Targeted central nervous system engineered exosome capable of efficiently enriching RNAi (Ribonucleic Acid Interference) as well as preparation method and application of engineered exosome

Abstract

The invention relates to a targeted central nervous system engineered exosome capable of efficiently enriching RNAi as well as a preparation method and application of the targeted central nervous system engineered exosome. The invention particularly relates to a sorting signal for regulating and controlling RNAi to be enriched into the exosome, and the RNAi enriched into the exosome through the sorting signal can be effectively combined with beta-amyloid protein mRNA expressed in a central nervous system after reaching a cortex and a hippocampal region. The invention also relates to a chimera comprising the sorting signal and the RNAi, and an exosome enriched with the RNAi. The exosome can efficiently target a central nervous system through overexpressed SIRP alpha and MCAM, and the contained siRNA can inhibit the production of beta-amyloid protein mRNA.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to engineered exosomes that efficiently enrich RNAi and target the central nervous system, their preparation methods, and applications. Background Technology

[0002] Currently, exosomes are widely developed and researched as a promising drug delivery carrier. As natural nanovesicles, exosomes have significant advantages over other traditional drug delivery systems: First, exosomes exhibit good biocompatibility, low immunogenicity, and low toxicity, avoiding the problems associated with synthetic nanocarriers (such as polyethylene glycol-modified nanoparticles) that can induce immune responses after repeated administration, leading to rapid clearance in vivo and adverse events such as allergic reactions. Second, exosomes are lipid vesicles, capable of effectively crossing major biological barriers, including the cell membrane and blood-brain barrier. This is the most important advantage of using exosomes for delivering RNAi drugs in central nervous system diseases. However, exosome-based brain-targeting RNAi drug delivery technology still faces many significant challenges, including low drug loading efficiency, low central nervous system targeting efficiency, and safety risks.

[0003] Patients typically exhibit cognitive impairments such as memory loss, confusion, and impaired judgment, which consequently affect their daily living abilities and have become a leading cause of death among the elderly. While some medications on the market can alleviate symptoms, such as cholinesterase inhibitors (e.g., donepezil, rivastigmine) and NMDA receptor antagonists (e.g., memantine), these drugs cannot cure the disease and only provide temporary symptom relief. Slowing the progression of Alzheimer's disease has become a serious public health challenge.

[0004] β-amyloid protein is closely related to Alzheimer's disease; its aggregation and deposition are considered one of the core pathological features of Alzheimer's disease, and inhibiting β-amyloid protein production is a key target for slowing the progression of dementia. Currently, strategies for inhibiting β-amyloid protein production involve various mechanisms and methods, including enzyme inhibition, gene therapy, immunotherapy, and dietary intervention. Although some strategies have shown promise in laboratory and clinical studies, the effectiveness and safety of these methods still require further refinement.

[0005] Recent studies have found that targeting BACE1 (siBACE) using RNA interference (RNAi) technology can effectively reduce the expression of β-amyloid protein. This strategy has shown good results in animal models, but issues such as effective siBACE delivery and safety still need to be addressed in its application, especially the lack of methods for preparing exosomes that efficiently inhibit β-amyloid protein production.

[0006] Therefore, it is urgent to effectively deliver siBACE to the central nervous system, increase the efficiency of exosome delivery systems in targeting the central nervous system, increase the effective working concentration of siBACE, degrade β-amyloid protein or inhibit its production, thereby delaying the progression of Alzheimer's disease, under the premise of increasing the efficiency of exosome delivery systems in targeting the central nervous system. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for the mass production of engineered exosomes capable of targeting and delivering siBACE1 to the central nervous system. This method produces engineered exosomes that efficiently target and deliver siBACE1 to the central nervous system, and these exosomes effectively inhibit the formation of β-amyloid protein deposits in the hippocampus and cortex.

[0008] Based on this, a first aspect of this application is to provide a sorting signal for efficiently enriching RNAi, the sorting signal consisting of a first portion located at the 5' end of the RNAi and a second portion located at the 3' end of the RNAi, for promoting the packaging of RNAi into extracellular vesicles, wherein the nucleotide sequence of the first portion is as shown in SEQ ID No. 1, and the nucleotide sequence of the second portion is as shown in SEQ ID No. 2.

[0009] The sorting signal is a nucleotide sequence that can be specifically recognized by sorting proteins on exosomes, thereby regulating the selective entry of RNAi into exosomes. When it chimerates with RNAi, it promotes RNAi loading into exosomes, allowing RNAi to accumulate there. The sorting signal can also enhance regulation of RNAi loading into exosomes by cleaving the RNAi fragment, releasing the RNAi from the chimera. Once the exosome reaches its target site, the RNAi can then inhibit mRNA expression.

[0010] The first part of the sorting signal of this application includes a nucleotide sequence having ≥75% homology with SEQ ID No.1, 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.

[0011] The second part of the sorting signal of this application includes a nucleotide sequence having ≥75% homology with SEQ ID No. 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.

[0012] 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.

[0013] 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 pathogenic gene mutations, and developing treatments for hereditary diseases.

[0014] As some embodiments of this application, the RNAi is selected from one or more of dsRNA, siRNA, shRNA, and miRNA.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] In some embodiments of this application, the RNAi is a siRNA that reduces BACE1 expression.

[0020] As some embodiments of this application, the siRNA is shown in SEQ ID No. 3.

