Engineered exosome for promoting leptin sensitivity by targeting central nervous system as well as preparation method and application of engineered exosome

Abstract

The invention provides an engineered exosome for promoting leptin sensitivity by targeting a central nervous system as well as a preparation method and application of the engineered exosome, and particularly provides RNAi for inhibiting fat synthesis, the engineered exosome for promoting leptin sensitivity by targeting the central nervous system, a preparation method of the engineered exosome and application of the engineered exosome in preparation of medicines for treating obesity. The engineered exosome prepared by the invention has SIRP alpha and MCAM proteins and can efficiently target a central nervous system, and meanwhile, the surface of the exosome is provided with a targeting peptide targeting leptin receptor neurons and LEP (61-90)-Lam2b fusion protein of leptin fragments, so that the exosome is finally combined with the leptin receptor neurons after efficiently targeting the central nervous system; the miRNAs which are loaded on the exosome and have the leptin sensitization effect can obviously improve the sensitivity of central leptin and play a role in reducing weight.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to RNAi that inhibits fat synthesis, engineered exosomes that target the central nervous system to promote leptin sensitivity, their preparation methods and applications. Background Technology

[0002] Obesity has become a growing public health problem worldwide. It is closely linked to a variety of chronic diseases, including type 2 diabetes, cardiovascular disease, hypertension, certain types of cancer, and metabolic syndrome, placing a significant economic burden on healthcare systems. Current intervention strategies for obesity mainly include lifestyle interventions, drug therapy, and bariatric surgery. However, the efficacy of lifestyle interventions is often limited, and while bariatric surgery is highly effective, its applicability is relatively limited. Drug therapy, as a commonly used obesity intervention strategy, can help some patients lose weight, but the efficacy of existing drugs varies from person to person, and many patients experience weight regain after weight loss. Therefore, there is an urgent need to explore new obesity treatment strategies to more effectively address this global challenge.

[0003] Leptin is a hormone secreted by adipocytes that plays a crucial role in regulating energy balance and appetite as a negative feedback signal for energy homeostasis. Leptin signals the brain, through its receptors in the hypothalamus, that sufficient body fat reserves are present, thereby suppressing appetite and increasing energy expenditure, ultimately leading to weight loss. However, in obese individuals, leptin resistance often occurs; despite high blood leptin levels, the hypothalamus's signaling response to leptin is weakened, resulting in a reduced effect of leptin in suppressing appetite and increasing energy expenditure, thus impacting weight loss. Therefore, targeting the hypothalamus to increase leptin sensitivity is an important strategy for effective weight loss.

[0004] The blood-brain barrier restricts the efficiency of drug delivery targeting the central nervous system, leading to less than ideal intervention outcomes. Exosomes, with their ability to cross the blood-brain barrier, offer a novel strategy for delivering drugs targeting the central nervous system. However, existing unmodified exosomes have limited efficiency in targeting central nervous system neurons, particularly leptin receptor neurons.

[0005] In existing technologies, the effectiveness of leptin as a therapeutic agent for treating obesity is reduced due to factors such as leptin resistance in the body. Therefore, sensitizers that improve leptin sensitivity are needed to overcome the effects of leptin resistance in treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing engineered exosomes that can target hypothalamic leptin receptor neurons and promote their leptin sensitivity, as well as their applications. The preparation method generates engineered exosomes that can efficiently target and deliver leptin receptor neurons to promote leptin signal transduction. The exosomes deliver their encapsulated miRNAs to receptor cells. The miRNAs can target negative feedback signaling molecules that inhibit leptin signal transduction, including SOCS3, PIAS, and PTEN.

[0007] Based on this, a first aspect of this application is to provide an RNAi that enhances leptin sensitivity by inhibiting the expression of at least one of the SOCS3, PIAS3, and PTEN genes.

[0008] RNA interference (RNAi) is a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). RNAi is a commonly used gene knockdown technique that downregulates gene expression by degrading mRNA or inhibiting its translation. It is applied in research on silencing oncogenes, correcting mutations in pathogenic genes, and developing treatments for hereditary diseases.

[0009] The SOCS3 gene is widely expressed in various tissues. Encoding SOCS3, or Cytokine Signaling Inhibitor 3, it negatively regulates cytokine signaling by binding to tyrosine kinase receptors in the JAK / STAT signaling pathway and inhibiting their activity. It can inhibit the signaling of various cytokines, including gp130, LIF, erythropoietin, insulin, IL12, GCSF, and leptin receptors. SOCS3 participates in regulating immune responses, inflammatory processes, and metabolic processes, playing a crucial role in maintaining immune homeostasis and inhibiting tumor growth. Abnormal expression of SOCS3 is associated with various diseases, including cancer, autoimmune diseases, and metabolic disorders. As a negative feedback inhibitor of the JAK-STAT3 pathway, SOCS3 inhibits the kinase activity of JAK by binding to it, thereby blocking the phosphorylation and activation of STAT3. SOCS3 can also directly bind to phosphorylated STAT3, preventing it from forming dimers and entering the nucleus, thus inhibiting the transcription of target genes.

[0010] The PIAS3 gene encodes a member of the PIAS (Protein Inhibitor of STAT Activation) family of transcriptional regulators. These proteins are signal transduction and transcriptional activators, acting as SUMO (small ubiquitin-like modifier)-E3 ligases that catalyze the covalent linking of SUMO proteins to specific target substrates. PIAS3 directly binds to several transcription factors, blocking or enhancing their activity. PIAS3 can inhibit STAT3 nuclear transport; by binding to phosphorylated STAT3, it prevents its entry into the nucleus, thereby suppressing the expression of related genes in the leptin signaling pathway. PIAS3 can also promote STAT3 dephosphorylation, potentially further attenuating its activity by promoting STAT3 dephosphorylation.

[0011] The PTEN gene encodes a 403-amino acid protein with phosphatase activity. PTEN protein can also indirectly inhibit insulin-induced phosphoinositol-3 kinase activity by specifically dephosphorylating the third phosphate group of IP3. IP3 is an important second messenger in the insulin-regulated cell growth signaling pathway, indicating that PTEN protein plays a role in this pathway. PTEN can regulate the PI3K / Akt pathway; by breaking down PIP3, PTEN inhibits the activity of the PI3K / Akt pathway, thereby reducing lipolysis and energy expenditure. PTEN also affects energy metabolism; in adipocytes, PTEN activity may influence energy metabolism and fat storage, thus affecting leptin sensitivity.

