. PEI-40K dilution was added to plasmid dilution, gently mixed, and incubated at room temperature for 10-15 minutes to form a plasmid / PEI-40K complex.

[0057] (3) Cell transfection: The plasmid / PEI-40K complex was slowly added dropwise to the cell culture medium in step (1), and the mixture was gently shaken while adding. Then the cell was placed back into the shaking incubator for further culture to achieve the expression of the fusion protein and the secretion of the engineered EV.

[0058] 2. Detection of GFP expression level (1) Fluorescence microscopy detection: 72 hours after transfection, the EGFP fluorescence signal of cells was observed using a fluorescence microscope, and the mean fluorescence intensity (MFI) was quantitatively analyzed using ImageJ software. The results are as follows: Figure 4 As shown in A and B, after ARF6 was expressed as a scaffold protein fused with EGFP (EGFP-ARF6), the green fluorescence signal produced in the cells was significantly higher than that of EGFP-MARCKS, EGFP-SDCBP, EGFP-ARRDC1 and EGFP-BASP1, and the MFI was the highest.

[0059] (2) Flow cytometry analysis: 72 hours after transfection, cells and cell culture supernatant were collected (at this time, the expression of cell fusion protein reached its peak, and EVs accumulated sufficiently in the culture medium). The proportion of EGFP-positive cells was detected by flow cytometry. The results are as follows: Figure 4 As shown in C, consistent with the results of step (1), the EGFP positivity rate of cells transfected with EGFP-ARF6 is significantly higher than that of other fusion plasmids.

[0060] (3) EV Nanoflow Cytometry Detection: The cell culture supernatant collected in step (2) was purified by differential ultracentrifugation combined with size exclusion chromatography (method as in step 1 of Example 1). Then, the EGFP positivity rate in EV was determined using a nanoflow cytometer (NanoFCM, Fuliu Biotechnology), and MFI was quantitatively analyzed using FlowJo software. The results are as follows: Figure 4 As shown in D~F, EVs transfected with EGFP-ARF6 exhibited the highest EGFP positivity rate (up to 79.6%) and MFI, significantly better than EGFP-MARCKS, EGFP-SDCBP, EGFP-ARRDC1, and EGFP-BASP1, indicating that ARF6 is the most efficient in promoting EGFP loading into EVs.

[0061] (4) Western blot analysis: Cells transfected for 72 hours and their derived EVs were collected, and the expression level of the EGFP fusion protein was detected. Results are as follows: Figure 4 As shown in G, the expression level of EGFP fusion protein was highest in cells transfected with EGFP-ARF6 and in EVs from which it was derived, and the expression level in EVs was higher than that in cells, further verifying the advantage of ARF6 in mediating POI loading into EVs.

[0062] The results of this embodiment demonstrate that ARF6 is significantly more efficient than MARCKS, SDCBP, ARRDC1, and BASP1 in mediating EGFP loading into EVs. Therefore, ARF6 has been identified as the scaffold protein with the optimal EV loading efficiency and can be used for subsequent engineering EV construction and application research.

[0063] Example 5 ARF6 engineered EV-mediated loading and positioning detection of different functional protein cargoes To systematically evaluate the universality of ARF6-mediated loading of different points of interest (POIs) into endothelial cells (EVs), this embodiment selected three representative target proteins of different sizes: IL1R2 (approximately 45 kDa, amino acid sequence as shown in SEQ ID NO. 6), TNFR1 (approximately 50 kDa, amino acid sequence as shown in SEQ ID NO. 7), and IL6ST (approximately 103 kDa, amino acid sequence as shown in SEQ ID NO. 8) as POIs for validation. All three proteins are single-transmembrane proteins. After fusing with ARF6, they are loaded onto the outer surface of the EV membrane and act as decoy protein receptors, specifically binding to and neutralizing their corresponding inflammatory ligands (IL-1β, TNF-α, and the IL-6 / sIL-6R complex), thereby blocking downstream pro-inflammatory signaling pathways and providing a novel anti-inflammatory strategy based on engineered EVs for the treatment of inflammation-related diseases.

