A medicament for treating huntington's disease

Abstract

The application discloses an engineered intracellular antibody, which is fused by a Nef protein sequence of HIV-1 and an intracellular antibody sequence for targeting and degrading mutant huntingtin protein, and has simple structure and is easy to artificially synthesize. The application further discloses an exosome carrying the intracellular antibody, wherein a rabies virus glycoprotein peptide segment RVG is expressed on the membrane of the exosome, so that the exosome can specifically target nerve cells, and has high stability, low immunogenicity, and high penetration, and can be injected intravenously and cut to target and efficiently remove the existing mutant huntingtin protein and its aggregates in the brain of a patient, solve the off-target problem of gene therapy, and have important significance for removing the existing mutant huntingtin protein and its aggregates in the brain of a patient with late Huntington's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug for treating Huntington's disease. Background Technology

[0002] Huntington's disease is an autosomal dominant inherited neurodegenerative disorder. Its pathogenic mechanism involves an abnormal amplification of the CAG trinucleotide repeat sequence in the Huntingtin gene (HTT) on chromosome 4, leading to an abnormal increase in the length of the polyglutamine sequence in the translated huntingtin protein. This results in a toxic mutant huntingtin protein, which produces large amounts of abnormal protein aggregates in the patient's neurons, causing neurotoxicity and neuronal death, ultimately leading to neurodegenerative changes. Currently, there is no cure for Huntington's disease. Approved medications for Huntington's disease, such as buphenazine and clonazepam, are used to alleviate symptoms. Research on fundamental treatments for Huntington's disease focuses primarily on virus-mediated gene editing therapies, such as directly targeting and knocking out the abnormal Huntingtin gene using the CRISPR-Cas system, and gene silencing therapies, such as antisense oligonucleotide technology and RNA interference technology, to block the expression of the abnormal Huntingtin gene. However, these technologies all have drawbacks. On the one hand, they cannot achieve high specificity in modifying gene expression, and the risk of off-target effects cannot be ignored. On the other hand, for patients with advanced Huntington's disease, gene therapy cannot eliminate the large number of mutated proteins and their aggregates already present in the brain. Furthermore, these therapies require administration via viral vectors, using stereotactic injection or intrathecal injection. Viral vectors may induce inflammatory responses in patients, while stereotactic injection or intrathecal injection are highly invasive methods of administration. Summary of the Invention

[0003] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a medicine for treating Huntington's disease.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides an engineered intracellular antibody, said intracellular antibody being formed by fusing the Nef protein sequence of HIV-1 with a sequence that targets and degrades a variant of the huntingtin protein.

[0006] In some embodiments of the invention, the fusion includes inserting the HIV-1 Nef protein sequence at both ends or in the middle of the sequence that targets the degradation of the mutant huntingtin protein.

[0007] In some embodiments of the present invention, the fused sequence for targeting and degrading the mutant huntingtin protein is coupled at both ends to the Nef protein sequence of HIV-1.

[0008] In some embodiments of the present invention, the Nef protein sequence of HIV-1 is its full length or a partial fragment.

[0009] In some embodiments of the present invention, the Nef protein sequence of HIV-1 is a partial fragment thereof.

[0010] In some embodiments of the present invention, the sequence encoding the targeted degradation variant huntingtin protein has the sequence shown in SEQ ID NO:5.

[0011] In some embodiments of the present invention, the intracellular antibody can be more extensively encapsulated within the cell and enter the exosome.

[0012] A second aspect of the present invention provides a nucleic acid molecule encoding an intracellular antibody of the first aspect.

[0013] In some embodiments of the present invention, the nucleic acid molecule further includes a modifying sequence.

[0014] In some embodiments of the present invention, the modified sequence includes a sequence encoding an HA tag protein.

[0015] In some embodiments of the present invention, the nucleic acid molecule is as shown in SEQ ID NO:1.

[0016] A third aspect of the present invention provides a biological material containing the intracellular antibody described in the first aspect or any of the nucleic acid molecules described in the preceding aspects, said biological material comprising:

[0017] (1) An expression cassette containing the nucleic acid molecules described above;

[0018] (2) A recombinant vector containing the nucleic acid molecules described above, or a recombinant vector containing the expression cassette described in (1);

[0019] (3) Recombinant cells containing the nucleic acid molecules described above, or recombinant cells containing the expression cassette described in (1), or recombinant cells containing the recombinant vector described in (2), or recombinant cells containing the intracellular antibodies described above.

