An engineered exosome targeting liver antagonizing pcsk9 and specifically binding ldl-c and construction method and application thereof

Abstract

The application provides an engineered exosome targeting liver, antagonizing PCSK9 and specifically combining LDL-C, and a construction method and application thereof, and belongs to the technical field of exosomes. The transmembrane protein PTGFRN is reformed, the PTGFRN sequence containing the L3-L7 segment of LDLR is expressed in the membrane outside, and then can specifically combine LDL-C. Meanwhile, the PCSK9 inhibiting drug Inclisiran is connected with the surface of the exosome. The engineered exosome has the dual effects of antagonizing PCSK9 and promoting LDL-C degradation, can efficiently target liver and inhibit the expression of PCSK9 of liver cells, can specifically combine LDL-C to the liver for degradation, provides a new way for reducing the blood lipid content, and has wide application value. Meanwhile, the engineered exosome provided by the application has a simple preparation method, can realize large-scale production of the engineered exosome, and provides a basis for wide application.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically relating to an engineered exosome that targets the liver to antagonize PCSK9 and specifically binds to LDL-C, its construction method, and its application. Background Technology

[0002] Elevated blood lipids are an important independent risk factor for cardiovascular disease, primarily manifested as elevated levels of low-density lipoprotein cholesterol (LDL-C) in plasma. Under normal conditions, plasma LDL-C first binds to the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes to form a complex, which then enters the cell. In the acidic environment of the endosome, the complex dissociates into LDLR and LDL-C. LDLR usually cycles back to the cell surface, while LDL-C is degraded in lysosomes, and its metabolites are reused in the cell. Therefore, LDLR expressed on the surface of hepatocytes is the primary factor determining LDL-C levels; the more LDLR that can bind, the lower the plasma LDL-C level. Studies have shown that the cell surface LDLR is a single-chain, single-transmembrane glycoprotein composed of 839 amino acids, consisting of five functional domains: a ligand-binding domain, an epidermal growth factor (EGF) precursor domain, an O-linked glycoside domain, a transmembrane domain, and a cytoplasmic domain. The ligand-binding domain is composed of a repeating sequence of seven amino acid residues, L1-L7, and is the main domain for LDLR to specifically bind to LDL-C.

[0003] Meanwhile, proprotein convertase subtilisin 9 (PCSK9) has become a target for novel lipid-lowering drugs. Studies have shown that PCSK9 can not only competitively bind to LDLR with LDL-C, leading to elevated LDL-C levels in the blood, but also "hijack" LDLR to enter lysosomes for degradation, disrupting the LDLR cycle. Therefore, PCSK9 is a key molecule leading to accelerated LDLR degradation and elevated LDL-C levels. Inhibiting PCSK9 plays an important role in restoring the LDLR cycle, enhancing LDL-C clearance, and maintaining lipid homeostasis. Inclisiran is a chemically synthesized PCSK9 small interfering RNA (siRNA) drug composed of GalNAc and PCSK9 siRNA. It lowers PCSK9 levels by targeting and inhibiting PCSK9 mRNA. GalNAc is a targeting ligand for the hepatocyte-specific desialyl glycoprotein receptor (ASGPR), exhibiting high affinity for hepatocytes and rapid internalization, thus ensuring that the PCSK9 siRNA drug specifically binds to membrane proteins before entering the cell. This siRNA drug can not only reduce circulating PCSK9 levels, but also effectively reduce intracellular PCSK9 levels. It has a strong sustained-release effect, low off-target rate, almost no adverse reactions, and can continuously reduce LDL-C levels.

[0004] Exosomes are vesicle-like nanostructures derived from the intracellular membrane system, with diameters ranging from 50 to 130 nm, and can be secreted and released by various cell types. Exosomes can carry a variety of molecules, such as proteins, RNA, and lipids, participating in physiological and pathological processes. Studies have shown that PTGFRN is a single-pass transmembrane protein abundantly accumulated on the surface of exosomes, with its N-terminus outside the membrane and its C-terminus inside, making it particularly suitable for constructing exosome-loaded fusion proteins through genetic engineering. As natural carriers of bioactive substances, exosomes possess advantages such as high stability, good permeability, low toxicity, and non-immunogenicity, showing promising application value and market prospects in drug loading and targeted delivery.