[0021] The siRNA of this application may have a nucleotide sequence with ≥75% homology to SEQ ID No.3, 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.

[0022] A second aspect of this application is to provide an RNAi chimera comprising the sorting signal and RNAi located between a first portion and a second portion of the sorting signal.

[0023] As some embodiments of this application, RNAi chimeras are shown in SEQ ID No. 4.

[0024] The RNAi chimera of this application may have a nucleotide sequence with ≥75% homology to SEQ ID No. 4, 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.

[0025] As some embodiments of this application, the RNAi is selected from one or more of dsRNA, siRNA, shRNA, and miRNA.

[0026] In some embodiments of this application, the RNAi is a siRNA that reduces BACE1 expression.

[0027] As some embodiments of this application, the siRNA is shown in SEQ ID No. 3.

[0028] A third aspect of this application is to provide an extracellular vesicle comprising the RNAi chimera or a nucleic acid encoding the RNAi chimera.

[0029] 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.

[0030] As some embodiments of this application, the extracellular vesicles include one or more of exosomes, apoptotic bodies, and microvesicles.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In some embodiments of this application, the extracellular vesicles are exosomes.

[0035] In some embodiments of this application, the exosomes are derived from adipocytes 3T3-L1.

[0036] 3T3-L1 is a subline of 3T3 cells (Swissalbino) that has been cloned and passaged continuously. This cell line has undergone a transformation from preadipocytes to adipoid cells from a state of rapid division to confluence and contact inhibition. It can produce triglycerides, and high concentrations of serum can enhance intracellular fat accumulation.

[0037] As some embodiments of this application, the exosomes are loaded with the RNAi in vitro.

[0038] In some embodiments of this application, the exosomes are adipose exosomes.

[0039] As some embodiments of this application, the extracellular vesicles are derived from cells overexpressing SIRPα and / or MCAM.

[0040] 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.

[0041] 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.

[0042] As some embodiments of this application, the amino acid sequence of SIRPα is shown in SEQ ID No. 5.

[0043] SIRPα can have an amino acid sequence that is ≥75% homologous to SEQ ID No. 5, for example, it can be 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.

[0044] As some embodiments of this application, the amino acid sequence of MCAM is shown in SEQ ID No. 6.

[0045] MCAM can have an amino acid sequence that is ≥75% homologous to SEQ ID No. 6, for example, it can be 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.

[0046] A fourth aspect of this application is to provide a pharmaceutical composition comprising the RNAi chimera or the extracellular vesicles described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0047] 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.

[0048] As some embodiments of this application, the pharmaceutical composition further includes a second drug for treating the inhibition of β-amyloid production or a drug for treating Alzheimer's disease.

[0049] The fifth aspect of this application is to provide a method for preparing the extracellular vesicles, the method comprising the steps of: (1) overexpressing SIRPα and MCAM in adipocytes; (2) loading the RNAi chimera into exosomes secreted by the adipocytes; and (3) isolating and collecting the exosomes.

[0050] In some embodiments of this application, the adipocytes in step (1) are 3T3-L1 cells.

[0051] In some embodiments of this application, SIRPα and MCAM are overexpressed in the adipocytes via lentivirus in step (1).

[0052] As some embodiments of this application, in step (2), the RNAi chimera is loaded into the exosomes secreted by the adipocytes via lentivirus.

[0053] As some embodiments of this application, the adipocytes in step (2) express RNAi chimeras.

[0054] As some embodiments of this application, the amino acid sequence of SIRPα is shown in SEQ ID No. 5.

[0055] As some embodiments of this application, the amino acid sequence of MCAM is shown in SEQ ID No. 6.

[0056] As some embodiments of this application, the nucleotide sequence of the RNAi chimera is shown in SEQ ID No. 4.

[0057] This application provides a method for preparing engineered exosomes that target the central nervous system and inhibit the production of β-amyloid protein. The exosomes are generated from engineered 3T3-L1 cells. The engineered 3T3-L1 cells are stable cells that can secrete highly efficient exosomes targeting the central nervous system, established by overexpressing SIRPα and MCAM through lentivirus. Overexpression of SIRPα and MCAM can increase the efficiency of exosomes targeting the central nervous system. By expressing sorting signals, the sorting of siBACE fragments by exosomes is increased, achieving efficient endogenous loading of siBACE. The technical means to achieve this effect can be by introducing sorting signal sequence structures through lentivirus.

[0058] A sixth aspect of this application is to provide the use of one or more of the sorting signal, the RNAi chimera, the extracellular vesicle, and the pharmaceutical composition in the preparation of a medicament.

[0059] As some embodiments of this application, the drug is a drug that inhibits β-amyloid protein and / or treats Alzheimer's disease.

[0060] As described above, the engineered exosomes for the central nervous system that efficiently enrich RNAi, their preparation method, and their applications have the following beneficial effects:

[0061] The technology of this application uses genetic engineering methods to prepare stable cells that can continuously produce engineered exosomes that deliver siBACE1 to the central nervous system. These stable cells overexpress SIRPα and MCAM, and the overexpression of SIRPα and MCAM enables the exosomes to efficiently target the central nervous system.

[0062] This application utilizes sorting signals to regulate RNAi delivery to exosomes, efficiently loading siBACE1 into exosomes.