[0064] 1. Construction of expression vector Expression vectors for the fusion proteins IL1R2-ARF6, TNFR1-ARF6, and IL6ST-ARF6 were constructed, respectively, using the same method as in Example 2, except that the nucleotide sequence encoding EGFP was replaced with the nucleotide sequences encoding IL1R2, TNFR1, and IL6ST, respectively. The sequence of the flexible peptide is GGGGGCGGGTCC (SEQ ID NO. 4), where the nucleotide sequence encoding the fusion protein IL1R2-ARF6 is shown in SEQ ID NO. 10, the nucleotide sequence encoding the fusion protein TNFR1-ARF6 is shown in SEQ ID NO. 11, and the nucleotide sequence encoding the fusion protein IL6ST-ARF6 is shown in SEQ ID NO. 12.

[0065] 2. Fabrication of ARF6 engineered EVs Following the procedure described in step 1 of Example 4, the recombinant plasmids of the above-mentioned fusion proteins were transiently transfected into HEK293F cells. Cells and culture supernatants were collected 72 hours after transfection. The culture supernatants were purified by differential ultracentrifugation combined with size exclusion chromatography (the method is the same as step 1 of Example 1) to obtain engineered IL1R2-ARF6-EV (hereinafter referred to as IL1R2-EV), TNFR1-ARF6-EV (hereinafter referred to as TNFR1-EV), and IL6ST-ARF6-EV (hereinafter referred to as IL6ST-EV). EVs transfected with empty plasmids were used as the control group (control-EV).

[0066] 3. Characterization of the ARF6 engineered EV (1) Particle size detection: The particle size distribution of EVs was detected using a ZetaView PMX 110 nanoparticle tracking analyzer (Particle Metrix, Germany) (NTA). The results are as follows: Figure 5As shown in Figure A, the median particle sizes of engineered IL1R2-EV, TNFR1-EV, and IL6ST-EV were 149.2 nm, 141.5 nm, and 142.1 nm, respectively. These were not significantly different from the control EV transfected with an empty plasmid (control-EV, median particle size 146.8 nm), indicating that the engineering modification based on the ARF6 scaffold protein and the loading of POIs with different molecular weights had no significant effect on the particle size distribution of EVs.

[0067] (2) Morphological examination: The morphology of EVs was observed using a transmission electron microscope (TEM). 10 μL of EV sample was added to a copper mesh and incubated at room temperature for 10 minutes. After removing the remaining liquid with filter paper, 10 μL of 3% uranium acetate was added and incubated for 2 minutes. After drying, images were taken using a TEM (JEM-1200EX, JEOL). The results are as follows: Figure 5 As shown in B, the control-EV and all engineered EVs exhibit typical circular vesicle structures, consistent with the morphological characteristics of EVs.

[0068] 4. Detection of ARF6 engineered EV fusion protein expression levels (1) mRNA expression level detection: Cells were collected 72 hours after transfection, and the relative mRNA expression levels of IL1R2, TNFR1, and IL6ST were detected by qRT-PCR. The results are as follows: Figure 5 As shown in C, compared with the control group (cell-control) transfected with empty plasmids, the mRNA expression level of the corresponding POI in the cells transfected with each fusion protein recombinant plasmid was significantly increased, indicating that the fusion protein recombinant plasmids were successfully transfected and expressed efficiently.

[0069] (2) Protein expression level detection: 72 hours after transfection, cells and their derived EVs were collected, and the expression levels of IL1R2, TNFR1, and IL6ST fusion proteins were detected by Western blot. Results are as follows: Figure 5 As shown in D, the protein expression levels of the corresponding POIs in cells transfected with each fusion plasmid and in their EVs were significantly higher than those in the control group, and the enrichment degree in EVs was higher. The molecular weight of the fusion protein bands was consistent with expectations, proving that ARF6 can efficiently mediate the loading of POIs of different molecular weights into EVs.

[0070] 5. Localization detection of fusion proteins in ARF6 engineered EVs To further verify the correct localization of ARF6-mediated POI loading into EVs, IL6ST, the largest molecular weight protein with a transmembrane structure, was used as a representative. Exosomal immunogold electron microscopy was employed to detect the localization of the IL6ST fusion protein in IL6ST-ARF6-EV (IL6ST-EV). The results are as follows: Figure 5As shown in E, no obvious gold particle signal was observed on the outer surface of the control-EV membrane, while a large number of gold particles were observed on the outer surface of the IL6ST-EV membrane. This indicates that after IL6ST is fused with ARF6, it can be correctly located on the outer surface of the EV membrane, and the fusion with ARF6 does not affect the inherent transmembrane properties of IL6ST.