[0020] A fourth aspect of the present invention provides a method for preparing exosomes, the method comprising the following steps: constructing a plasmid based on the nucleic acid molecule described above and / or the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane; transfecting the plasmid into cells; inducing expression; and collecting the exosomes.

[0021] In some embodiments of the present invention, the steps are as follows: constructing a plasmid based on the nucleic acid molecules described above, transfecting the plasmid into cells, expressing the plasmid, and collecting exosomes.

[0022] In some embodiments of the present invention, the steps are as follows: constructing plasmids based on the nucleic acid molecules described above and the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane, transfecting the two plasmids into cells simultaneously, inducing expression, and collecting exosomes.

[0023] In some embodiments of the present invention, the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane is its full length or a fragment thereof.

[0024] In some embodiments of the present invention, the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane is a fragment thereof.

[0025] In some embodiments of the present invention, the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane further includes a modifying sequence.

[0026] In some embodiments of the present invention, the modified sequence is a sequence encoding the tag protein FLAG.

[0027] In some embodiments of the present invention, the sequence encoding the rabies virus glycoprotein peptide RVG that can be expressed on the exosome membrane is shown in SEQ ID NO:2.

[0028] In some embodiments of the present invention, the sequence plasmid vector encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane comprises pcDNA3.1, pKJE7, and pGro7.

[0029] In some embodiments of the present invention, the plasmid vector encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane is pcDNA3.1.

[0030] In some embodiments of the present invention, the nucleic acid molecule constructing plasmid vector based on the above aspects is the PRK5 plasmid.

[0031] In some embodiments of the present invention, the cells comprise HEK 293T cells and mesenchymal stem cells.

[0032] In some embodiments of the present invention, the cells are HEK 293T cells.

[0033] In some embodiments of the present invention, the method for collecting exosomes includes ultrafiltration, gradient centrifugation, and magnetic bead immunoassay.

[0034] In some embodiments of the present invention, the method for collecting exosomes is gradient centrifugation.

[0035] In some embodiments of the present invention, the centrifugal force used in the gradient centrifugation method is 500×g to 124000×g.

[0036] A fifth aspect of the present invention provides exosomes prepared according to the above aspects, the exosomes comprising the intracellular antibody described in the first aspect.

[0037] In some embodiments of the present invention, the membrane of the exosome has the rabies virus glycoprotein peptide RVG.

[0038] A sixth aspect of the present invention provides the use of the intracellular antibodies, nucleic acid molecules, biomaterials or exosomes described above in the preparation of a medicament for treating Huntington's disease.

[0039] In some embodiments of the present invention, the administration method of the drug includes intravenous injection or intravenous infusion.

[0040] In some embodiments of the invention, the drug also contains other pharmaceutically acceptable excipients.

[0041] In some embodiments of the present invention, the adjuvants include pH adjusters, isotonic adjusters, stabilizers, and buffers.

[0042] The beneficial effects of this invention are:

[0043] 1. The present invention provides an intracellular antibody, wherein the intracellular antibody is formed by fusing the Nef protein sequence of HIV-1 and the sequence of targeted degradation mutant huntingtin protein, and can be more encapsulated in the cell and enter the exosome. The intracellular antibody has a simple structure, is easy to synthesize artificially, and has a low cost.