[0063] The exosomes enriched with RNAi in this application, when introduced into the central nervous system, have significant therapeutic and physiological effects.

[0064] The method provided in this application can efficiently and stably generate engineered exosomes that can target siBACE1 in the central nervous system. The method is simple and easy to implement, and the process can be effectively scaled up, providing a new strategy for achieving targeted inhibition of β-amyloid protein production in the central nervous system. Attached Figure Description

[0065] Figure 1 The engineered cell lines of this application stably express MCAM and SIRPα;

[0066] Figure 2 This application demonstrates that the sorting signal modulates the enrichment of siBACE1 into engineered exosomes EN-siBACE1-EVs;

[0067] Figure 3 The study demonstrated that the engineered exosomes EN-siBACE1-EVs of this application, when injected via the tail vein into Alzheimer's disease model mice, significantly increased the level of siBACE1 in the hippocampus and cortex of the mice and had a therapeutic effect.

[0068] Figure 4 The study demonstrated the biocompatibility and safety of mice treated with engineered exosomes and control exosomes, by assessing circulating inflammatory factors, detecting inflammatory factors and microglial cell activation in the hippocampus and cortex. Detailed Implementation

[0069] 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.

[0070] [Experimental Materials]

[0071] 3T3-L1 cells (ATCC; Catalog No.: CL-173)

[0072] Lentiviral (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0073] mCherry fluorescent protein (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0074] EGFP fluorescent protein (Shumi Technology Wuhan Shumi Brain Science Technology Co., Ltd.)

[0075] Puromycin (MCE; Catalog No.: HY-K1057)

[0076] Methylphenidate S (MCE Company; Product No.: HY-HY-103401)

[0077] Alzheimer's disease mouse model (Jicui Yaokang)

[0078] 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.

[0079] Example 1

[0080] 1.1 A stable cell line overexpressing SIRPα and MCAM (targeted central nervous system cell line) was constructed through viral infection and other methods.

[0081] Lentivirals overexpressed MCAM and SIRPα proteins, respectively, and carried puromycin and cyproterone S resistance genes as markers. The same 3T3-L1 cell line was transfected with these two viruses, and a stable cell line simultaneously overexpressing MCAM and SIRPα proteins was constructed after puromycin and cyproterone S drug screening, resulting in the engineered cell line used in this application.

[0082] The specific method is as follows:

[0083] 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.

[0084] 2. Construct viruses overexpressing MCAM and SIRPα, both with a titer of 10^8. The protein sequence of SIRPα is shown in SEQ ID No. 5, and the gene sequence encoding it is shown in SEQ ID No. 7. The protein sequence of MCAM is shown in SEQ ID No. 6, and the gene sequence encoding it is shown in SEQ ID No. 8.

[0085] 3. Set the MOI of the two viruses to 20 each, for a total MOI of 40.

[0086] 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.

[0087] 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.

[0088] 6. Observe the fluorescence carried by the virus after 24-48 hours.

[0089] 7. Immunoblot analysis of MCAM and SIRPα expression can be performed 48 hours later.

[0090] Table 1

[0091] 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

[0092] 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 mCherry and EGFP fluorescent proteins in the stable transgenic strain. When both fluorescence signals appear simultaneously in a single cell line, it indicates that both MCAM and SIRPα proteins are overexpressed in the same cell line. 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, and the cell lines that stably overexpress membrane proteins MCAM and SIRPα have been successfully constructed.

[0093] Example 2

[0094] Engineered 3T3-L1 cells capable of efficiently encapsulating siBACE1 were constructed. The encapsulation of siBACE1 depends on a sortable RNAi motif. The sorting signal consists of a first part located at the 5' end of siBACE1 and a second part located at the 3' end of siBACE1. The nucleotide sequence of the first part is shown in SEQ ID No. 1, and the nucleotide sequence of the second part is shown in SEQ ID No. 2.

[0095] Based on the stable cell line constructed in Example 1 that can secrete exosomes targeting the central nervous system, a 3T3-L1 stable cell line capable of efficiently loading siBACE1 into exosomes was further constructed through viral infection and other methods. Specifically, the chimera of the sorting sequence and siBACE1 described above was introduced into the stable cell line constructed in Example 1 that can secrete exosomes targeting the central nervous system. The transfection method was the same as in Example 1. The sequence of siBACE1 is shown in SEQ ID No. 3. The nucleotide sequences of the constructed sorting signal motif and the siBACE1 chimera are shown in SEQ ID No. 4. The sorting signal, after being chimeric with RNAi, promotes RNAi loading into exosomes, allowing siBACE1 to accumulate in the exosomes. The sorting signal also enhances the regulation of RNAi loading into exosomes, causing siBACE1 to be released from the chimera. Therefore, after the exosomes reach the target site, siBACE1 exerts its inhibitory effect on mRNA expression.

[0096] Subsequently, exosomes secreted by engineered 3T3-L1 cells were separated by differential centrifugation, their particle size was measured, and they were characterized by immunoblotting, transmission electron microscopy, and other methods. The content of siBACE1 in the exosomes was detected by qRT-PCR technology, and they were identified as exosomes EN-siBACE1-EVs encapsulated with siBACE1.