[0005] However, there are currently no reports of drugs that simultaneously possess the dual effects of antagonizing PCSK9 and promoting LDL-C degradation, hindering the treatment process of hypercholesterolemia or hyperlipidemia. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide an engineered exosome that targets the liver to antagonize PCSK9 and specifically binds to LDL-C, as well as its construction method and application, which has a good lipid-lowering effect and provides a new means for the treatment of hypercholesterolemia or hyperlipidemia.

[0007] This invention provides an engineered exosome that targets and antagonizes PCSK9 in the liver and specifically binds to LDL-C. The engineered exosome expresses the fusion protein LDLR L3-L7-PTGFRN extracellularly, and the surface of the engineered exosome is attached with Inclisiran. The amino acid sequence of LDLR L3-L7 in the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:1; the amino acid sequence of PTGFRN is shown in SEQ ID NO:2.

[0008] Preferably, the fusion protein LDLR L3-L7-PTGFRN further includes a linker peptide; The amino acid sequence of the linker peptide is shown in SEQ ID NO:3.

[0009] Preferably, the amino acid sequence of the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:4.

[0010] Preferably, the inclisiran is composed of PCSK9 siRNA and N-acetylgalactosamine; The inclisiran is cholesterol-modified inclisiran.

[0011] This invention provides a method for constructing the engineered exosomes, comprising the following steps: 1) Construct a recombinant vector expressing the fusion gene LDLR L3-L7-PTGFRN; 2) Transfect the recombinant vector described in step 1) into mammalian cells to obtain recombinant cells; 3) Culture the recombinant cells described in step 2) and collect exosomes; 4) Co-incubate the exosomes described in step 3) with Inclisiran to obtain engineered exosomes.

[0012] Preferably, the method for expressing the recombinant vector of the fusion gene LDLR L3-L7-PTGFRN in step 1) includes the following steps: The fusion gene LDLR L3-L7-PTGFRN sequence with restriction enzyme sites at both ends was digested with enzymes, and the resulting digested fragment was cloned into a plasmid digested with the same type of restriction endonuclease to obtain a recombinant vector.

[0013] Preferably, the nucleotide sequence of the fusion gene LDLR L3-L7-PTGFRN is shown in SEQ ID NO:5; The restriction endonuclease includes Nhe Ⅰ / Hind III.

[0014] Preferably, the transfection method involves treating HEK293T cells with the recombinant plasmid under the action of a transfection reagent; The culture involves culturing transfected HEK293T cells in antibiotic-free fetal bovine serum medium for 4-6 h, then replacing it with serum-free complete medium, culturing for 48 h, collecting the cell supernatant, and obtaining a solution containing exosomes.

[0015] This invention provides the application of the engineered exosomes or the engineered exosomes obtained by the construction method in the preparation of lipid-lowering drugs.

[0016] Preferably, the diseases treated by the drug include one or more of the following: primary hypercholesterolemia (heterozygous familial and non-familial), refractory hyperlipidemia, fatty liver and myocardial infarction, stroke and unstable angina, and other atherosclerotic derivative diseases. The beneficiary population includes patients who are intolerant to or have limited efficacy with statins, and the risk of cardiovascular events in patients with atherosclerotic cardiovascular diseases is reduced.

[0017] This invention provides an engineered exosome that targets the liver, antagonizes PCSK9, and specifically binds to LDL-C. The engineered exosome expresses the fusion protein LDLR L3-L7-PTGFRN extracellularly, and inclisiran is attached to the surface of the engineered exosome. The amino acid sequence of LDLR L3-L7 in the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:1; the amino acid sequence of PTGFRN is shown in SEQ ID NO:2. This invention modifies the transmembrane protein PTGFRN, specifically by fusing an extracellular PTGFRN sequence containing the L3-L7 region of LDLR, thereby enabling specific binding to LDL-C. Simultaneously, the PCSK9 inhibitor inclisiran is attached to the exosome surface. The engineered exosome possesses dual functions of antagonizing PCSK9 and promoting LDL-C degradation, efficiently targeting the liver and inhibiting PCSK9 expression in hepatocytes, while also specifically binding to LDL-C for liver degradation, providing a novel approach to reducing blood lipid levels and possessing broad application value. Meanwhile, the engineered exosomes provided by this invention have a simple preparation method and can realize large-scale production of engineered exosomes, providing a foundation for wide application. Attached Figure Description