[0012] SOCS3 and PIAS3 together form a negative feedback network to prevent excessive activation of leptin signaling and maintain signal balance. PTEN affects fat breakdown and energy metabolism by regulating the PI3K / Akt pathway and is closely related to the metabolic effects of leptin.

[0013] As some embodiments of this application, the RNAi includes one or more of dsRNA, siRNA, shRNA, and miRNA.

[0014] dsRNA, or double-stranded RNA, is an RNA molecule formed by the renaturation of two complementary strands. It can be cleaved by the Dicer enzyme to form siRNA. Double-stranded RNA can be used to inhibit cancer and reduce protein expression. The principle is to construct antisense RNA that is the mRNA obtained after transcription of cell-specific proteins, thereby blocking the expression of the protein through base binding, which ultimately leads to the obstruction of the normal metabolic pathway of cancer cells or the protein to be processed.

[0015] siRNA, or small interfering RNA, also known as short interfering RNA or silencing RNA, is a double-stranded RNA that is 20 to 25 nucleotides long. It has many different uses in biology. siRNA is mainly involved in RNA interference (RNAi) to regulate gene expression in a specific way.

[0016] shRNA, or short hairpin RNA, consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. It is then followed by 5-6 T molecules as a transcription terminator for RNA polymerase III. In vivo, siRNA can be cloned into a plasmid vector using the siRNA sequence as a "short hairpin." When introduced into an animal, this hairpin sequence is expressed, forming a "double-stranded RNA" (dsRNA), which is then processed by RNAi channels.

[0017] microRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules, approximately 22 nucleotides in length, that participate in post-transcriptional gene expression regulation. miRNAs exert their regulatory effects on target mRNAs by disrupting their stability or inhibiting their translation. Each miRNA can target multiple genes, and several miRNAs can regulate the same gene. This complex regulatory network allows for the regulation of multiple gene expression by a single miRNA, as well as the precise regulation of a specific gene expression through combinations of several miRNAs.

[0018] As some embodiments of this application, the RNAi inhibits the expression of SOCS3, PIAS3, and PTEN genes.

[0019] In some embodiments of this application, the RNAi is a miRNA, and the nucleotide sequence of the miRNA is shown in any one of SEQ ID No. 1 to 2. The miRNA of this application inhibits the expression of SOCS3, PIAS3, and PTEN genes, suppresses the negative feedback signal of leptin signal transduction, reduces leptin antagonism, and increases leptin sensitivity, thereby treating obesity. In some embodiments of this application, the nucleotide sequence of the miRNA is shown in SEQ ID No. 1. In some embodiments of this application, the nucleotide sequence of the miRNA is shown in SEQ ID No. 2. In some embodiments of this application, the nucleotide sequence of the miRNA is shown in SEQ ID No. 1 to 2.

[0020] The miRNA of this application may have a nucleotide sequence with ≥75% homology to SEQ ID No. 1-2, for example, it may have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0021] Homology refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules. Sequence analysis is the ultimate method for determining the degree of homology, and DNA-DNA hybridization or DNA-RNA hybridization are also useful estimation methods.

[0022] A second aspect of this application is to provide extracellular vesicles containing the aforementioned RNAi.

[0023] Extracellular vesicles (EVs) are diverse membrane-bound vesicles originating from endosomes and cell membranes that are released from cells into the extracellular environment. They are collectively referred to as extracellular vesicles and mediate interactions between the environment and organisms, as well as among organisms, by carrying and transmitting functional molecules such as RNA, proteins, and metabolites. They have become widely concerned signal communication carriers. Based on differences in their generation process, release pathway, size, contents, and functions, they are divided into three types: microvesicles, exosomes, and apoptosis bodies.

[0024] As some embodiments of this application, the extracellular vesicles are one or more of exosomes, apoptotic bodies, and microvesicles.

[0025] Exosomes are extracellular vesicles surrounded by a phospholipid bilayer, with a diameter of 50-100 nm. They originate from inward budding of the cell membrane to form endosomes. Within the cell, they undergo early endosome formation, multivesicle complex formation, directed assembly, and migration before fusing with the cell membrane and being exocytotically released from the cell. Due to their origin, exosomes have a more complex composition and structure, even containing components "ingested" from the extracellular matrix or culture medium. Exosomes can not only translocate mRNA and miRNA, oncogenetic receptors, and HIV particles, but also possess antigen presentation, immune activation, and immunosuppressive activities.

[0026] Apoptotic bodies are produced by bubbling during apoptosis. They are 1-5 μm in diameter and can horizontally transfer DNA and oncogenes. When taken up by phagocytes, they present T-cell antigenic determinants and have an immunosuppressive effect.

[0027] Microvesicles are extracellular vesicles formed by cells directly budding outwards or "squeezing out" of the cell. They contain a cell membrane and some cytoplasmic components. The particle size of microvesicles ranges from 100 nm to 1000 nm, and their surface contains proteins such as CD40, integrins, and selectins, with a high content of phosphatidylserine.

[0028] In some embodiments of this application, the extracellular vesicles are exosomes.

[0029] As some embodiments of this application, the exosomes are loaded with the RNAi in vitro.

[0030] In some embodiments of this application, the exosomes are adipose exosomes.

[0031] In some embodiments of this application, the exosomes are secreted by 3T3-L1 cells.

[0032] In some embodiments of this application, the extracellular vesicles are derived from cells overexpressing SIRPα and MCAM.

[0033] SIRPα (Signal Regulatory Protein Alpha) is an immunomodulatory receptor belonging to the immunoglobulin superfamily, and plays an important role in tumor immunity and autoimmune diseases.

[0034] Melanoma cell adhesion molecule (MCAM), also known as CD146 (differentiation cluster 146) or cell surface glycoprotein MUC18, is a whole membrane glycoprotein that promotes tumor growth, angiogenesis, and metastasis, and is considered a promising target for cancer treatment. Its function is as a receptor for laminin α4, a matrix molecule widely expressed within the blood vessel wall.