[0097] Exosomes without overexpression of membrane proteins MCAM and SIRPα were used, along with a chimeric sequence composed of a siRNA overexpressing in cells and siBACE1, which is present in the art. The nucleic acid sequence of the chimeric sequence is shown in SEQ ID No. 9. Control exosomes Con-siBACE1-EVs were prepared by encapsulating siBACE1. The control exosomes did not express the sorting signal of this application.

[0098] Figure 2 Image A shows a transmission electron microscopy (TEM) comparison of engineered exosomes encapsulating siBACE1 in this application with control exosomes. Figure 2 As can be seen, overexpression of MCAM and SIRP on engineered exosomes did not disrupt the exosome structure (EN-siBACE1-EVs), and the engineered exosomes maintained the same size and morphology as the control exosomes.

[0099] Detection of specific markers related to exosomes (extracellular vesicles) CD63, CD9, and TSG101 showed that the engineered exosomes EN-siBACE1-EVs of this application, like the control exosomes Con-siBACE1-EVs, are both exosomes. The detection results are shown in [see attached image]. Figure 2 B.

[0100] Figure 2 C shows a comparison of siBACE1 concentration in the engineered exosomes EN-siBACE1-EVs and the control exosomes Con-siBACE1-EVs. It can be seen that the siBACE1 content in the engineered exosomes EN-siBACE1-EVs is significantly higher than that in the control exosomes Con-siBACE1-EVs. The sorting signal of this application was used, which greatly increased the ability of exosomes to encapsulate siBACE1, enriching the encapsulated siBACE1 in the exosomes. The concentration was increased from the conventional 100 fmol / μg to more than 15000 fmol / μg, which is more than 100 times higher.

[0101] The above experimental results demonstrate that the engineered exosomes EN-siBACE1-EVs of this application successfully and efficiently enriched siBACE1.

[0102] Example 3

[0103] After injecting the engineered exosomes EN-siBACE1-EVs from Example 2 into Alzheimer's disease model mice via tail vein, the levels of siBACE1 in the hippocampus and cortex of the mice were significantly increased. The levels of siBACE1 in the cortex and hippocampus of the mice were detected using qRT-PCR, and the results are shown below. Figure 3As shown in A-B, the engineered exosomes of this application can effectively deliver siBACE1 to specific brain regions in the central nervous system, namely the hippocampus and cortex. The control exosome (vehicle) in this embodiment was prepared using cell lines overexpressing membrane proteins MCAM and SIRPα, and encapsulated siBACE1 using a chimeric sequence composed of a sequence of siRNA overexpressed in cells and siBACE1, as existing in the art. The nucleic acid sequence of the chimeric sequence is shown in SEQ ID No. 9. The control exosome (vehicle) overexpresses membrane proteins MCAM and SIRPα, but it encapsulates very little siBACE1.

[0104] After engineered exosomes efficiently delivered siBACE1 to the central cortex and hippocampus, immunoblotting analysis of BACE1 protein levels in the mouse cortex and hippocampus revealed that these engineered exosomes significantly reduced BACE1 expression in both regions. (See attached results). Figure 3 C~D.

[0105] Figure 3 E–F show that after engineered exosomes efficiently delivered siBACE1 to the central cortex and hippocampus, Aβ deposition in the cortex and hippocampus was shown in green fluorescence. Compared with control exosomes without RNAi chimeras, EN-siBACE1-EVs significantly reduced Aβ deposition in the cortex and hippocampus.

[0106] Figure 3 G-H show the latency to stage in recipient mice after tail vein injection of engineered exosomes EN-siBACE1-EVs from Example 2, from... Figure 3 As can be seen from G, after tail vein injection of engineered exosomes EN-siBACE1-EVs, the latency to reach the platform in mice gradually decreased each day thereafter. By day 5 after administration, the latency to reach the platform in mice had decreased from 50 seconds to 20 seconds. Figure 3 H showed that on day 5 after administration, the proportion of time spent in the target quadrant in mice was significantly higher than that in the control group, which together indicate that EN-siBACE1-Evs can exert a sustained therapeutic effect and improve cognition in dementia model mice.

[0107] Example 4

[0108] Mice were treated with engineered exosomes and control exosomes. Circulating inflammatory factors, hippocampal and cortical inflammatory factors, and microglial cell activation were assessed to evaluate the biocompatibility and safety of the engineered exosomes. In this embodiment, the control exosome (vehicle) was prepared using a cell line overexpressing the membrane proteins MCAM and SIRPα, and encapsulated with a chimeric sequence consisting of a sequence of siRNA overexpressed in cells and siBACE1, as described in SEQ ID No. 9. The control exosome (vehicle) overexpressed the membrane proteins MCAM and SIRPα, but encapsulated very little siBACE1.

[0109] The test results are shown below. Figure 4 A~G, from Figure 4 As can be seen, there was no significant difference in serum concentrations of peripheral blood inflammatory factors IL-6, IL-1β, and TNF-α between mice treated with engineered exosomes EN-siBACE1-Evs and mice treated with control exosomes. Figure 4 B shows that there were no significant differences in the levels of IL-6, IL-1β, and TNF-α mRNA in the cortex of mice treated with engineered exosomes EN-siBACE1-Evs and mice treated with control exosomes. Figure 4 C shows that there was no significant difference in the mRNA levels of IL-6, IL-1β, and TNF-α between the two groups in the hippocampus of mice treated with engineered exosomes EN-siBACE1-Evs and mice treated with control exosomes. Figure 4 D-4G showed that the activation status of microglia in the cortex and hippocampus of mice treated with engineered exosomes EN-siBACE1-Evs and mice treated with control exosomes did not change, indicating that engineered exosomes EN-siBACE1-Evs did not induce a central immune response, demonstrating that engineered exosomes EN-siBACE1-Evs have good safety.