[0044] 2. The present invention provides an exosome containing an intracellular antibody that targets and degrades mutant huntingtin protein, and expressing the rabies virus glycoprotein peptide RVG on its membrane. The exosome exhibits good stability, low immunogenicity, and high permeability, allowing it to cross the blood-brain barrier via intravenous injection to reach the designated site, avoiding the inflammatory reactions and trauma caused by brain-targeted injection or intrathecal injection. Furthermore, the exosome possesses good targeting ability, directly clearing mutant huntingtin protein and its aggregates already present in the patient's brain, thus solving the off-target problem of gene therapy. This is of great significance for effectively clearing mutant huntingtin protein and its aggregates in the brains of patients with advanced Huntington's disease. Attached Figure Description

[0045] Figure 1This section compares the results of exosomes prepared from cells transfected with SM3 plasmid and Nef-SM3 plasmid in Example 3. HA represents the tag proteins carried by Nef-SM3 and SM3; Vinculin is a cytoskeletal protein used as an internal control for loading amount; Calnexin is an endoplasmic reticulum membrane marker; and CD63 is an exosome membrane marker. A shows the results of Western blotting (WB) experiments on cell lysates and purified exosomes; WT represents exosomes generated by 293T cells without plasmid transfection, used as a negative control; B shows the quantitative analysis of SM3 protein levels in exosomes from A (n=4), with significance analysis performed using a two-tailed unpaired t-test, *p<0.05.

[0046] Figure 2 The exosomes prepared in Example 4 were tested. MOCK represents HEK293T cells without any plasmid transfection as the control group; S-SM3 represents exosomes simultaneously expressing Nef-Scramble and RVG-LAMP2b; N-SM3 represents exosomes simultaneously expressing Nef-SM3 and RVG-LAMP2b; A shows the results of Western blotting experiments on cell lysates, cell culture supernatant, and purified exosomes; HA represents the tag proteins carried by Nef-scramble and Nef-SM3; FLAG represents the tag protein of RVG-LAMP2b; Calnexin represents the endoplasmic reticulum membrane marker; CD9 and CD63 represent exosome membrane markers; TSG101 represents the exosome content marker; B shows the transmission electron microscopy results of the prepared N-SM3 exosomes, with a scale bar of 1 μm at 7000× and 500 nm at 20000×; C shows the results of nanoparticle tracking analysis (NTA) of the prepared N-SM3 exosomes.

[0047] Figure 3 The image shows the results of an in vitro cell uptake experiment of N-SM3 exosomes prepared in Example 4 with fluorescent labeling. Green fluorescence represents exosomes labeled with the fluorescent dye PKH67, and blue represents cell nuclei labeled with the fluorescent dye DAPI. Scale bar: 30 μm.

[0048] Figure 4 The figure shows the results of an in vivo brain cell uptake experiment of N-SM3 exosomes prepared in Example 4, which are fluorescently labeled. In the figure, red fluorescence represents exosomes labeled with the fluorescent dye Dil, blue represents cell nuclei labeled with the fluorescent dye DAPI, and green represents neurons labeled with NeuN antibody. Scale bar: 15 μm.

[0049] Figure 5Figure A shows the results of detecting the level and number of mutant huntingtin protein (mHTT) aggregates in Huntington's disease model mice after intravenous injection of exosomes. WT mice were used as controls. A shows the detection of mHTT levels and aggregates in the striatum of Huntington's disease model mice after treatment with S-SM3 and N-SM3 exosomes prepared in Example 4 using mEM48 antibody and 1C2 antibody; B shows the quantitative analysis of the mHTT aggregate level detection results using mEM48 antibody in A (n=4), with significance analysis performed using a two-tailed unpaired t test, *p<0.05; C shows the quantitative analysis of the mHTT level detection results using 1C2 antibody in A (n=4), with significance analysis performed using a two-tailed unpaired t test, *p<0.05.

[0050] Figure 6 The images show the levels and aggregates of mutant huntingtin protein (mHTT) in a Huntington's disease model mouse after intravenous injection of exosomes. WT mice were used as a control, and CAG140Q KI was used to represent a mouse model with the human HTT gene containing 140 glutamine repeat sequences knocked in. Image A shows the immunohistochemical staining results of frozen sections of striatum tissue from Huntington's disease model mice treated with mEM48 antibody-treated with S-SM3 and N-SM3 exosomes. The black dotted structures in the image represent mHTT aggregates. The left image is a normal photograph, and the right image is a magnified view. Scale bar: 40 μm. Image B shows the quantitative analysis of the number of mHTT aggregates in image A (n=3), analyzed using one-way ANOVA with Tukey's multiple comparisons test. *p<0.05, **p<0.01. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0052] Example 1: Establishment of a method for preparing exosomes

[0053] (1) Design the target sequence and clone it into a plasmid vector to drive its expression.