[0035] As some embodiments of this application, the extracellular vesicles are derived from cells expressing a fusion protein of a targeting peptide and a leptin peptide that targets leptin receptor neurons.

[0036] As some embodiments of this application, the amino acid sequence of SIRPα is shown in SEQ ID No. 3.

[0037] The amino acid sequence of SIRPα can have ≥75% homology with SEQ ID No.3, for example, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0038] As some embodiments of this application, the amino acid sequence of MCAM is shown in SEQ ID No. 4.

[0039] The amino acid sequence of MCAM can have ≥75% homology with SEQ ID No. 4, for example, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0040] As some embodiments of this application, the targeting peptide for leptin receptor neurons is the LEP(61-90)-Lam2b fusion protein.

[0041] As some embodiments of this application, the amino acid sequence of the LEP(61-90)-Lam2b fusion protein is shown in SEQ ID No. 5.

[0042] The amino acid sequence of the LEP(61-90)-Lam2b fusion protein can have ≥75% homology with SEQ ID No. 5, for example, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0043] A third aspect of this application is to provide a pharmaceutical composition comprising the aforementioned RNAi or the aforementioned extracellular vesicles, and a pharmaceutically acceptable carrier and / or excipient.

[0044] As some embodiments of this application, the pharmaceutical composition is formulated into a dosage form that can be administered via intranasal, intravenous, intramuscular, subcutaneous, sublingual, oral, transdermal, local, or intracerebral administration.

[0045] As some embodiments of this application, the pharmaceutical composition further includes a second drug for treating obesity.

[0046] As some embodiments of this application, the second drug includes leptin.

[0047] A fourth aspect of this application is to provide a method for preparing the extracellular vesicles, the method comprising the steps of: (1) expressing a fusion protein of a targeting peptide and a leptin peptide targeting leptin receptor neurons in adipocytes, while overexpressing SIRPα and MCAM; (2) loading the RNAi into exosomes secreted by the adipocytes; and (3) isolating and collecting the exosomes.

[0048] In some embodiments of this application, the adipocytes in step (1) are 3T3-L1 cells.

[0049] The exosomes of this application are secreted by engineered 3T3-L1 cells, which include: 1) proteins that enhance the central targeting of exosomes, 2) tags that target leptin receptor neurons, and 3) the ability to load the miRNA into the exosomes.

[0050] As some embodiments of this application, step (1) involves introducing the overexpressed SIRPα and MCAM genes into the adipocytes via lentivirus.

[0051] As some embodiments of this application, step (2) involves loading RNAi into exosomes secreted by the adipocytes via lentivirus.

[0052] Stable cells capable of secreting highly efficient exosomes targeting the central nervous system were established by overexpressing SIRPα and MCAM via lentivirus. Furthermore, siBACE1 was efficiently loaded into exosomes via lentiviral delivery of functional sequences that endogenously load siSOCS3, siPIAS3, and siPTEN into the exosomes. Additionally, the LEP(61-90)-Lam2b fusion protein was expressed in the exosomes, resulting in exosomes containing targeting peptides and leptin fragments targeting leptin receptor neurons. The resulting exosomes efficiently targeted the central nervous system and bound to leptin receptor neurons, exerting the therapeutic effect of leptin on obesity under the action of sensitizers siSOCS3, siPIAS3, and siPTEN, which enhance leptin sensitivity.

[0053] As some embodiments of this application, the amino acid sequence of SIRPα is shown in SEQ ID No. 3.

[0054] As some embodiments of this application, the amino acid sequence of MCAM is shown in SEQ ID No. 4.

[0055] As some embodiments of this application, the fusion protein of the targeting peptide and leptin peptide targeting leptin receptor neurons is a LEP(61-90)-Lam2b fusion protein.

[0056] The present application provides a technical route for expressing and sorting proteins that target the central nervous system, fusion proteins that target leptin receptor neurons and leptin fragments into exosomes. Among them, the proteins that target the central nervous system are SIRPα and MCAM, and the proteins that target the central nervous system are overexpressed via lentivirus; the tag that targets leptin receptor neurons is a LEPR targeting peptide fused with Lam2b protein.

[0057] As some embodiments of this application, the amino acid sequence of the LEP(61-90)-Lam2b fusion protein is shown in SEQ ID No. 5.

[0058] In some embodiments of this application, the RNAi is a miRNA, and the nucleotide sequences of the miRNA are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0059] The fifth aspect of this application is to provide the use of the said RNAi or the said extracellular vesicles or the said pharmaceutical composition in the preparation of a medicament for treating obesity.

[0060] As some embodiments of this application, the drug for treating obesity includes any of the following: a. a drug for reducing weight; b. a drug for reducing body fat percentage; c. a drug for reducing food intake; d. a drug for increasing energy metabolism; e. a drug for activating pSTAT3; and f. a drug for increasing leptin sensitivity in the central nervous system.

[0061] A sixth aspect of this application is to provide a method for treating obesity, the method comprising administering the miRNA or the extracellular vesicles or the pharmaceutical composition to an obese patient.

[0062] As some embodiments of this application, the treatment of obesity includes one or more of the following indicators: reducing total weight, reducing body mass index, reducing waist circumference, and reducing total fat.

[0063] As described above, the engineered exosomes that target the central nervous system and promote leptin sensitivity, the preparation method thereof, and their applications have the following beneficial effects:

[0064] The exosomes of this application have overexpressed SIRPα and MCAM proteins, which enable the exosomes to efficiently target the central nervous system. At the same time, the surface of the exosomes has a targeting peptide that targets leptin receptor neurons and a leptin fragment LEP(61-90)-Lam2b fusion protein, which ultimately enables the exosomes to efficiently target the central nervous system and bind to leptin receptor neurons.

[0065] The technology of this application uses genetic engineering methods to prepare stable cells that can continuously produce engineered exosomes that deliver miRNA to the central nervous system. These exosomes can efficiently target the central nervous system and endogenously load miRNA.

[0066] The method provided in this application can efficiently and stably generate engineered exosomes that can target miRNAs in the central nervous system. The method is simple and easy to implement, and the process can be effectively scaled up. It provides a new strategy for improving leptin sensitivity by targeting the central nervous system and inhibiting SOCS3, PIAS3 and PTEN.