[0018] Figure 1 The fusion protein sequence provided by this invention; Figure 2 A schematic diagram of the construction of a recombinant vector plasmid for expressing the fusion gene LDLR L3-L7-PTGFRN; Figure 3 A schematic diagram of engineered exosomes that target and antagonize PCSK9 in the liver and specifically bind to LDL-C; Figure 4 This shows the results of Western blot analysis of exosomes in cells and exosomes. Ctrl represents the control group, and Vec represents the exosome group. ctrl Group L3-L7 was the empty plasmid control group, L3-L7 was the plasmid modification group, FLAG was the FLAG antibody, and GAPDH was the internal control. Figure 5 For engineered exosome therapy after ApoE - / - Mouse plasma metabolic parameters and lipid-lowering results; Figure 6 For engineered exosome therapy after ApoE - / - The results showed that atherosclerotic plaques in mice were significantly reduced in size, inhibiting the progression of atherosclerosis. Detailed Implementation

[0019] This invention provides an engineered exosome that targets and antagonizes PCSK9 in the liver and specifically binds to LDL-C. The engineered exosome expresses the fusion protein LDLR L3-L7-PTGFRN extracellularly, and the surface of the engineered exosome is attached with Inclisiran. The amino acid sequence of LDLR L3-L7 in the fusion protein LDLR L3-L7-PTGFRN is as shown in SEQ ID NO:1 (PPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASC) The amino acid sequence of PTGFRN is shown in SEQ ID NO:2 (GPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSW). FAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD).

[0020] In this invention, the fusion protein LDLR L3-L7-PTGFRN preferably has LDLR L3-L7 expressed at the N-terminus of the PTGFRN protein, ensuring the extracellular expression of the LDLR L3-L7 motif on exosomes. Simultaneously, the LDLR L3-L7 motif is a necessary region for binding LDL-C in plasma; therefore, extracellular expression of LDLR L3-L7 is beneficial for reducing LDL-C levels, thereby lowering blood lipid levels and preventing and / or treating the development of atherosclerosis. The fusion protein LDLR L3-L7-PTGFRN preferably also includes a linker peptide. The amino acid sequence of the linker peptide is shown in SEQ ID NO:3 (GGGGSGGGGSGGGGS). The addition of the linker peptide allows the proteins on both sides to perform their independent functions, avoiding adverse results such as misfolding of the fusion protein, low protein yield, or impaired biological activity. The amino acid sequence of the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:3 (GGGGSGGGGSGGGS). NO:4 (PPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRRCRGLYVFQGDSSPCSAFEFHCL SGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKE CGGGGSGGGGSGGGGSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEF LLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEEAIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD).

[0021] In this invention, the inclisiran is preferably composed of PCSK9 siRNA and N-acetylgalactosamine. This invention does not impose any particular limitation on the source of the inclisiran; any source of inclisiran known in the art can be used. In this embodiment, the inclisiran was purchased from Suzhou Beixin Biotechnology Co., Ltd. The inclisiran is cholesterol-modified. The preparation of the cholesterol-modified inclisiran was entrusted to Suzhou Beixin Biotechnology Co., Ltd. Cholesterol modification of the inclisiran facilitates its conjugation with exosomes.

[0022] This invention provides a method for constructing the engineered exosomes, comprising the following steps: 1) Construct a recombinant vector expressing the fusion gene LDLR L3-L7-PTGFRN; 2) Transfect the recombinant vector described in step 1) into mammalian cells to obtain recombinant cells; 3) Culture the recombinant cells described in step 2) and collect exosomes; 4) Co-incubate the exosomes described in step 3) with Inclisiran to obtain engineered exosomes.

[0023] This invention constructs a recombinant vector expressing the fusion gene LDLR L3-L7-PTGFRN.

[0024] In this invention, the method for expressing the recombinant vector of the fusion gene LDLR L3-L7-PTGFRN preferably includes the following steps: The fusion gene LDLR L3-L7-PTGFRN sequence with restriction enzyme sites at both ends was digested with enzymes, and the resulting digested fragment was cloned into a plasmid digested with the same type of restriction endonuclease to obtain a recombinant vector.

[0025] In this invention, the gene sequence of the fusion gene LDLR L3-L7-PTGFRN is as follows: Nhe I-Kozak-PTGFRN signal peptide-L3-L7-LINKER-PTGFRN-Flag- Hind III. The Kozak sequence enhances the translation efficiency of the fusion gene, and the PTGFRN signal peptide guides the fusion protein to the cell membrane, with the insertion of a flag facilitating tracking. The preferred nucleotide sequence of the fusion gene LDLR L3-L7-PTGFRN is shown in SEQ ID NO:5.