[0054] (2) Transfect HEK 293T cells with plasmids expressing intracellular antibodies. After 24 hours of transfection, replace the culture medium with DMEM cell culture medium without exosomes and continue culturing for another 24 hours before collecting the culture medium.

[0055] (3) HEK 293T cell exosomes were collected using a gradient centrifugation method. The specific steps included: centrifuging at 500×g for 10 minutes at low temperature and collecting the supernatant; then centrifuging the supernatant at 2000×g for 10 minutes at low temperature and collecting the supernatant; then centrifuging the supernatant at 10000×g for 30 minutes at low temperature and collecting the supernatant. The collected supernatant was transferred to a dedicated ultracentrifuge tube and centrifuged at 124000×g at 4℃ for 135 minutes. After centrifugation, the supernatant was aspirated, and the precipitate was resuspended in sterile PBS to obtain the purified target exosomes. All centrifugation operations were performed in a clean environment.

[0056] Example 2: Construction of Nef-SM3 plasmid, RVG-LAMP2b plasmid, SM3 plasmid, and Nef-Scramble plasmid

[0057] Methods for preparing Nef-SM3 plasmid, RVG-LAMP2b plasmid, SM3 plasmid, and Nef-Scramble plasmid, wherein the RVG-LAMP2b plasmid is a commercially available pcDNA3.1 plasmid (from Jinan University) carrying sequence fragments encoding RVG (rabies virus glycoprotein) and LAMP2b (exosome-associated membrane glycoprotein 2b), and also carrying a FLAG-tagged protein sequence; the SM3 plasmid carries the sequence encoded by the SEQ ID shown in the table below. NO:5 shows a commercially available PRK5 plasmid (from Jinan University) encoding an intracellular antibody sequence capable of targeting and degrading the variant huntingtin protein; the Nef-SM3 plasmid is a commercially available PRK5 plasmid (from Jinan University) carrying a fragment encoding the HIV-1 Nef protein sequence and the aforementioned intracellular antibody sequence capable of targeting and degrading the variant huntingtin protein; the Nef-Scramble plasmid is a commercially available PRK5 plasmid (from Jinan University) carrying a fragment encoding the HIV-1 Nef protein sequence and a scramble sequence that expresses the intracellular antibody sequence targeting and degrading the variant huntingtin protein in a disordered manner. In other words, the scramble sequence is a segment with the same base ratio as the intracellular antibody sequence targeting and degrading the variant huntingtin protein, but expresses a non-functional protein, serving as a comparison for subsequent experiments. The SM3, Nef-SM3, and Nef-Scramble plasmids all carry two HA-tagged protein sequences. The inserted fragments of the Nef-SM3 plasmid, RVG-LAMP2b plasmid, SM3 plasmid, and Nef-Scramble plasmid are shown in the table below.

[0058] Plasmid construction includes the following steps:

[0059] The insert fragments encoding the Nef-SM3 plasmid, SM3 plasmid, and Nef-Scramble plasmid were designed and chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the PRK5 plasmid vector to drive their expression. The RVG-LAMP2b sequence was synthesized and cloned into the pcDNA3.1 plasmid for expression.

[0060]

[0061]

[0062] Note: In the insert fragment of the Nef-SM3 plasmid, the bolded sequence is the HIV Nef sequence, and the underlined sequence is the sequence that targets and degrades the mutant huntingtin protein; in the insert fragment of the RVG-LAMP2b plasmid, the bolded sequence is the LAMP2b sequence, and the underlined sequence is the RVG sequence; in the insert fragment of the Nef-Scramble plasmid, the bolded sequence is the Nef sequence, and the underlined sequence is the Scramble sequence.

[0063] Example 3: Comparison of exosome preparation using SM3 plasmid and Nef-SM3 plasmid

[0064] Based on the exosome preparation steps in Example 1, exosomes were prepared using the Nef-SM3 plasmid and SM3 plasmid obtained in Example 2, and wild-type HEK 293T cells without transfection with any plasmids were set as the WT control group.