[0067] The miRNA of this application, after being delivered to leptin receptor neurons via exosomes, can significantly increase leptin sensitivity, showing significant differences from the control in terms of body weight, body composition, food intake, and energy expenditure levels. Attached Figure Description

[0068] Figure 1 The engineered cell lines of this application stably express MCAM and SIRPα;

[0069] Figure 2 This application demonstrates that the engineered exosome membrane is rich in the LEP(61-90)-Lam2b fusion protein;

[0070] Figure 3 The study demonstrated that miRNA was introduced into engineered exosomes via electroporation without significantly affecting the structure and function of the engineered exosomes. Exosomes loaded with miRNA significantly downregulated the expression of SOCS3, PIAS3, and PTEN genes.

[0071] Figure 4 The study showed that engineered exosomes loaded with leptin-sensitizing miRNAs, when injected intravenously into obese recipient mice, significantly increased central leptin sensitivity. Detailed Implementation

[0072] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0073] [Experimental Materials]

[0074] 3T3-L1 cells (ATCC; Catalog No.: CL-173)

[0075] Lentiviral (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0076] mCherry fluorescent protein (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0077] EGFP fluorescent protein (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0078] Puromycin (MCE; Catalog No.: HY-K1057)

[0079] Methylphenidate S (MCE Company; Product No.: HY-HY-103401)

[0080] PKH26 dye (MCE Company; Product No.: HY-D1451)

[0081] C57 / BL6 mice (Jicui Yaokang)

[0082] Obese mice (Jicui Yaokang)

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0084] Example 1

[0085] 1.1 Lentivirals overexpressing MCAM and SIRPα proteins were constructed, respectively. These lentiviruses co-expressed mCherry and EGFP fluorescent proteins, and carried puromycin and cytomegalovirus S resistance genes as markers, respectively. The amino acid sequence of the membrane protein SIRPα is shown in SEQ ID No. 3, and the amino acid sequence of the membrane protein MCAM is shown in SEQ ID No. 4.

[0086] The same 3T3-L1 cell line was transfected with both viruses, and a stable cell line simultaneously overexpressing MCAM and SIRPα proteins was constructed after screening with puromycin and cypermethrin S, thus obtaining the engineered cell line used in this application. The specific methods are as follows:

[0087] 1. Seed 3T3-L1 cells in 24-well plates (2×10^5 cells / well), ensuring that the cell confluence is between 30% and 50% before transfection.

[0088] 2. Viruses overexpressing MCAM and SIRPα were constructed, respectively, with titers of 10^8.

[0089] 3. Set the MOI of the two viruses to 20 each, for a total MOI of 40.

[0090] 4. Add virus solution to each well according to the amount in Table 1, and make up the volume to 500 μl with complete culture medium, and shake gently.

[0091] 5. 8-12 hours after transfection, extract the virus-containing culture medium, wash twice with PBS, and then add 500 μl of complete culture medium.

[0092] 6. Observe the fluorescence carried by the virus after 24-48 hours.

[0093] 7. Immunoblot analysis of MCAM and SIRPα expression can be performed 48 hours later.

[0094] Table 1

[0095] Cell count MOI Virus titer Virus volume MCAM 2×10^5 20 10^8 cells / ml 25μl SIRPα 2×10^5 20 10^8 cells / ml 25μl

[0096] See results Figure 1 , Figure 1 A and 1B are the inactivation curves of puromycin and cypermethrin S, respectively. Figure 1 C represents the expression of the stable transgenic mCherry and EGFP fluorescent protein. Figure 1 D and 1E show the overexpression of MCAM and SIRPα proteins in the stable mutant strain compared to the control group. Figure 1 The experimental results demonstrate that the engineered cell lines of this application stably express MCAM and SIRPα.

[0097] Example 2

[0098] The tag targeting leptin receptor neurons is a LEPR targeting peptide fused with the Lam2b protein, the amino acid sequence of which is shown in SEQ ID No. 5. A lentivirus expressing the leptin receptor neuron tag was constructed and used to infect the engineered cell line prepared in Example 1 that stably expresses MCAM and SIRPα. The method for constructing engineered cells that efficiently target leptin receptor neurons for exosomes is the same as that for constructing engineered cell lines that stably express MCAM and SIRPα in Example 1. This yields engineered cells that produce exosomes that efficiently target leptin receptor neurons.

[0099] Exosomes from the engineered 3T3-L1 cells described above were isolated by ultracentrifugation. Their particle size was measured, and they were characterized by immunoblotting and transmission electron microscopy. The results of the constructed exosomes expressing the LEPR-targeting peptide are shown below. Figure 2 . Figure 2 A shows the concentration distribution of exosome particle size. Figure 2 B detected marker proteins on the exosome membrane. Figure 2 C is its electron microscope image, and its size falls within the range of exosome size, confirming that exosomes were obtained. Figure 2 The scale bar in C is 100 nm.

[0100] Figure 2 The expression of the LEPR-targeting peptide on the exosomes expressing the LEPR-targeting peptide was detected by D-test in the control group (Native) and the constructed exosomes. It was observed that the exosomes expressing the LEPR-targeting peptide constructed in this embodiment were rich in LEPR. The control group (Native) consisted of exosomes that overexpressed only MCAM and SIRPα proteins but did not express the LEPR-targeting peptide.

[0101] Engineered exosomes were stained with PKH26 dye and injected via tail vein into C57 / BL6 mice to detect their ability to target leptin receptor neurons in the central nervous system and hypothalamus. Results are shown below. Figure 2 E. In the figure, the green markers are neurons that express leptin receptors, and the red markers are exosomes that target neurons with leptin receptors in the central nervous system and hypothalamus. It can be seen that, compared with the control group, the presence of LEPR-targeting peptides significantly increases the efficiency of exosomes entering neurons with leptin receptors. Figure 2 F shows a comparison of the fluorescence intensity of exosomes from the two groups in neurons with leptin receptors, confirming that the engineered exosomes obtained through this embodiment can efficiently target the central nervous system and accumulate in leptin receptor neurons. That is, the engineered exosomes obtained can not only efficiently target the central nervous system but also significantly increase the efficiency of targeting leptin receptor neurons.

