Medicine for treating Huntington's disease

Abstract

The invention discloses an engineered intracellular antibody, the engineered intracellular antibody is formed by fusing a Nef protein sequence of HIV-1 and an intracellular antibody sequence of targeted degradation variation huntingtin, and the intracellular antibody is simple in structure and easy to artificially synthesize. A rabies virus glycoprotein peptide fragment RVG is expressed on a membrane of the exosome, so that the exosome specifically targets nerve cells, and has high stability, low immunogenicity and high penetrability; the mutant Huntington protein and the aggregate thereof existing in the brain of a patient can be efficiently cleared in a targeted manner through intravenous injection, the off-target problem of gene therapy is solved, and the mutant Huntington protein and the aggregate thereof existing in the brain of a patient suffering from late Huntington disease are of great significance in clearing away the mutant Huntington protein and the aggregate thereof existing in the brain of the patient suffering from late Huntington disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a drug for treating Huntington's disease. Background Art

[0002] Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of the CAG trinucleotide repeat sequence in the Huntingtin gene (HTT) on chromosome 4. This leads to an abnormal increase in the length of the polyglutamine sequence in the translated huntingtin protein, resulting in a toxic mutant huntingtin protein. This mutant huntingtin protein forms large amounts of abnormal protein aggregates in the patient's neurons, causing neuronal toxicity and neuronal death, leading to neuronal degeneration. Currently, there is no clinical cure for Huntington's disease. Approved drugs for Huntington's disease, such as tetrabenazine and clonazepam, are intended to alleviate Huntington's disease symptoms. Current research on fundamental treatments for Huntington's disease focuses primarily on viral-mediated gene editing therapies, such as the CRISPR-Cas system, which directly targets and knocks out the abnormal Huntingtin gene, and gene silencing therapies, such as antisense oligonucleotides and RNA interference, which block the expression of the abnormal Huntingtin gene. However, these technologies all have drawbacks. For one thing, they lack high specificity in modifying gene expression, and the risk of off-target effects cannot be ignored. Furthermore, for patients with advanced Huntington's disease, gene therapy cannot eliminate the large number of mutant proteins and their aggregates already present in the brain. Furthermore, these therapies require administration via viral vectors, either through stereotactic or intrathecal injections, which can cause inflammatory reactions in patients. Stereotactic or intrathecal injections are highly invasive methods of administration. Summary of the Invention

[0003] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a drug for treating Huntington's disease.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides an engineered intracellular antibody, wherein the intracellular antibody is formed by fusion of the HIV-1 Nef protein sequence and a sequence that targets and degrades mutant Huntington protein.

[0006] In some embodiments of the present invention, the fusion comprises inserting HIV-1 Nef protein sequences at both ends or in the middle of the sequence targeting degradation of variant huntingtin protein.

[0007] In some embodiments of the present invention, the fusion is to connect the two ends of the sequence that targets the degradation of the mutant huntingtin protein to the Nef protein sequence of HIV-1.

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

[0009] In some embodiments of the present invention, the HIV-1 Nef protein sequence 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 packaged into exosomes within the cell.

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

[0013] In some embodiments of the present invention, the nucleic acid molecule further comprises a modified 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 has a sequence as shown in SEQ ID NO: 1.

[0016] The third aspect of the present invention provides a biomaterial containing the intracellular antibody described in the first aspect or the nucleic acid molecule described in any one of the above aspects, wherein the biomaterial comprises:

[0017] (1) an expression cassette containing the nucleic acid molecule described in the above aspects;

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

[0019] (3) A recombinant cell containing the nucleic acid molecule described in the above aspects, or a recombinant cell containing the expression cassette described in (1), or a recombinant cell containing the recombinant vector described in (2), or a recombinant cell containing the intracellular antibody described in the above aspects.

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

[0021] In some embodiments of the present invention, the steps are: constructing a plasmid based on the nucleic acid molecule of the above aspects, transfecting the plasmid into cells, and collecting exosomes after expression.

[0022] In some embodiments of the present invention, the steps are: constructing plasmids based on the nucleic acid molecule of the above aspect and the sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane, transfecting the two plasmids into cells at the same time, and collecting exosomes after inducing expression.

[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.

[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 comprises a modified sequence.

[0026] In some embodiments of the present invention, the modified sequence is a sequence encoding a 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 as SEQ ID NO: 2.