[0065] Meanwhile, RIPA lysis buffer (Beyotime Biotechnology) was used to extract cell lysis buffer from transfected and cultured cells. The specific steps were as follows: centrifugation was used to collect cell pellet, an appropriate amount of RIPA lysis buffer containing protease inhibitor was added to the cell pellet, the cells were resuspended by pipetting, the ultrasonic cell disruptor was adjusted to 100W power for ultrasonic disruption, and the cells were reacted on ice for 30 minutes. The lysis buffer was then collected for testing.

[0066] The prepared exosomes and cell lysates were analyzed by Western blotting, and the results are as follows: Figure 1 As shown in Figure A, a large amount of Nef-SM3 and SM3 intracellular antibodies were generated in the cell lysate. However, in the purified exosomes, only a small amount of SM3 intracellular antibody appeared, while most of the Nef-SM3 intracellular antibody entered the exosomes. Figure B shows that the content of Nef-SM3 intracellular antibody in exosomes was significantly higher than that of SM3 peptide. Therefore, Nef-SM3 intracellular antibody has a better ability to be encapsulated and enter exosomes than SM3 intracellular antibody.

[0067] Example 4: Preparation and Validation of S-SM3 and N-SM3 Exosomes

[0068] Two experimental groups were set up: S-SM3, containing Nef-Scramble plasmid and RVG-LAMP2b plasmid prepared in Example 2; and N-SM3, containing Nef-SM3 plasmid and RVG-LAMP2b plasmid prepared in Example 2. The plasmids of the experimental groups were transfected into HEK 293T cells according to the experimental steps of Example 1 to prepare exosomes, and HEK293T cells without any plasmid transfection were set up as the control group MOCK.

[0069] Cell culture supernatant (cell culture medium before purification), purified exosomes of each group, and cell lysates obtained using the method for extracting cell lysates in Example 3 were subjected to Western blotting and nanoparticle size detection. MOCK exosomes and N-SM3 exosomes were detected by transmission electron microscopy.

[0070] Experimental results are as follows Figure 2 As shown in Figure A, large amounts of FLAG-labeled RVG-LAMP2b protein, HA-labeled Nef-SM3 intracellular antibody, and Nef-Scramble protein of the same size as Nef-SM3 were produced in the cell lysate. Figure 2 In Figure A (corresponding band to Nef-SM3-HA), the purified exosomes showed detectable exosome markers such as CD9, CD63, and TSG101, but the intracellular membrane component marker Calnexin was not detected. Simultaneously, exosomes containing FLAG-labeled RVG-LAMP2b protein, as well as HA-labeled Nef-SM3 and Nef-Scramble proteins, were detected. Transmission electron microscopy (B) images showed the vesicle structure and phospholipid bilayer structure of the exosomes. Figure C showed that the exosome particles in the sample had a uniform diameter, mainly around 110 nm. In summary, this embodiment successfully constructed exosomes containing both Nef-SM3 intracellular antibody and RVG-LAMP2b protein, and exosomes containing both Nef-Scramble protein and RVG-LAMP2b protein.

[0071] Example 5: In vitro cell absorption experiment of exosome drugs

[0072] The N-SM3 exosomes prepared in Example 4 were subjected to in vitro HEK 293T cell uptake assay. The control group consisted of HEK 293T cells incubated with PBS.

[0073] The specific experimental steps are as follows.

[0074] (1) Quantification of exosome protein: The amount of exosome protein was determined using a commercially available BCA (Bicinchoninic Acid) protein concentration assay kit (according to the instructions for use).

[0075] (2) Preparation of dye working solution: Dilute the fluorescent dye PKH67 (Merck Life Sciences) storage diluent 10 times with universal membrane labeling diluent C (Merck Life Sciences) to prepare a dye working solution with a concentration of 100 μM.

[0076] (3) Exosome staining: Add the dye working solution to the exosomes according to the instructions (10-200 μg of exosome protein to 50 μL of staining working solution). After adding the dye working solution, mix by vortexing for 1 min, then incubate for 10 min. Add 10 mL of 1×PBS to the incubated exosome-dye complex and mix well. Centrifuge at 124000×g, 4℃ for 135 min to extract the exosomes again to remove excess dye. Finally, resuspend the precipitate in 1×PBS. The precipitate is the fluorescently labeled exosome.