[0102] Example 3

[0103] Two miRNAs with leptin-sensitizing effects were simultaneously loaded into engineered exosomes prepared in Example 2 using electroporation. The loading efficiency of the miRNAs was quantitatively analyzed. The sequences of the two miRNAs, miR-148a-3p and miR-30a-3p, are shown in SEQ ID No. 1 and SEQ ID No., respectively. The control exosomes (EVs) in this example were EVs that did not encapsulate the leptin-sensitizing miRNAs but overexpressed MCAM and SIRPα proteins and expressed the LEPR targeting peptide.

[0104] For detailed test results, please see [link / details]. Figure 3 , Figure 3 A shows a comparison of exosome encapsulation efficiency without the addition of miRNAs via electroporation versus exosome encapsulation efficiency with the addition of miRNAs. Figure 3 B shows electron micrographs of control exosomes without miRNAs that sensitize leptin and engineered exosomes loaded with miRNAs. Figure 3 Electron microscopy images of B show that the engineered exosomes loaded with miRNAs and the control exosomes do not differ significantly in appearance. Figure 3 The scale size in B is 100 nm.

[0105] Figure 3C shows the Western blotting results of different exosomal marker proteins TSG101, CD9, and CD81 in control exosomes without miRNAs loading leptin-sensitizing agents and engineered exosomes loaded with miRNAs. Figure 3 In diagram C, the left lane shows the detection of control exosomes (EVs), and the right lane shows engineered exosomes loaded with miRNAs. Figure 3 C shows that the engineered exosomes loaded with miRNAs in this application retained exosome marker proteins and were not significantly different from control exosomes.

[0106] Figure 3 D and 3E show the fold changes of the two miRNAs in exosomes, reflecting the relative changes in the levels of miR-148a-3p and miR-30a-3p. Figure 3 Figures D through E show that, compared to control exosomes, the miRNA content in the engineered exosomes loaded with miRNAs in this application is significantly increased.

[0107] The figures above illustrate that electroporation has no significant effect on the structure and function of exosomes.

[0108] Example 4

[0109] When engineered exosomes prepared in Example 3, carrying two leptin-sensitizing miRNAs, were injected intravenously into obese recipient mice (C57 / BL6 mice induced into an obese model by high-fat diet for 8 weeks), the levels of these miRNAs in the hypothalamus were significantly increased, indicating that the engineered exosomes could carry miRNAs into the brain and significantly downregulate the genes SOCS3, PIAS, and PTEN. After continuous treatment with these engineered exosomes for 4 weeks, the body weight, body composition, food intake, and energy expenditure of the obese mice were measured. The sensitivity of hypothalamic neurons to leptin was also measured. The results confirmed that the engineered exosomes could significantly increase central leptin sensitivity. Experimental results are shown below. Figure 4 In this embodiment, the blank control Vehicle was PBS as the solvent, and the control exosomes were EVs that did not encapsulate leptin-sensitizing miRNAs but overexpressed MCAM and SIRPα proteins and expressed LEPR-targeting peptides.

[0110] Figure 4 Western blotting results for A showed that exosomes encapsulating miRNAs downregulated the genes SOCS3, PIAS, and PTEN, significantly reducing protein expression. Figure 4 A indicates that exosomal miRNAs encapsulating miRNAs, when targeted to the central nervous system, can effectively reduce the expression of SOCS3, PIAS, and PTEN in the hypothalamus.

[0111] Figure 4 B shows the changes in body weight of mice in different experimental groups. It can be seen that the body weight of obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs gradually decreased from the beginning to the 4th week, while the body weight of obese mice in the blank control group and those injected with engineered exosomes without leptin-sensitizing miRNAs increased. Figure 4 C shows the body fat analysis of these three groups of mice at week 4 of the experiment. It can be seen that the body fat percentage of obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs was reduced.

[0112] Figure 4 D shows the food intake at week 4 of the experiment, indicating that obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs had reduced food intake. Figure 4 E shows a comparison of energy consumption over a day (24h), during the day and at night for the three experimental groups. Compared with the blank control group and the obese mouse group without engineered exosomes carrying leptin-sensitizing miRNAs, the obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs showed increased energy metabolism at all time points.

[0113] Figure 4 F shows the expression of pSTAT3 in the brains of the three groups of mice. It can be seen that pSTAT3 is activated only in the brains of obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs. It is a marker of leptin signaling activation, demonstrating the increased activation of leptin in mice. Figure 4 G analyzed the relative pSTAT3 levels in the three groups of mice, showing that obese mice injected with engineered exosomes carrying leptin-sensitizing miRNAs had significantly increased pSTAT3 levels in their brains and began to exert a weight-loss effect.

[0114] Sequence List:

[0115] miR-148a-3p (miRNA, SEQ ID 1):

[0116] UCAGUGCACUACAGAACUUUGU miR-30a-3p (miRNA, SEQ ID 2):

[0117] CUUUCAGUCGGAUGUUUGCAGC SIRPαsequence:

[0118] Amino acid sequence (SEQ ID NO.3):

[0119] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQ

[0120] WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVE

[0121] FKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVD

[0122] PVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAE

[0123] NQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKL

[0124] TCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLV

[0125] RIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQ

[0126] PASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

[0127] Nucleotide sequence of SIRPα (SEQ ID NO.6):

[0128] TCCGGCCCGCACCCACCCCCAAGAGGGGCCTTCAGCTTTGGGGCTCAGAGGCACGACCTCCTG

[0129] GGGAGGGTTAAAAGGCAGACGCCCCCCCGCCCCCCGCGCCCCCGCGCCCCGACTCCTTCGCCG

[0130] CCTCCAGCCTCTCGCCAGTGGGAAGCGGGGAGCAGCCGCGCGGCCGGAGTCCGGAGGCGAGG

[0131] GGAGGTCGGCCGCAACTTCCCCGGTCCACCTTAAGAGGACGATGTAGCCAGCTCGCAGCGCTG

[0132] ACCTTAGAAAAACAAGTTTGCGCAAAGTGGAGCGGGGACCCGGCCTCTGGGCAGCCCCGGCG

[0133] GCGCTTCCAGTGCCTTCCAGCCCTCGCGGGCGGCGCAGCCGCGGCCCATGGAGCCCGCCGGCC

[0134] CGGCCCCCGGCCGCCTCGGGCCGCTGCTCTGCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAG

[0135] GAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCT

[0136] GGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGG

[0137] TTCAGAGGAGCTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGG

[0138] GTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACA

[0139] TCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACG

[0140] TGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGG

[0141] TATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACG

[0142] GCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCC

[0143] AGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTG

[0144] GTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAG

[0145] GGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAG

[0146] GTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTT

[0147] CTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGG

[0148] CCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGT

[0149] ATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGT

[0150] CAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCG

[0151] CTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCT

[0152] GGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTC

[0153] CACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAA

[0154] TGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGA

[0155] GCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACA

[0156] CCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCA

[0157] AGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAGTGAATGGGACCGT

[0158] GGTTTGCTCTAGCACCCATCTCTACGCGCTTTCTTGTCCCACAGGGAGCCGCCGTGATGAGCAC

[0159] AGCCAACCCAGTTCCCGGAGGGCTGGGGCGGTGCAGGCTCTGGGACCCAGGGGCCAGGGTGG

[0160] CTCTTCTCTCCCCACCCCTCCTTGGCTCTCCAGCACTTCCTGGGCAGCCACGGCCCCCTCCCCC

[0161] CACATTGCCACATACCTGGAGGCTGACGTTGCCAAACCAGCCAGGGAACCAACCTGGGAAGTG

[0162] GCCAGAACTGCCTGGGGTCCAAGAACTCTTGTGCCTCCGTCCATCACCATGTGGGTTTTGAAG

[0163] ACCCTCGACTGCCTCCCCGATGCTCCGAAGCCTGATCTTCCAGGGTGGGGAGGAGAAAATCCC

[0164] ACCTCCCCTGACCTCCACCACCTCCACCACCACCACCACCACCACCACCACCACTACCACCAC

[0165] CACCCAACTGGGGCTAGAGTGGGGAAGATTTCCCCTTTAGATCAAACTGCCCCTTCCATGGAAA

[0166] AGCTGGAAAAAAACTCTGGAACCCATATCCAGGCTTGGTGAGGTTGCTGCCAACAGTCCTGGC

[0167] CTCCCCCATCCCTAGGCTAAAGAGCCATGAGTCCTGGAGGAGGAGAGGACCCCTCCCAAAGGA

[0168] CTGGAGACAAAACCCTCTGCTTCCTTGGGTCCCTCCAAGACTCCCTGGGGCCCAACTGTGTTG

[0169] CTCCACCCGGACCCATCTCTCCCTTCTAGACCTGAGCTTGCCCCTCCAGCTAGCACTAAGCAAC

[0170] ATCTCGCTGTGGACGCCTGTAAATTACTGAGAAATGTGAAACGTGCAATCTTGAAACTGAGGTG

[0171] TTAGAAAACTTGATCTGTGGTGTTTTGTTTTGTTTTTTTTCTTAAAACAACAGCAACGTGATCTT

[0172] GGCTGTCTGTCATGTGTTGAAGTCCATGGTTGGGTCTTGTGAAGTCTGAGGTTTAACAGTTTGT

[0173] TGTCCTGGAGGGATTTTCTTACAGCGAAGACTTGAGTTCCTCCAAGTCCCAGAACCCCAAGAAT

[0174] GGGCAAGAAGGATCAGGTCAGCCACTCCCTGGAGACACAGCCTTCTGGCTGGGACTGACTTG

[0175] GCCATGTTCTCAGCTGAGCCACGCGGCTGGTAGTGCAGCCTTCTGTGACCCCGCTGTGGTAAGT

[0176] CCAGCCTGCCCAGGGCTGCTGAGGGCTGCCTCTTGACAGTGCAGTCTTATCGAGACCCAATGC

[0177] CTCAGTCTGCTCATCCGTAAAGTGGGGATAGTGAAGATGACACCCCTCCCCACCACCTCTCATA

[0178] AGCACTTTAGGAACACACAGAGGGTAGGGATAGTGGCCCTGGCCGTCTATCCTACCCCTTTAGT

[0179] GACCGCCCCCATCCCGGCTTTCTGAGCTGATCCTTGAAGAAGAAATCTTCCATTTCTGCTCTCA

[0180] AACCCTACTGGGATCAAACTGGAATAAATTGAAGACAGCCAGGGGGATGGTGCAGCTGTGAAG

[0181] CTCGGGCTGATTCCCCCTCTGTCCCAGAAGGTTGGCCAGAGGGTGTGACCCAGTTACCCTTTAA

[0182] CCCCCACCCTTCCAGTCGGGTGTGAGGGCCTGACCGGGCCCAGGGCAAGCAGATGTCGCAAG

[0183] CCCTATTTATTCAGTCTTCACTATAACTCTTAGAGTTGAGACGCTAATGTTCATGACTCCTGGCCT

[0184] TGGGATGCCCAAGGGATTTCTGGCTCAGGCTGTAAAAGTAGCTGAGCCATCCTGCCCATTCCTG

[0185] GAGGTCCTACAGGTGAAACTGCAGGAGCTCAGCATAGACCCAGCTCTCTGGGGGATGGTCACC

[0186] TGGTGATTTCAATGATGGCATCCAGGAATTAGCTGAGCCAACAGACCATGTGGACAGCTTTGGC

[0187] CAGAGCTCCCGTGTGGCATCTGGGAGCCACAGTGACCCAGCCACCTGGCTCAGGCTAGTTCCA

[0188] AATTCCAAAAGATTGGCTTGTAAACCTTCGTCTCCCTCTCTTTTACCCAGAGACAGCACATACG

[0189] TGTGCACACGCATGCACACACACATTCAGTATTTTAAAAGAATGTTTTCTTGGTGCCATTTTCAT

[0190] TTTATTTTATTTTTTAATTCTTGGAGGGGGAAATAAGGGAATAAGGCCAAGGAAGATGTATAGCT

[0191] TTAGCTTTAGCCTGGCAACCTGGAGAATCCACATACCTTGTGTATTGAACCCCAGGAAAAGGAA

[0192] GAGGTCGAACCAACCCTGCGGAAGGAGCATGGTTTCAGGAGTTTATTTTAAGACTGCTGGGAA

[0193] GGAAACAGGCCCCATTTTGTATATAGTTGCAACTTAAACTTTTTGGCTTGCAAAATATTTTTGTAA

[0194] TAAAGATTTCTGGGTAATAATGA

[0195] MCAM sequence:

[0196] Amino acid sequence (SEQ ID NO.4):

[0197] MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVDWFSV

[0198] HKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRV

[0199] YKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNRVHIQSSQTVESS

[0200] GLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKE

[0201] GDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISL

[0202] LSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQWLREETGQVLERGPVLQLHDL

[0203] KREAGGGYRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTI

[0204] SWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSNTT

[0205] TGLSTSTASPHTRANSTSTERKLPEPESRGVVIVAVIVCILVLAVLGAVLYFLYKKGKLPCRRSGKQEI

[0206] TLPPSRKSELVVEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRHMCAM nucleotide sequence (SEQ ID NO.7):

[0207] ACTTGGCTCTCGCCCTCCGGCCGGGAAGCATGGGGCTTCCCAGGCTGGTCTGCGCCTTCTTGCT

[0208] CGCCGCCTGCTGCTGCTGTCCTCGCGTCGCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCC

[0209] TGAGCTGGTGGAGGTGGAAGTGGGCAGCACAGCCCTTCTGAAGTGCGGCCTCTCCCAGTCCCA

[0210] AGGCAACCTCAGCCATGTCGACTGGTTTTCTGTCCACAAGGAGAAGCGGACGCTCATCTTCCG

[0211] TGTGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGGCTCAGCCTCCAGG

[0212] ACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCATCTTCTTGTGCC

[0213] AGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGCGTCTACAAAGCTCCGGAGG

[0214] AGCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGTGAACAGTAAGGAGCCTGAGGAGGTCG

[0215] CTACCTGTGTAGGGAGGAACGGGTACCCCATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCC

[0216] TCTGAAGGAGGAGAAGAACCGGGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTT

[0217] GTACACCTTGCAGAGTATTCTGAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTA

[0218] CTGTGAGCTCAACTACCGGCTGCCCAGTGGGAACCACATGAAGGAGTCCAGGGAAGTCACCGT

[0219] CCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGGAAGTGGAGCCCGTGGGAATGCTGAAGGA

[0220] AGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCCTCCACCACACTTCAGCATCAG

[0221] CAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAACCAACGACAACGGGGTCCTG

[0222] GTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCGCTATGAATGTCAGGGCCTGGACTTGGAC

[0223] ACCATGATATCGCTGCTGAGTGAACCACAGGAACTACTGGTGAACTATGTGTCTGACGTCCGAG

[0224] TGAGTCCCGCAGCCCCTGAGAGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAG

[0225] AGTAGCCAGGACCTCGAGTTCCAGTGGCTGAGAGAAGAGACAGGCCAGGTGCTGGAAAGGGG

[0226] GCCTGTGCTTCAGTTGCATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGTC

[0227] TGTGCCCAGCATACCCGGCCTGAACCGCACACAGCTGGTCAACGTGGCCATTTTTGGCCCCCCT

[0228] TGGATGGCATTCAAGGAGAGGAAGGTGTGGGTGAAAGAGAATATGGTGTTGAATCTGTCTTGT

[0229] GAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGAACGTCAACGGCACGGCAAGTGAACA

[0230] AGACCAAGATCCACAGCGAGTCCTGAGCACCCTGAATGTCCTCGTGACCCCGGAGCTGTTGGA

[0231] GACAGGTGTTGAATGCACGGCCTCCAACGACCTGGGCAAAAACACCAGCATCCTCTTCCTGGA

[0232] GCTGGTCAATTTAACCACCCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTTCCAC

[0233] TGCCAGTCCTCATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGA

[0234] GCCGGGGCGTGGTCATCGTGGCTGTGATTGTGTGCATCCTGGTCCTGGCGGTGCTGGGCGCTGT

[0235] CCTCTATTTCCTCTATAAGAAGGGCAAGCTGCCGTGCAGGCGCTCAGGGAAGCAGGAGATCAC

[0236] GCTACCCCCGTCTCGTAAGAGCGAACTTGTAGTTGAAGTTAAGTCAGATAAGCTCCCAGAAGA

[0237] GATGGGCCTCCTGCAGGGCAGCAGCGGTGACAAGAGGGCTCCGGGAGACCAGGGAGAGAAAT

[0238] ACATCGATCTGAGGCATTAGCCCCGAATCACTTCAGCTCCCTTCCCTGCCTGGACCATTCCCAG

[0239] CTCCCTGCTCACTCTTCTCTCAGCCAAAGCCTCCAAAGGGACTAGAGAGAAGCCTCCTGCTCC

[0240] CCTCGCCTGCACACCCCCTTTCAGAGGGCCACTGGGTTAGGACCTGAGGACCTCACTTGGCCC

[0241] TGCAAGGCCCGCTTTTCAGGGACCAGTCCACCACCATCTCCTCCACGTTGAGTGAAGCTCATCC

[0242] CAAGCAAGGAGCCCCAGTCTCCCGAGCGGGTAGGAGAGTTTCTTGCAGAACGTGTTTTTTCTT

[0243] TACACACATTATGGCTGTAAATACCTGGCTCCTGCCAGCAGCTGAGCTGGGTAGCCTCTCTGAG

[0244] CTGGTTTCCTGCCCCAAAGGCTGGCTTCCACCATCCAGGTGCACCACTGAAGTGAGGACACAC

[0245] CGGAGCCAGGCGCCTGCTCATGTTGAAGTGCGCTGTTCACACCCGCTCCGGAGAGCACCCCAG

[0246] CAGCATCCAGAAGCAGCTGCAGTGTTGCTGCCACCACCCTCCTGTCTGCCTCTTCAAAGTCTCC

[0247] TGTGACATTTTTTCTTTGGTCAGAAGCCAGGAACTGGTGTCATTCCTTAAAAGATACGTGCCGG

[0248] GGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCA

[0249] CAAAGTCAGGACGAGACCATCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAA

[0250] AAAAAATTAGCTAGGCGTAGTGGTTGGCACCTATAGTCCCAGCTACTCGGAAGGCTGAAGCAG

[0251] GAGAATGGTATGAATCCAGGAGGTGGAGCTTGCAGTGAGCCGAGACCGTGCCACTGCACTCCA

[0252] GCCTGGGCAACACAGCGAGACTCCGTCTCGAGGAAAAAAAAAGAAAAGATACGTGCCTGCGG

[0253] TGAGGAAGCTGGGCGCTGTTTTCGAGTTCAGGTGAATTAGCCTCAATCCCCCGTGTTCACTTGG

[0254] CTCCCATAGCCCTCTTGATGGATCACGTAAAACTGAAAGGCAGCGGGGAGCAGACAAAGATGA

[0255] GGTCTACACTGTCCTTCATGGGGATTAAAGCTATGGTTATATTAGCACCAAACTTCTACAAACCA

[0256] AGCTCAGGGCCCCAACCCTAGAAGGGCCCAAATGAGAGAATGGTACTTAGGGATGGAAAACG

[0257] GGCCTGGCTAGAGCTTCGGGTGTGTGTGTCTGTCTGTGTGTATGCATACATATGTGTGTATATATG

[0258] GTTTTGTCAGGTGTGTAAATTTGCAAATTGTTTCCTTTATATATGTATGTATATATATATATGAAAAT

[0259] ATATATATATATGAAAAATAAAGCTTAATTGTCCCAGAAA

[0260] The amino acid sequence of the LEP(61-90)-Lam2b fusion protein (SEQ ID NO.5):

[0261] The nucleotide sequence of the YQQILTSMPSRNVIQISNDLENLRDLLHVL LEP(61-90)-Lam2b fusion protein (SEQ ID NO.8):

[0262] ttcattcctgggctccaccccatcctgaccttatccaagatggaccagacactggcagtctaccaacagatcctcaccagtatgccttcc

[0263] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. RNAi, characterized in that, The RNAi enhances leptin sensitivity by inhibiting the expression of at least one of the SOCS3, PIAS3, and PTEN genes. Preferably, the RNAi includes one or more of dsRNA, siRNA, shRNA, and miRNA; Preferably, the RNAi inhibits the expression of SOCS3, PIAS3, and PTEN genes.

2. The RNAi according to claim 1, characterized in that, The RNAi is a miRNA, and the nucleotide sequence of the miRNA is shown in any one of SEQ ID No. 1 to 2.

3. Extracellular vesicles, characterized in that, The extracellular vesicles comprise the RNAi as described in any one of claims 1 to 2; Preferably, the extracellular vesicles are one or more of exosomes, apoptotic bodies, and microvesicles.

4. The extracellular vesicle according to claim 3, characterized in that, The extracellular vesicles are exosomes; Preferably, the exosomes load the RNAi in vitro; Preferably, the exosomes are adipose exosomes; More preferably, the exosomes are secreted by 3T3-L1 cells.

5. The extracellular vesicle according to claim 3 or 4, characterized in that, The exosomes were derived from cells overexpressing SIRPα and MCAM; And / or, the extracellular vesicles are derived from cells expressing a fusion protein of a targeting peptide and a leptin peptide that targets leptin receptor neurons; Preferably, the amino acid sequence of SIRPα is shown in SEQ ID No. 3; Preferably, the amino acid sequence of MCAM is shown in SEQ ID No. 4; Preferably, the targeting peptide targeting leptin receptor neurons is the LEP(61-90)-Lam2b fusion protein; More preferably, the amino acid sequence of the LEP(61-90)-Lam2b fusion protein is shown in SEQ ID No.

5.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the RNAi of claim 1 or the extracellular vesicles of any one of claims 3 to 5, and a pharmaceutically acceptable carrier and / or excipient.

7. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is formulated into a dosage form that can be administered via intranasal, intravenous, intramuscular, subcutaneous, sublingual, oral, transdermal, local, or intracerebral administration. Alternatively, the pharmaceutical composition may further include a second drug for treating obesity; Preferably, the second drug includes leptin.

8. A method for preparing extracellular vesicles according to any one of claims 3 to 5, characterized in that, The method includes the following steps: (1) Express the fusion protein of targeting peptide and leptin peptide targeting leptin receptor neurons in adipocytes, while overexpressing SIRPα and MCAM; (2) The RNAi is loaded into exosomes secreted by the adipocytes; (3) The exosomes were separated and collected.

9. The method according to claim 8, characterized in that, The adipocytes in step (1) are 3T3-L1 cells; And / or, step (1) involves introducing the overexpressed SIRPα and MCAM genes into the adipocytes via lentivirus; And / or, step (2) involves loading RNAi into exosomes secreted by the adipocytes via lentivirus; And / or, the RNAi is a miRNA, and the nucleotide sequences of the miRNA are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; Preferably, the amino acid sequence of SIRPα is shown in SEQ ID No. 3; Preferably, the amino acid sequence of MCAM is shown in SEQ ID No. 4; Preferably, the fusion protein of the targeting peptide and leptin peptide targeting leptin receptor neurons is a LEP(61-90)-Lam2b fusion protein; More preferably, the amino acid sequence of the LEP(61-90)-Lam2b fusion protein is shown in SEQ ID No.

5.

10. The use of the RNAi according to claim 1, the extracellular vesicles according to any one of claims 3 to 5, and the pharmaceutical composition according to any one of claims 6 to 7 in the preparation of a medicament for treating obesity.

11. The application according to claim 10, characterized in that, The medication for treating obesity includes any one of the following: a. Medications for reducing weight; b. Medications that reduce body fat percentage; c. Medications that reduce food intake; d. Drugs that increase energy metabolism; e. Drugs that activate pSTAT3; And f. Drugs that increase the sensitivity of the central nervous system to leptin.

12. A method for treating obesity, characterized in that, The method includes administering the miRNA of claim 1, the extracellular vesicles of any one of claims 3 to 5, or the pharmaceutical composition of any one of claims 6 to 7 to an obese patient.

13. The method according to claim 12, characterized in that, The treatment of obesity includes one or more of the following indicators: reducing total weight, reducing body mass index, reducing waist circumference, and reducing total fat.

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