[0026] In this invention, based on the restriction enzyme sites at both ends of the fusion gene, the restriction endonucleases used for plasmid digestion preferably include... Nhe Ⅰ / HindIII. This invention does not impose any particular limitation on the type of plasmid used; any mammalian cell expression vector well-known in the art can be used. In this embodiment, the plasmid is a pcDNA3.1(-) vector. The construction of the recombinant vector was commissioned to Nanjing Genscript Biotech Co., Ltd.

[0027] After obtaining the recombinant vector, the present invention transfects the recombinant vector into mammalian cells to obtain recombinant cells.

[0028] In this invention, the transfection method preferably involves treating HEK293T cells with the recombinant plasmid under the action of a transfection reagent. The mass ratio of the recombinant plasmid to the volume ratio of the transfection reagent is 2-4 µg: 4-8 µL. This invention does not impose any particular limitation on the type of transfection reagent; any transfection reagent well-known in the art can be used, such as HighGene (ABclonal Technology, China).

[0029] After obtaining the recombinant cells, the present invention cultured the recombinant cells and collected exosomes.

[0030] In this invention, the culture includes culturing transfected HEK293T cells in antibiotic-free fetal bovine serum medium for 4-6 h, then replacing it with serum-free complete medium, culturing for 48 h, collecting the cell supernatant, and obtaining a solution containing exosomes.

[0031] In this invention, the antibiotic-free fetal bovine serum culture medium is preferably DMEM medium with a fetal bovine serum volume concentration of 10%. The culture conditions are preferably 37 °C and 5% CO2.

[0032] In this invention, the serum-free complete culture medium is preferably DMEM culture medium containing 1% penicillin-streptomycin double antibiotics.

[0033] In this invention, the engineered exosomes, after administration, can effectively bind to LDL-C in the blood, enhancing the body's ability to clear LDL-C and thus achieving the goal of lowering blood lipids. At the same time, Inclisiran exerts a targeted inhibition of PCSK9 expression by interfering with PCSK9 mRNA, further inhibiting PCSK9 synthesis and secretion, and further enhancing the ability to clear LDL-C by reducing competitive binding to LDLR. In addition, Inclisiran can specifically target hepatocytes, thereby further increasing the efficiency of hepatocyte degradation of LDL-C, ultimately achieving the effect of controlling blood lipid levels.

[0034] Given that engineered exosomes have the lipid-lowering effect of targeting the liver to antagonize PCSK9 and specifically binding to and degrading LDL-C, this invention provides the application of the engineered exosomes or the engineered exosomes obtained by the construction method in the preparation of lipid-lowering drugs.

[0035] In this invention, the diseases treated by the drug preferably include one or more of the following: heterozygous familial hypercholesterolemia, refractory hyperlipidemia, fatty liver and cardiovascular and cerebrovascular diseases caused by hyperlipidemia, and the beneficiary population includes patients who are intolerant to or have limited efficacy with statins.

[0036] In this invention, the drug comprises an injectable formulation. Animal-level experiments: Each mouse was injected intravenously with 150 μg of engineered exosomes twice a week for four weeks.

[0037] The following detailed description, in conjunction with embodiments, illustrates an engineered exosome that targets the liver, antagonizes PCSK9, and specifically binds to LDL-C, along with its construction method and applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0038] Example 1 A method for constructing a recombinant vector 1) Using pcDNA3.1(-) as a vector, the LDL-C L3-L7 fragment was inserted into the extramembrane region of the transmembrane protein PTGFRN to form a co-expression vector. The exogenously inserted fusion gene fragment is shown in [reference needed]. Figure 1 The sequence of the fragment is shown in SEQ ID NO:1, and the gene synthesis was completed by Nanjing Genscript Biotech Co., Ltd.

[0039] Following enzyme digestion and ligation, the humanized L3-L7 PTGFRN motif was loaded into the pcDNA3.1(-) vector. The recombinant vector construction was performed by Nanjing Genscript Biotech Co., Ltd. A positive recombinant vector was obtained upon verification.