[0077] (4) Cell incubation: 50 μL of fluorescently labeled exosomes were added to the cell culture medium and co-cultured with the target cells for 42 hours. After the culture was completed, the culture medium was removed, and DAPI dye diluted 1:1000 with PBS was added. After incubation at room temperature for 5 minutes, the dye was removed and the cells were washed 3 times with PBS. The fluorescence enrichment was then observed under a confocal microscope.

[0078] Experimental results are as follows Figure 3 As shown, exosomes labeled with the green fluorescent dye PKH67 appear near the cell nuclei labeled with the blue fluorescent dye DAPI, indicating that exosomes can be absorbed into cells in vitro.

[0079] Example 6: In vivo cellular absorption experiment of exosome drugs

[0080] In vivo cell uptake of the N-SM3 exosomes prepared in Example 4 was measured. The control group consisted of C57 mice injected with PBS. The mice were purchased from Zhaoqing Ruisheng Biotechnology Co., Ltd. and housed in a standardized barrier environment with a 12-hour light / dark cycle at the Experimental Animal Management Center of Jinan University. All animal experimental procedures and husbandry management strictly followed the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals," and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Jinan University (Approval No.: IACUC20221117-03).

[0081] The specific steps are as follows.

[0082] (1) Based on the experimental steps in Example 5, the exosomes were stained with the fluorescent dye Dil (Thermo Fisher Scientific);

[0083] (2) 50 μL of the exosomes obtained in step (1) were injected into the retro-orbital venous plexus of mice. (4.9 × 10⁻⁶) 11Particles / mL;

[0084] (3) 24 hours after injection, mice were anesthetized with isoflurane, and 0.9% saline was perfused through the heart. Then the brain was dissected, and the dissected brain was fixed with 10 mL of 4% paraformaldehyde for 24 hours. Then it was dehydrated with 30% sucrose at 4°C for 48 hours.

[0085] (4) After embedding the dehydrated brain in (3) with tissue cryoprotectant (OCT), the brain was cut into 20μm slices and attached to a glass slide using a cryostat (Leica CM1950);

[0086] (5) Soak the brain slices on the slide in PBS containing 0.3% Triton X-100 for 1 hour. After removing the liquid, draw a circular hydrophobic circle around the brain tissue using an immunohistochemical pen.

[0087] (6) Add blocking solution (PBS solution containing 3% BSA and 2% commercially available normal goat serum) and incubate at room temperature for 1 hour;

[0088] (7) Remove the blocking solution, add NeuN antibody primary antibody solution diluted 1:1000 with the blocking solution (purchased from abcam, ab177487), and incubate overnight in a 4°C refrigerator;

[0089] (8) Remove the primary antibody solution, wash the tissue three times with PBS, and then add fluorescent secondary antibody solution diluted 1:1000 with blocking buffer (purchased from Invitrogen, A-11008), and incubate at room temperature in the dark for 1 hour.

[0090] (9) Remove the fluorescent secondary antibody solution, wash 3 times with PBS, then add DAPI solution diluted 1:1000 with PBS, and incubate at room temperature in the dark for 5 minutes.

[0091] (10) Remove the DAPI solution, wash 3 times with PBS, and then cover with a coverslip;

[0092] (11) Observe the fluorescence enrichment under a confocal microscope.

[0093] Experimental results are as follows Figure 4 As shown, exosomes labeled with the red fluorescent dye Dil appear near the nucleus (labeled with the blue fluorescent dye DAPI) of neuronal cells (labeled with green fluorescence by NeuN antibody), indicating that exosomes bind to mouse brain cells in vivo and are absorbed into the cells.

[0094] Example 7: Immunoblot experiment on the effect of exosomal drugs on mHTT levels and aggregate clearance in the brain of Huntington's disease model rats.

[0095] The mHTT levels and aggregate clearance effects of S-SM3 and N-SM3 exosomes from Example 4 in the brains of Huntington's disease model mice were tested using mEM48 and 1C2 antibodies, with WT mice as controls. The mEM48 antibody was an anti-mutant huntingtin protein antibody (anti-HTT antibody, MAB5374, Millipore); the 1C2 antibody was an anti-polyglutamine antibody (MAB1574, Millipore).