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

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

[0031] In some embodiments of the present invention, the cells include 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 comprises 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 124,000×g.

[0036] The fifth aspect of the present invention provides exosomes prepared according to the above aspect, wherein the exosomes contain the intracellular antibody according to the first aspect.

[0037] In some embodiments of the present invention, the membrane of the exosomes contains the rabies virus glycoprotein peptide segment RVG.

[0038] A sixth aspect of the present invention provides use of the intracellular antibody, nucleic acid molecule, biomaterial or exosome described in the above aspects in the preparation of a drug for treating Huntington's disease.

[0039] In some embodiments of the present invention, the administration of the drug includes intravenous injection and intravenous drip.

[0040] In some embodiments of the present invention, the drug further contains other pharmaceutically acceptable adjuvants.

[0041] In some embodiments of the present invention, the adjuvant includes a pH adjuster, an isotonicity adjuster, a stabilizer, and a buffer.

[0042] The beneficial effects of the present invention are:

[0043] 1. The present invention provides an intracellular antibody, which is composed of a fusion of the HIV-1 Nef protein sequence and a sequence that targets and degrades a mutant huntingtin protein. It can be more encapsulated and incorporated into exosomes within cells. The intracellular antibody has a simple structure, is easy to synthesize artificially, and has low cost.

[0044] 2. The present invention provides an exosome, which contains intracellular antibodies that target the degradation of variant huntingtin protein and expresses the rabies virus glycoprotein peptide RVG on the membrane. The exosome has good stability, low immunogenicity, and high penetrability. It can cross the blood-brain barrier through intravenous injection to reach the designated site, avoiding the inflammatory response and trauma caused by brain-targeted injection or intrathecal injection. The exosome also has good targeting and can directly eliminate the variant huntingtin protein and its aggregates already in the patient's brain, solving the off-target problem of gene therapy. It is of great significance for effectively eliminating the variant huntingtin protein and its aggregates in the brain of patients with late-stage Huntington's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1Comparison of exosomes produced from cells transfected with the SM3 plasmid and the Nef-SM3 plasmid in Example 3. HA is the tag protein carried by Nef-SM3 and SM3; Vinculin is a cytoskeletal protein used as an internal control for sample loading; Calnexin is an endoplasmic reticulum membrane marker; and CD63 is an exosome membrane marker. A shows Western blotting of cell lysates and purified exosomes. WT is exosomes produced by 293T cells without plasmid transfection, serving as a negative control. B shows quantitative analysis of SM3 protein levels in exosomes from A (n = 4). Two-tailed unpaired t-test was used for significance analysis. *p < 0.05.

[0046] Figure 2 To detect the exosomes prepared in Example 4. MOCK is the control group of HEK293T cells without any plasmid transfection, S-SM3 is the exosome expressing Nef-Scramble and RVG-LAMP2b simultaneously, and N-SM3 is the exosome expressing Nef-SM3 and RVG-LAMP2b simultaneously; A is the WB experimental results of cell lysate, cell culture supernatant and purified exosomes, HA is the tag protein carried by Nef-scramble and Nef-SM3; FLAG is the tag protein of RVG-LAMP2b; Calnexin is the endoplasmic reticulum membrane marker; CD9 and CD63 are exosome membrane markers; TSG101 is the exosome content marker; B is the transmission electron microscopy detection result of the prepared N-SM3 exosomes, scale bar at 7000×: 1μm, scale bar at 20000×: 500nm; C is the nanoparticle tracking analysis (NTA) result of the prepared N-SM3 exosomes.

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

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

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

[0050] Figure 6 Figure 1 shows the results of mutant huntingtin (mHTT) protein levels and aggregates in Huntington's disease model mice after intravenous injection of exosomes. WT mice served as controls, while CAG140Q KI mice were knocked in with a human HTT gene containing 140 glutamine repeats. A shows immunohistochemical staining of frozen sections of striatal tissue from Huntington's disease model mice treated with S-SM3 and N-SM3 exosomes using the mEM48 antibody. The black punctate structures in the image are mHTT aggregates. The left image is a normal image, and the right image is a magnified view. Scale bar: 40 μm. B shows quantification of mHTT aggregates in panel A (n = 3). Differences were analyzed using one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0052] Example 1 Establishment of Exosome Preparation Method

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

[0054] (2) HEK 293T cells were transfected with a plasmid expressing intracellular antibodies. 24 hours after transfection, the cells were replaced with DMEM cell culture medium without exosomes. The culture medium was collected after another 24 hours of culture.