[0071] The results of this embodiment demonstrate that ARF6, as a scaffold protein, can stably and efficiently load functional proteins of different molecular weights (such as IL1R2, TNFR1, and IL6ST) into EVs without affecting the correct localization of the target protein on the EV. This verifies the universality and robustness of ARF6-mediated protein cargo loading and provides a reliable technical basis for the EV delivery of various types of functional proteins.

[0072] Example 6 Anti-inflammatory effects of IL6ST-EV in an LPS-induced RAW 264.7 macrophage inflammation model Based on the exosome immunogold electron microscopy results from step 5 of Example 5, the IL6ST fusion protein has been confirmed to be correctly located on the outer surface of the IL6ST-ARF6-EV (IL6ST-EV) membrane. To further verify the functional integrity and biological activity of IL6ST-EV, this example constructs an LPS-induced RAW 264.7 macrophage inflammation model and evaluates its in vitro anti-inflammatory effect.

[0073] 1. Experimental grouping and treatment RAW 264.7 macrophages were seeded in 12-well plates. After the cells reached 80% confluence, either control-EV (prepared in Example 5) or IL6ST-EV (prepared in Example 5) was added (both at a dose of 1×10⁻⁶). 9 Cells were tested per well, and LPS (final concentration 1 μg / mL) was added to stimulate the cells for 24 hours. Cells and supernatant were then collected for subsequent detection. The group without LPS and EV was used as the normal control group (control), and the group with LPS and an equal volume of PBS (EV solvent) was used as the positive control group (LPS+PBS).

[0074] 2. Evaluation of in vitro anti-inflammatory activity (1) mRNA level detection: The mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α, and CCL2 in cells were detected by qRT-PCR. The results are as follows: Figure 6 As shown in A, the mRNA expression levels of each inflammatory factor in the LPS+IL6ST-EV group were significantly lower than those in the LPS+PBS group and the LPS+control-EV group.

[0075] (2) Protein level detection: The secretion levels of IL-6 and TNF-α proteins in the cell supernatant were detected by ELISA. The results are as follows: Figure 6 As shown in B, the protein secretion levels of IL-6 and TNF-α in the supernatant of the LPS+IL6ST-EV group were significantly lower than those in the LPS+PBS group and the LPS+control-EV group.

[0076] The results of this embodiment show that IL6ST-EV can significantly reduce the transcription and secretion levels of inflammatory factors in an LPS-induced RAW 264.7 macrophage inflammation model, exhibiting significant in vitro anti-inflammatory activity.

[0077] Example 7 Therapeutic effects of IL6ST-EV in a mouse model of sepsis In this embodiment, a mouse model of sepsis was established using the cecum ligation and puncture (CLP) method to further evaluate the in vivo therapeutic potential of engineered IL6ST-ARF6-EV (IL6ST-EV).

[0078] 1. Animal model establishment and grouping Eight-week-old male C57BL / 6J mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were randomly divided into four groups: sham-operated group, CLP+PBS group, CLP+control-EV group, and CLP+IL6ST-EV group. The CLP sepsis model was established as follows: Mice were anesthetized with tribromoethanol and fixed in a supine position. Abdominal hair was shaved and disinfected. A 1.5-2 cm incision was made along the midline of the abdomen to access the abdominal cavity and expose the cecum (usually located in the left lower quadrant). The cecum was ligated approximately halfway between the distal end of the cecum and the ileocecal valve. An 18-gauge needle was used to pierce the ligation point, and a small amount of feces was gently squeezed out before repositioning the cecum. The abdomen was then sutured closed layer by layer. Immediately after surgery, physiological saline (50 mL / kg) was injected subcutaneously to combat shock. In the sham-operated group, only the cecum was exposed without ligation or puncture; the remaining procedures were the same. Mice in each group were administered the corresponding treatments via tail vein immediately after surgery, at 24 hours, and at 48 hours: the CLP+control-EV group was injected with control-EV (prepared in Example 5), and the CLP+IL6ST-EV group was injected with IL6ST-EV (prepared in Example 5), at a dose of 1.0 × 10⁻⁶. 10 Particles per mouse per dose (dissolved in 500 μL PBS), CLP+PBS group injected with an equal volume of PBS. All mice were sacrificed 72 hours post-surgery and samples were collected.