[0096] The HTT gene knock-in (140Q) mouse model used in this embodiment was derived from the Jackson Laboratory (strain number #027409). The experimental animals were housed in a standardized barrier environment at the Laboratory Animal Management Center of Jinan University, with a 12-hour light / dark cycle. All animal experimental procedures and husbandry management strictly followed the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals," and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Jinan University (Approval No.: IACUC20221117-03).

[0097] The specific experimental steps are as follows.

[0098] (1) The S-SM3 and N-SM3 exosomes prepared in Example 4 were administered to Huntington's disease model mice via suborbital vein injection. Each mouse was injected with 50 μL of the corresponding exosomes daily, at a concentration of 4.9 × 10⁻⁶. 11 The dose was administered at a concentration of granules / mL for 7 consecutive days. After 7 days of injection, a 3-day waiting period was followed by an evaluation of the treatment effect.

[0099] (2) Anesthetize the mice in step (1) with isoflurane, perfuse 0.9% saline through the heart, and then dissect the brain.

[0100] (3) The mouse brain tissue obtained in (2) was ground using a Luka Grinding instrument (LUKYM-II) and then placed in a cold container containing Halt. TM Protease inhibitor mixture (Thermo Scientific), 50 mmol·L -1 NaF and PMSF were lysed in RIPA buffer (50 mm Tris pH 8.0, 150 mm NaCl, 1 mm EDTA pH 8.0, 1 mm EGTA pH 8.0, 0.1% SDS, 0.5% DOC and 1% Triton X-100).

[0101] (4) The tissue lysate was shaken and incubated at 4°C for 30 min, then the sample was sonicated and centrifuged at 12000 rpm for 10 min. The supernatant was then used for subsequent experiments.

[0102] (5) Load the supernatant from (4) onto an SDS-PAGE gel and transfer it to a nitrocellulose (NC) membrane. Block the membrane with 5% milk / TBST (50 mm Tris-HCl, pH 7.4, containing 20 mm Tween 20) for 1 hour at room temperature. Dilute the primary antibody (purchased from Merck Biotechnology) in 3% BSA / TBST and incubate it with the NC membrane overnight at 4°C.

[0103] (6) Take the overnight NC membrane from (5) and wash it three times in TBST (10 minutes each time), then incubate it with the second antibody (purchased from Boster) conjugated with horseradish peroxidase (HRP) at room temperature in 5% milk / TBST for 1 hour.

[0104] (7) Subsequently, ECL was used for development and Western blot images were obtained using ChemiScop 6000 (CLiNqinxiang). The images were quantified using the optical density quantification method and analyzed using ImageJ software.

[0105] Experimental results are as follows Figure 5 As shown, compared to S-SM3 exosomes which lack clearance ability, N-SM3 exosomes exhibited higher mHTT levels and aggregates in mice with Huntington's disease model treated with drugs. Figure 5 The clearance effect of A) was observed, and this clearance effect was significant for mHTT levels and aggregates. Figure 5 (B and C in the middle).

[0106] Example 8: Immunohistochemical staining experiment on the effect of exosomal drugs on mHTT levels and aggregate clearance in the brain of Huntington's disease model rats.

[0107] Immunohistochemical staining experiments were performed using mEM48 antibody to investigate the mHTT levels and aggregate clearance effects of S-SM3 and N-SM3 exosomes from Example 4 in the brains of Huntington's disease model mice, with WT mice as controls.

[0108] The specific steps are as follows.

[0109] (1) Based on steps (1) and (2) of Example 7, the brains of Huntington's disease model mice treated with injection of S-SM3 and N-SM3 exosomes were obtained;

[0110] (2) The dissected brain was fixed with 10 mL of 4% paraformaldehyde for 24 hours, and then dehydrated with 30% sucrose at 4°C for 48 hours. After dehydration, the brain was embedded with tissue cryoprotectant (OCT) and then cut into 20 μm sections using a cryostat (Leica CM1950).