[0040] Example 2 A method for constructing and validating engineered exosomes 1) Mix 3 µg of the recombinant vector prepared in Example 1 with 6 µL of transfection reagent, let stand for 20 min, and then slowly add it to a HEK293T cell culture dish containing 10% fetal bovine serum (DMEM, Gibco, USA; fetal bovine serum, Gibco, USA) without antibiotics. 2) Place the transfected cells in a cell incubator at 37 ℃ containing 5% CO2. After 5 h, replace with serum-free complete culture medium (DMEM, Gibco, USA). Collect the cell supernatant after 48 h. (6) Filter the cell supernatant using a 0.22 μm filter to remove cell debris; (7) The cell supernatant was centrifuged at 100,000 g for 10 h, and the resulting precipitate was the exosome. The obtained exosome was detected by Western blot.

[0041] Simultaneously, exosomes derived from normal HEK293T cells were collected, following the same procedure as above, i.e., control exosomes (exo ctrl Exosomes were collected after transfecting HEK293T cells with an empty plasmid, following the same procedure as above, i.e., empty plasmid control exosomes (exo Vec-ctrl ).

[0042] The obtained exosomes were subjected to Western blot analysis to detect FLAG tags.

[0043] Following the aforementioned exosome extraction method, after centrifugation, the residual liquid in the precipitated exosomes was removed using a vacuum pump. 60 µL of RIPA lysis buffer (Beyotime, China) was added, and the mixture was pipetted until the exosomes were completely dissolved. The mixture was then placed on ice for 30 min to allow for complete exosome protein lysis. Protein quantification was performed using a serial dilution method (Thermo, USA), and protein sample preparation was carried out. A 12% gel (Beyotime, China) was prepared, and the exosome protein sample was added for electrophoresis. The electrophoresis voltage was set to 120 V for 25 min, and electrophoresis was started. Once the protein sample entered the lower separating gel, the electrophoresis voltage was adjusted to 160 V for 60 min. The position of the protein sample was observed; once it reached the expected position, electrophoresis was stopped, and transfer was performed. A constant current of 200 mA was set for 2 h. Antibody incubation was then performed. According to the antibody instructions, an appropriate amount of antibody was diluted proportionally. FLAG antibody (Sigma, USA) was selected as the primary antibody. The mixture was gently shaken on a shaker for 30 min and then incubated overnight at 4°C. The secondary antibody was goat anti-rabbit IgG HRP (BBI Sangon Biotech, China), which was placed in a dark box and slowly shaken on a shaker at room temperature for 1 hour. Finally, chemiluminescence was performed (ECL chemiluminescence solution, GE, USA), with GAPDH as an internal control.

[0044] The results are as follows Figure 2 As shown, exosomes derived from HEK293T carry the LDLR L3-L7 sequence.

[0045] Example 3 animal experiments The ApoE model of atherosclerosis - / -Mice (purchased from Jicui Pharmaceutical Co., Ltd.) were randomly divided into 5 groups: PBS group (PBS purchased from Kaiji Biotechnology, KGB5001, as a control), control exosome group (exosomes derived from normal cells extracted in Example 1), empty plasmid transfected cell-derived exosome group (exosomes transfected with empty plasmids extracted in Example 1), L3-L7 modified exosome group (L3-L7 modified exosomes extracted in Example 1), and Alirocumab group (PCSK9 inhibitor purchased from Meilun Biotechnology, MB2787, as a positive control).

[0046] The PBS group consisted of individuals who received sterile PBS via tail vein injection into ApoE. - / - In mice, the dose was 200 μL / mouse, once a week. After 4 weeks of injection, blood samples were collected and sections of the aortic root of the mice were stained with hematoxylin and eosin (HE). The control exosome group consisted of control exosomes injected into ApoE via tail vein. - / - In mice, the exosome dosage was 150 μg (200 μL PBS suspension) per mouse, once a week for 4 weeks. After that, blood samples were collected and sections of the aortic root of the mice were stained with HE. The procedure for transfecting cell-derived exosomes with empty plasmids and L3-L7 modified exosomes is the same as above; The Alirocumab group consisted of patients who received a PCSK9 inhibitor via tail vein injection into ApoE. - / - In mice, the dose was 150 μg (200 μL PBS suspension) per mouse, once a week. After 4 weeks, blood samples were collected and sections of the aortic root of the mice were stained with hematoxylin and eosin (HE).

[0047] Metabolic indicators were measured in blood samples from each group, and HE staining was performed on the aortic root of mice to assess the effect of the drug on atherosclerotic plaques.