[0079] 2. Evaluation of in vivo therapeutic effect (1) Survival analysis: The survival rate of mice within 72 hours after surgery was recorded and survival curves were plotted. The results are as follows: Figure 7As shown in A, the survival rate of the CLP+IL6ST-EV group was 86.67%, which was significantly higher than that of the CLP+PBS group (53.33%) and the CLP+control-EV group (53.33%), indicating that IL6ST-EV can significantly improve the survival outcome of septic mice.

[0080] (2) Serum biochemical marker detection: Serum renal function markers (Scr, BUN), liver function markers (ALT, AST), lactate dehydrogenase (LDH), and C-reactive protein (CRP) levels were detected using appropriate commercial kits. Results are as follows: Figure 7 As shown in B, the above indicators in the CLP+IL6ST-EV group were significantly lower than those in the CLP+PBS group and the CLP+control-EV group, suggesting that IL6ST-EV can effectively reduce multi-organ damage and systemic inflammatory response caused by sepsis.

[0081] (3) Detection of serum inflammatory factors and chemokines: The expression levels of serum inflammatory factors and chemokines were measured using the Luminex multifactor detection platform. Results are as follows: Figure 7 As shown in Figure C, compared with the CLP+PBS group and the CLP+control-EV group, the levels of multiple inflammatory factors and chemokines in the CLP+IL6ST-EV group were significantly reduced, further confirming its systemic anti-inflammatory effect.

[0082] The results of this embodiment show that engineered IL6ST-EV can significantly improve the survival rate of septic mice, improve liver and kidney function damage, and effectively inhibit systemic inflammatory response, demonstrating good in vivo therapeutic potential.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of ARF6 as a scaffold protein in the preparation of engineered extracellular vesicles, characterized in that, The amino acid sequence of ARF6 is shown in SEQ ID NO.

1.

2. An engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles include an IL6ST-ARF6 fusion protein, wherein the IL6ST-ARF6 fusion protein is formed by linking IL6ST to the scaffold protein ARF6 using a flexible peptide. The amino acid sequence of the IL6ST is shown in SEQ ID NO. 8, the amino acid sequence of the scaffold protein ARF6 is shown in SEQ ID NO. 1, and the nucleotide sequence of the flexible peptide is shown in SEQ ID NO.

4. The nucleotide sequence encoding the IL6ST-ARF6 fusion protein is shown in SEQ ID NO.

12. The IL6ST is anchored to the outer membrane surface of the engineered extracellular vesicles.

3. The engineered extracellular vesicles according to claim 2, characterized in that, The extracellular vesicles are exosomes.

4. An engineered cell, characterized in that, Used for producing engineered extracellular vesicles as described in claim 2 or 3.

5. The engineered cell according to claim 4, characterized in that, The engineered cells include human embryonic kidney HEK293 cells, which include HEK293F cells.

6. The method for preparing engineered extracellular vesicles according to claim 2 or 3, characterized in that, The method includes the following steps: separating and purifying the culture supernatant of the engineered cells according to claim 4 or 5 to obtain engineered extracellular vesicles; the method for preparing the engineered cells includes the following steps: constructing an expression vector encoding the IL6ST-ARF6 fusion protein of claim 2, transfecting the expression vector into host cells and culturing them to obtain engineered cells.

7. The preparation method according to claim 6, characterized in that, The separation and purification method employs differential ultracentrifugation combined with size exclusion chromatography, including the following steps: centrifugation at 300g for 5 minutes, followed by centrifugation of the supernatant at 2000g for 20 minutes, then centrifugation of the supernatant at 15000g for 30 minutes, and finally ultracentrifugation of the supernatant at 100000g for 2 hours to obtain extracellular vesicle precipitate; after resuspending the extracellular vesicle precipitate, purification is performed using size exclusion chromatography, and the enriched extracellular vesicle components are collected to obtain engineered extracellular vesicles.

8. The use of the engineered extracellular vesicles according to claim 2 or 3 in the preparation of drugs for treating sepsis.

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