[0111] (3) Immerse the tissue sections obtained in (2) in 4% PFA pre-cooled to 4°C for 10 min for fixation;

[0112] (4) Remove the fixative and wash with PBS 3 times, 10 min each time;

[0113] (5) Add 3% H2O2 to the tissue section to remove background catalase and reduce background. After 10 min, remove the liquid and add new 3% H2O2. Repeat this several times until no bubbles are generated on the tissue section. Wash with PBS 3 times, 10 min each time.

[0114] (6) Immerse the tissue sections in sodium citrate (pH 6.0) antigen retrieval solution and heat at 98°C for 10 min;

[0115] (7) Remove the tissue sections, wash them 3 times with PBS for 10 minutes each time, and draw a hydrophobic circle around the tissue sections on the slide using an immunohistochemical pen.

[0116] (8) Add the membrane blocking solution (3% BSA + 2% commercially available normal goat serum + 0.3% Triton X-100in PBS) and block at room temperature for 1 h;

[0117] (9) Remove the blocking solution, add the primary antibody solution diluted with the blocking solution as needed, and incubate overnight at 4°C;

[0118] (10) Take out the primary antibody solution and wash it with PBS 3 times, 10 min each time;

[0119] (11) Add secondary antibody (abcam Biotinylated Goat anti-Mouse & Rabbit IgG (H+L)), incubate at room temperature for 10 min, wash with PBS 4 times, 5 min each time;

[0120] (12) Add triple antibody (abcam Streptavidin HRP), incubate at room temperature for 10 min, wash with PBS 4 times, 5 min each time;

[0121] (13) Mix DAB Substrate and DAB Chromogen in the abcam DAB Substrate Kit at a ratio of 50:1, then add the mixture to the tissue section and incubate at room temperature for 1-5 min until a specific signal can be observed without a dark background. Add PBS to stop the reaction and wash with PBS 4 times for 10 min each time.

[0122] (14) The tissue sections were immersed in the corresponding liquids in the order of 70% ethanol-80% ethanol-95% ethanol-95% ethanol-anhydrous ethanol-anhydrous ethanol for 5 minutes in order to dehydrate them in a gradient manner.

[0123] (15) After dehydration, the tissue sections are soaked in the liquid in the order of xylene-xylene to clear the sections.

[0124] (16) Add an appropriate amount of Canada acetone to the tissue section, cover it with a coverslip, and after the resin solidifies, image it under a microscope.

[0125] The results are as follows Figure 6 As shown, after intravenous injection of non-therapeutic S-SM3 exosomes into Huntington's disease model mice, the levels of mHTT and aggregates in the brain remained high. Figure 6 (A), and significantly compared with WT mice ( Figure 6 In mice treated with N-SM3 exosomes, the levels of mHTT and the number of aggregates in the brain were reduced (B). Figure 6 (A) and is significant ( Figure 6 (See Figure B). As can be seen, the Nef-SM3 fusion protein sequence prepared in this application can significantly reduce the level of mHTT and aggregates in the brain of Huntington's disease model mice, and the exosomes are modified to express RVG protein on the membrane surface, giving them good targeting ability.

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An exosome, characterized in that, The exosomes contain intracellular antibodies, and the membrane of the exosomes has the rabies virus glycoprotein peptide RVG. The intracellular antibody is composed of the HIV-1 Nef protein sequence and a sequence that targets and degrades the mutant huntingtin protein. The sequence of the nucleic acid molecule encoding the intracellular antibody is shown in SEQ ID NO:

1.

2. The method for preparing exosomes according to claim 1, characterized in that, The preparation method includes the following steps: Plasmids were constructed based on nucleic acid molecules encoding intracellular antibodies and sequences encoding RVG, a glycoprotein peptide of rabies virus expressed on exosome membranes. Both plasmids were simultaneously transfected into cells, and exosomes were collected after expression.

3. The preparation method according to claim 2, characterized in that, The sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane is shown in SEQ ID NO:

2.

4. The preparation method according to claim 2, characterized in that, The plasmid vector constructed based on the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane is pcDNA3.1 plasmid.

5. The preparation method according to claim 2, characterized in that, The cells in question are HEK293T cells.

6. The use of the exosomes according to claim 1 in the preparation of a medicament for treating Huntington's disease.

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