[0110] Sequence List:

[0111] The first part of the sorting signal (SEQ ID No. 1):

[0112] ACACACCTGCAGGAAGAAACTTCTGGAGGAAGACAGCACGTTTGGTCTTTTGAGGCAGACGA

[0113] CTGTTACAAGTTTGC

[0114] The second part of the sorting signal (SEQ ID No. 2):

[0115] AAACTTGTAACAGTCGTCTTGCTCTAGAGGCTGTGGTCGCCGATGCCGCCAGTGCTCCAGACG

[0116] CCAGGCCGAGTGGCCCTGGTGGCCACGTCGCTGCGCCTTTCCCTTGGGGGAGGGGCAAGGC siBACE1 (SEQ ID No.3):

[0117] CTGAGTATGATAGAAG Si BACE1 chimera (SEQ ID No.4):

[0118] ACACACCTGCAGGAAGAAACTTCTGGAGGAAGACAGCACGTTTGGTCTTTTGAGGCAGACGA

[0119] CTGTTACAAGTTTGCCTGAGTATGATAGAAGAAACTTGTAACAGTCGTCTTGCTCTAGAGGCTG

[0120] TGGTCGCCGATGCCGCCAGTGCTCCAGACGCCAGGCCGAGTGGCCCTGGTGGCCACGTCGCTG

[0121] CGCCTTTCCCTTGGGGGAGGGGCAAGGC

[0122] SIRPα amino acid sequence (SEQ ID No.5):

[0123] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQ

[0124] WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVE

[0125] FKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVD

[0126] PVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAE

[0127] NQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKL

[0128] TCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLV

[0129] RIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQ

[0130] PASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

[0131] Nucleotide sequence of SIRPα (SEQ ID No.7):

[0132] TCCGGCCCGCACCCACCCCCAAGAGGGGCCTTCAGCTTTGGGGCTCAGAGGCACGACCTCCTG

[0133] GGGAGGGTTAAAAGGCAGACGCCCCCCCGCCCCCCGCGCCCCCGCGCCCCGACTCCTTCGCCG<able>

[0134] CCTCCAGCCTCTCGCCAGTGGGAAGCGGGGAGCAGCCGCGCGGCCGGAGTCCGGAGGCGAGG

[0135] GGAGGTCGGCCGCAACTTCCCCGGTCCACCTTAAGAGGACGATGTAGCCAGCTCGCAGCGCTG<00003

[0136] ACCTTAGAAAAACAAGTTTGCGCAAAGTGGAGCGGGGACCCGGCCTCTGGGCAGCCCCGGCG

[0137] GCGCTTCCAGTGCCTTCCAGCCCTCGCGGGCGGCGCAGCCGCGGCCCATGGAGCCCGCCGGCC

[0138] CGGCCCCCGGCCGCCTCGGGCCGCTGCTCTGCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAG It should be noted that there seem to be some incorrect or inconsistent tags in the original text (such as <able> which might be a formatting error). This translation is done as accurately as possible based on the provided text.