[0048] Blood lipid and biochemical tests: Mice were fasted for 8 hours, and blood was collected from the eyeballs after anesthesia. Whole blood was instilled into 1.5 mL EP tubes and allowed to stand at room temperature for 20 minutes. (4℃, 3,000...) g Centrifuge for 20 min. Carefully collect the upper layer of pale yellow, clear liquid (serum) and perform subsequent total cholesterol (TC) and triglyceride (TG) tests using a fully automated biochemical analyzer.

[0049] Paraffin sectioning and HE staining: Mouse aortas with surrounding connective tissue removed were fixed in 4% paraformaldehyde for 24 h. The tissue was removed, trimmed, and dehydrated using a gradient of alcohols. Melted paraffin was dropped into an embedding frame, and the dehydrated tissue was quickly placed in before it solidified. The frame was then cooled to -20°C. After the paraffin block cooled and solidified, it was removed and sectioned. The thickness was set to approximately 3 μm. The tissue sections were flattened in warm water (40°C), gently lifted with a glass slide, and dried to ensure close adhesion between the tissue and the slide. The sections were then dewaxed in xylene I and II for 20 min each, followed by immersion in anhydrous ethanol I, anhydrous ethanol II, and 75% ethanol for 5 min each, and washed for 1 min. Hematoxylin staining was performed for 5 min, followed by rapid differentiation with 1% hydrochloric acid alcohol for 5 s, and then washing. The sections were then dehydrated in 85% and 95% ethanol, and stained with eosin for 5 min each. The sections were then immersed in anhydrous ethanol I, II, and III for 5 min each, followed by immersion in xylene I and II for 5 min each. Finally, seal the film with neutral resin.

[0050] The results are as follows Figure 5 As shown, plasma cholesterol in the L3-L7 modified exosome treatment group ( Figure 5 A) and triglycerides ( Figure 5 The levels of B in the group were significantly lower than those in the control group, and the decrease in each indicator was similar to that in the PCKS9 inhibitor Alirocumab group.

[0051] The results are as follows Figure 6 As shown, the L3-L7 modified exosome treatment group ApoE - / - The plaques in mice were significantly smaller than those in the control group, and the treatment effect was similar to that of the PCKS9 inhibitor Alirocumab group, indicating that the modified exosomes can effectively inhibit the formation of atherosclerotic plaques.

[0052] 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. An exosome, characterized in that, The exosomes express the fusion protein LDLR L3-L7-PTGFRN extracellularly. The amino acid sequence of LDLR L3-L7 in the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:1; the amino acid sequence of PTGFRN is shown in SEQ ID NO:2; and the amino acid sequence of the fusion protein LDLR L3-L7-PTGFRN is shown in SEQ ID NO:

4.

2. The exosomes according to claim 1, characterized in that, The fusion protein LDLR L3-L7-PTGFRN also includes a linker peptide; The amino acid sequence of the linker peptide is shown in SEQ ID NO:

3.

3. A method for constructing exosomes according to claim 1 or 2, characterized in that, Includes the following steps: 1) Construct a recombinant vector expressing the fusion gene LDLR L3-L7-PTGFRN; 2) Transfect the recombinant vector described in step 1) into mammalian cells to obtain recombinant cells; 3) Culture the recombinant cells described in step 2) and collect exosomes.

4. The construction method according to claim 3, characterized in that, The method for expressing the recombinant vector of the fusion gene LDLR L3-L7-PTGFRN as described in step 1) includes the following steps: The fusion gene LDLR L3-L7-PTGFRN sequence with restriction enzyme sites at both ends was digested with enzymes, and the resulting digested fragment was cloned into a plasmid digested with the same type of restriction endonuclease to obtain a recombinant vector.

5. The construction method according to claim 4, characterized in that, The nucleotide sequence of the fusion gene LDLR L3-L7-PTGFRN is shown in SEQ ID NO:5; The restriction endonuclease includes Nhe Ⅰ / Hind III.

6. The construction method according to claim 3, characterized in that, The transfection method involves treating HEK293T cells with the recombinant plasmid under the action of a transfection reagent; The culture involves culturing transfected HEK293T cells in antibiotic-free fetal bovine serum medium for 4-6 hours, then replacing it with serum-free complete medium, culturing for 48 hours, collecting the cell supernatant, and obtaining a solution containing exosomes.

7. The use of the exosomes according to claim 1 or 2, or the exosomes obtained by the construction method according to any one of claims 3 to 6, in the preparation of a medicament for treating atherosclerosis.

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