[0139] GAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCT

[0140] GGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGG

[0141] TTCAGAGGAGCTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGG

[0142] GTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACA

[0143] TCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACG

[0144] TGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGG

[0145] TATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACG

[0146] GCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCC

[0147] AGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTG

[0148] GTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAG

[0149] GGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAG

[0150] GTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTT

[0151] CTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGG

[0152] CCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGT

[0153] ATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGT

[0154] CAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCG

[0155] CTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCT

[0156] GGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTC

[0157] CACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAA

[0158] TGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGA

[0159] GCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACA

[0160] CCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCA

[0161] AGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAGTGAATGGGACCGT

[0162] GGTTTGCTCTAGCACCCATCTCTACGCGCTTTCTTGTCCCACAGGGAGCCGCCGTGATGAGCAC

[0163] AGCCAACCCAGTTCCCGGAGGGCTGGGGCGGTGCAGGCTCTGGGACCCAGGGGCCAGGGTGG

[0164] CTCTTCTCTCCCCACCCCTCCTTGGCTCTCCAGCACTTCCTGGGCAGCCACGGCCCCCTCCCCC

[0165] CACATTGCCACATACCTGGAGGCTGACGTTGCCAAACCAGCCAGGGAACCAACCTGGGAAGTG

[0166] GCCAGAACTGCCTGGGGTCCAAGAACTCTTGTGCCTCCGTCCATCACCATGTGGGTTTTGAAG

[0167] ACCCTCGACTGCCTCCCCGATGCTCCGAAGCCTGATCTTCCAGGGTGGGGAGGAGAAAATCCC

[0168] ACCTCCCCTGACCTCCACCACCTCCACCACCACCACCACCACCACCACCACCACTACCACCAC

[0169] CACCCAACTGGGGCTAGAGTGGGGAAGATTTCCCCTTTAGATCAAACTGCCCCTTCCATGGAAA

[0170] AGCTGGAAAAAAACTCTGGAACCCATATCCAGGCTTGGTGAGGTTGCTGCCAACAGTCCTGGC

[0171] CTCCCCCATCCCTAGGCTAAAGAGCCATGAGTCCTGGAGGAGGAGAGGACCCCTCCCAAAGGA

[0172] CTGGAGACAAAACCCTCTGCTTCCTTGGGTCCCTCCAAGACTCCCTGGGGCCCAACTGTGTTG

[0173] CTCCACCCGGACCCATCTCTCCCTTCTAGACCTGAGCTTGCCCCTCCAGCTAGCACTAAGCAAC

[0174] ATCTCGCTGTGGACGCCTGTAAATTACTGAGAAATGTGAAACGTGCAATCTTGAAACTGAGGTG

[0175] TTAGAAAACTTGATCTGTGGTGTTTTGTTTTGTTTTTTTTCTTAAAACAACAGCAACGTGATCTT

[0176] GGCTGTCTGTCATGTGTTGAAGTCCATGGTTGGGTCTTGTGAAGTCTGAGGTTTAACAGTTTGT

[0177] TGTCCTGGAGGGATTTTCTTACAGCGAAGACTTGAGTTCCTCCAAGTCCCAGAACCCCAAGAAT

[0178] GGGCAAGAAGGATCAGGTCAGCCACTCCCTGGAGACACAGCCTTCTGGCTGGGACTGACTTG

[0179] GCCATGTTCTCAGCTGAGCCACGCGGCTGGTAGTGCAGCCTTCTGTGACCCCGCTGTGGTAAGT

[0180] CCAGCCTGCCCAGGGCTGCTGAGGGCTGCCTCTTGACAGTGCAGTCTTATCGAGACCCAATGC

[0181] CTCAGTCTGCTCATCCGTAAAGTGGGGATAGTGAAGATGACACCCCTCCCCACCACCTCTCATA

[0182] AGCACTTTAGGAACACACAGAGGGTAGGGATAGTGGCCCTGGCCGTCTATCCTACCCCTTTAGT

[0183] GACCGCCCCCATCCCGGCTTTCTGAGCTGATCCTTGAAGAAGAAATCTTCCATTTCTGCTCTCA

[0184] AACCCTACTGGGATCAAACTGGAATAAATTGAAGACAGCCAGGGGGATGGTGCAGCTGTGAAG

[0185] CTCGGGCTGATTCCCCCTCTGTCCCAGAAGGTTGGCCAGAGGGTGTGACCCAGTTACCCTTTAA

[0186] CCCCCACCCTTCCAGTCGGGTGTGAGGGCCTGACCGGGCCCAGGGCAAGCAGATGTCGCAAG

[0187] CCCTATTTATTCAGTCTTCACTATAACTCTTAGAGTTGAGACGCTAATGTTCATGACTCCTGGCCT

[0188] TGGGATGCCCAAGGGATTTCTGGCTCAGGCTGTAAAAGTAGCTGAGCCATCCTGCCCATTCCTG

[0189] GAGGTCCTACAGGTGAAACTGCAGGAGCTCAGCATAGACCCAGCTCTCTGGGGGATGGTCACC

[0190] TGGTGATTTCAATGATGGCATCCAGGAATTAGCTGAGCCAACAGACCATGTGGACAGCTTTGGC

[0191] CAGAGCTCCCGTGTGGCATCTGGGAGCCACAGTGACCCAGCCACCTGGCTCAGGCTAGTTCCA

[0192] AATTCCAAAAGATTGGCTTGTAAACCTTCGTCTCCCTCTCTTTTACCCAGAGACAGCACATACG

[0193] TGTGCACACGCATGCACACACACATTCAGTATTTTAAAAGAATGTTTTCTTGGTGCCATTTTCAT

[0194] TTTATTTTATTTTTTAATTCTTGGAGGGGGAAATAAGGGAATAAGGCCAAGGAAGATGTATAGCT

[0195] TTAGCTTTAGCCTGGCAACCTGGAGAATCCACATACCTTGTGTATTGAACCCCAGGAAAAGGAA

[0196] GAGGTCGAACCAACCCTGCGGAAGGAGCATGGTTTCAGGAGTTTATTTTAAGACTGCTGGGAA

[0197] GGAAACAGGCCCCATTTTGTATATAGTTGCAACTTAAACTTTTTGGCTTGCAAAATATTTTTGTAA

[0198] TAAAGATTTCTGGGTAATAATGA

[0199] MCAM amino acid sequence (SEQ ID No.6):

[0200] MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVDWFSV

[0201] HKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRV

[0202] YKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNRVHIQSSQTVESS

[0203] ​​​​​​​​

[0206] KREAGGGYRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTI

[0207] SWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSNTT

[0208] TGLSTSTASPHTRANSTSTERKLPEPESRGVVIVAVIVCILVLAVLGAVLYFLYKKGKLPCRRSGKQEI

[0209] TLPPSRKSELVVEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRHMCAM nucleotide sequence (SEQ ID No.8):

[0210] ACTTGGCTCTCGCCCTCCGGCCGGGAAGCATGGGGCTTCCCAGGCTGGTCTGCGCCTTCTTGCT

[0211] CGCCGCCTGCTGCTGCTGTCCTCGCGTCGCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCC

[0212] TGAGCTGGTGGAGGTGGAAGTGGGCAGCACAGCCCTTCTGAAGTGCGGCCTCTCCCAGTCCCA

[0213] AGGCAACCTCAGCCATGTCGACTGGTTTTCTGTCCACAAGGAGAAGCGGACGCTCATCTTCCG

[0214] TGTGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGGCTCAGCCTCCAGG

[0215] ACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCATCTTCTTGTGCC

[0216] AGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGCGTCTACAAAGCTCCGGAGG

[0217] AGCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGTGAACAGTAAGGAGCCTGAGGAGGTCG

[0218] CTACCTGTGTAGGGAGGAACGGGTACCCCATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCC

[0219] TCTGAAGGAGGAGAAGAACCGGGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTT

[0220] GTACACCTTGCAGAGTATTCTGAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTA

[0221] CTGTGAGCTCAACTACCGGCTGCCCAGTGGGAACCACATGAAGGAGTCCAGGGAAGTCACCGT

[0222] CCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGGAAGTGGAGCCCGTGGGAATGCTGAAGGA

[0223] AGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCCTCCACCACACTTCAGCATCAG

[0224] CAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAACCAACGACAACGGGGTCCTG

[0225] GTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCGCTATGAATGTCAGGGCCTGGACTTGGAC

[0226] ACCATGATATCGCTGCTGAGTGAACCACAGGAACTACTGGTGAACTATGTGTCTGACGTCCGAG

[0227] TGAGTCCCGCAGCCCCTGAGAGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAG

[0228] AGTAGCCAGGACCTCGAGTTCCAGTGGCTGAGAGAAGAGACAGGCCAGGTGCTGGAAAGGGG

[0229] GCCTGTGCTTCAGTTGCATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGTC

[0230] TGTGCCCAGCATACCCGGCCTGAACCGCACACAGCTGGTCAACGTGGCCATTTTTGGCCCCCCT

[0231] TGGATGGCATTCAAGGAGAGGAAGGTGTGGGTGAAAGAGAATATGGTGTTGAATCTGTCTTGT

[0232] GAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGAACGTCAACGGCACGGCAAGTGAACA

[0233] AGACCAAGATCCACAGCGAGTCCTGAGCACCCTGAATGTCCTCGTGACCCCGGAGCTGTTGGA

[0234] GACAGGTGTTGAATGCACGGCCTCCAACGACCTGGGCAAAAACACCAGCATCCTCTTCCTGGA

[0235] GCTGGTCAATTTAACCACCCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTTCCAC

[0236] TGCCAGTCCTCATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGA

[0237] GCCGGGGCGTGGTCATCGTGGCTGTGATTGTGTGCATCCTGGTCCTGGCGGTGCTGGGCGCTGT

[0238] CCTCTATTTCCTCTATAAGAAGGGCAAGCTGCCGTGCAGGCGCTCAGGGAAGCAGGAGATCAC

[0239] GCTACCCCCGTCTCGTAAGAGCGAACTTGTAGTTGAAGTTAAGTCAGATAAGCTCCCAGAAGA

[0240] GATGGGCCTCCTGCAGGGCAGCAGCGGTGACAAGAGGGCTCCGGGAGACCAGGGAGAGAAAT

[0241] ACATCGATCTGAGGCATTAGCCCCGAATCACTTCAGCTCCCTTCCCTGCCTGGACCATTCCCAG

[0242] CTCCCTGCTCACTCTTCTCTCAGCCAAAGCCTCCAAAGGGACTAGAGAGAAGCCTCCTGCTCC

[0243] CCTCGCCTGCACACCCCCTTTCAGAGGGCCACTGGGTTAGGACCTGAGGACCTCACTTGGCCC

[0244] TGCAAGGCCCGCTTTTCAGGGACCAGTCCACCACCATCTCCTCCACGTTGAGTGAAGCTCATCC

[0245] CAAGCAAGGAGCCCCAGTCTCCCGAGCGGGTAGGAGAGTTTCTTGCAGAACGTGTTTTTTCTT

[0246] TACACACATTATGGCTGTAAATACCTGGCTCCTGCCAGCAGCTGAGCTGGGTAGCCTCTCTGAG

[0247] CTGGTTTCCTGCCCCAAAGGCTGGCTTCCACCATCCAGGTGCACCACTGAAGTGAGGACACAC

[0248] CGGAGCCAGGCGCCTGCTCATGTTGAAGTGCGCTGTTCACACCCGCTCCGGAGAGCACCCCAG

[0249] CAGCATCCAGAAGCAGCTGCAGTGTTGCTGCCACCACCCTCCTGTCTGCCTCTTCAAAGTCTCC

[0250] TGTGACATTTTTTCTTTGGTCAGAAGCCAGGAACTGGTGTCATTCCTTAAAAGATACGTGCCGG

[0251] GGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCA

[0252] CAAAGTCAGGACGAGACCATCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAA

[0253] AAAAAATTAGCTAGGCGTAGTGGTTGGCACCTATAGTCCCAGCTACTCGGAAGGCTGAAGCAG

[0254] GAGAATGGTATGAATCCAGGAGGTGGAGCTTGCAGTGAGCCGAGACCGTGCCACTGCACTCCA

[0255] GCCTGGGCAACACAGCGAGACTCCGTCTCGAGGAAAAAAAAAGAAAAGATACGTGCCTGCGG

[0256] TGAGGAAGCTGGGCGCTGTTTTCGAGTTCAGGTGAATTAGCCTCAATCCCCCGTGTTCACTTGG

[0257] CTCCCATAGCCCTCTTGATGGATCACGTAAAACTGAAAGGCAGCGGGGAGCAGACAAAGATGA

[0258] GGTCTACACTGTCCTTCATGGGGATTAAAGCTATGGTTATATTAGCACCAAACTTCTACAAACCA

[0259] AGCTCAGGGCCCCAACCCTAGAAGGGCCCAAATGAGAGAATGGTACTTAGGGATGGAAAACG

[0260] GGCCTGGCTAGAGCTTCGGGTGTGTGTGTCTGTCTGTGTGTATGCATACATATGTGTGTATATATG

[0261] GTTTTGTCAGGTGTGTAAATTTGCAAATTGTTTCCTTTATATATGTATGTATATATATATATGAAAAT

[0262] ATATATATATATGAAAAATAAAGCTTAATTGTCCCAGAAA

[0263] Chimera containing a sequence of conventionally overexpressed siRNA (SEQ ID No. 9):

[0264] CAGGTCAGTAAGACAGAATGCTGAAGAAGGCATATCAACGTTGACAGTGA GCGAC TGTAAACA

[0265] TCCTCGACTGGAAGCTGAGTATGATAGAAGCTTTTCAGTCGGATGTTTGCAGCTGCCTACTGCCT

[0266] CCAACTTCAAGGAGTAATTATCTTGTTGGCTGAAACCAATACCTTGCTTTCTCTTTGATTTATTTT

[0267] TACAAAGCTTGGGTGGAAAAGATAAATTAAACAACTTCAAAACCACAGAAGTGTTCTTCCTGT

[0268] GCATAGAATATG

[0269] 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. A sorting signal for efficient enrichment of RNAi, characterized in that, The sorting signal consists of a first portion located at the 5' end of the RNAi and a second portion located at the 3' end of the RNAi, and is used to promote the packaging of the RNAi into extracellular vesicles, wherein the nucleotide sequence of the first portion is shown in SEQ ID No. 1 and the nucleotide sequence of the second portion is shown in SEQ ID No.

2.

2. The sorting signal according to claim 1, characterized in that, The RNAi is selected from one or more of dsRNA, siRNA, shRNA, and miRNA; Preferably, the RNAi is a siRNA that reduces BACE1 expression.

3. An RNAi chimera characterized in that, The RNAi chimera includes the sorting signal as described in any one of claims 1 to 2 and the RNAi located between the first and second portions of the sorting signal; Preferably, the RNAi is selected from one or more of dsRNA, siRNA, shRNA, and miRNA; Preferably, the RNAi is a siRNA that reduces BACE1 expression; More preferably, the siRNA is as shown in SEQ ID No.

3.

4. An extracellular vesicle, characterized in that, The extracellular vesicles comprise the RNAi chimera of claim 3 or the nucleic acid encoding the RNAi chimera; Preferably, the extracellular vesicles include one or more of exosomes, apoptotic bodies, and microvesicles.

5. Extracellular vesicle according to claim 4, characterized in that, The extracellular vesicles are exosomes; And / or, the extracellular vesicles are derived from cells that overexpress SIRPα and / or MCAM; Preferably, the exosomes are adipose exosomes; Preferably, the amino acid sequence of SIRPα is shown in SEQ ID No. 5; Preferably, the amino acid sequence of MCAM is shown in SEQ ID No. 6; More preferably, the exosomes are derived from adipocytes 3T3-L1.

6. A pharmaceutical composition, characterized in that, The drug combination comprises the RNAi chimera of claim 3 or the extracellular vesicles of any one of claims 4 to 5, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition is formulated into a dosage form that can be administered via intranasal, intravenous, intramuscular, subcutaneous, sublingual, oral, transdermal, topical, or intracerebral administration. Preferably, the pharmaceutical composition further includes a second drug for treating the inhibition of β-amyloid production or a drug for treating Alzheimer's disease.

7. A method for preparing the extracellular vesicles according to claim 4 or 5, characterized in that, The method includes the following steps: (1) Overexpression of SIRPα and MCAM in adipocytes; (2) Load the RNAi chimera into exosomes secreted by the adipocytes; (3) The exosomes were separated and collected.

8. The method according to claim 9, characterized in that, The adipocytes in step (1) are 3T3-L1 cells; And / or, in step (1), SIRPα and MCAM are overexpressed in the adipocytes via lentivirus; And / or, in step (2), the RNAi chimera is loaded into the exosomes secreted by the adipocytes via lentivirus; And / or, the adipocytes in step (2) express RNAi chimeras.

9. The method according to claim 8, characterized in that, The amino acid sequence of SIRPα is shown in SEQ ID No. 5; And / or, the amino acid sequence of MCAM is shown in SEQ ID No. 6; And / or, the nucleotide sequence of the RNAi chimera is shown in SEQ ID No.

4.

10. The use of one or more of the following in the preparation of a medicament: the sorting signal according to any one of claims 1 to 2, the RNAi chimera according to claim 3, the extracellular vesicle according to any one of claims 4 to 5, and the pharmaceutical composition according to claim 6; Preferably, the drug is a drug that inhibits β-amyloid protein and / or treats Alzheimer's disease.

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