[0055] (3) HEK 293T cell exosomes were collected by gradient centrifugation. The specific steps include: centrifugation at 500×g for 10 minutes to collect the supernatant; centrifugation at 2000×g for 10 minutes to collect the supernatant; centrifugation at 10000×g for 30 minutes to collect the supernatant. The collected supernatant was transferred to a centrifuge tube for ultracentrifugation and centrifuged at 124000×g at 4°C for 135 minutes. After centrifugation, the supernatant was aspirated and the precipitate was resuspended in sterile PBS to obtain the purified 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] Preparation methods of 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), which also carries a FLAG tag protein sequence; the SM3 plasmid is a plasmid carrying the following SEQ ID NO:5 shows a commercially available PRK5 plasmid (from Jinan University) encoding an intracellular antibody sequence capable of targeting and degrading variant huntingtin protein; Nef-SM3 plasmid is a commercially available PRK5 plasmid (from Jinan University) carrying a fragment encoding HIV-1 Nef protein sequence and the intracellular antibody sequence encoding the variant huntingtin protein; Nef-Scramble plasmid is a commercially available PRK5 plasmid (from Jinan University) carrying a fragment encoding HIV-1 Nef protein sequence and a scramble sequence encoding the intracellular antibody sequence encoding the variant huntingtin protein, i.e., the sequence encoding Scramble is a segment with the same base ratio as the intracellular antibody sequence encoding the variant huntingtin protein, but the protein expressed is non-functional, as a comparison for subsequent experiments. SM3 plasmid, Nef-SM3 plasmid, and Nef-Scramble plasmid all carry two HA tag protein sequences. The insert fragments of the Nef-SM3 plasmid, RVG-LAMP2b plasmid, SM3 plasmid and Nef-Scramble plasmid are shown in the following table.

[0058] The construction of the plasmid 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 synthesized RVG-LAMP2b sequence was cloned into the pcDNA3.1 plasmid for expression.

[0060]

[0061]

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

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

[0064] Based on the exosome preparation steps in Example 1, the Nef-SM3 plasmid and the SM3 plasmid obtained in Example 2 were used to prepare exosomes, respectively, and wild-type HEK 293T cells not transfected with any plasmid were set as the WT control group.

[0065] At the same time, RIPA lysis buffer (Biyuntian Biotechnology) was used to extract cell lysate from the transfected and cultured cells. The specific steps were as follows: collect the cell pellet by centrifugation, add an appropriate amount of RIPA lysis buffer containing protease inhibitors to the cell pellet, resuspend by blowing, adjust the power of the ultrasonic cell disruptor to 100 W for ultrasonic disruption, react on ice for 30 minutes, and then collect the lysate for detection.

[0066] The prepared exosomes and cell lysates were subjected to protein immunoblotting, and the results were as follows: Figure 1 As shown in Figure A, a large amount of Nef-SM3 and SM3 intracellular antibodies were produced in the cell lysate, but after purification, only a small amount of SM3 intracellular antibodies appeared in the exosomes, while most of the Nef-SM3 intracellular antibodies entered the exosomes. Figure B shows that the content of Nef-SM3 intracellular antibodies in exosomes was significantly higher than that of SM3 polypeptide. Therefore, Nef-SM3 intracellular antibodies have a better ability to be encapsulated into exosomes than SM3 intracellular antibodies.

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

[0068] Two experimental groups were set up, S-SM3: containing the Nef-Scramble plasmid and RVG-LAMP2b plasmid prepared in Example 2; N-SM3: containing the 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 not transfected with any plasmid were set as the control group MOCK.

[0069] The cell culture supernatant (cell culture medium before purification), the purified exosomes of each group, and the cell lysate obtained by the cell lysate extraction method in Example 3 were subjected to protein immunoblotting and nanoparticle size detection, and MOCK exosomes and N-SM3 exosomes were detected by transmission electron microscopy.

[0070] The experimental results are as follows Figure 2 As shown in Figure A, a large amount of FLAG-tagged RVG-LAMP2b protein, HA-tagged Nef-SM3 intracellular antibody, and Nef-Scramble protein with the same size as Nef-SM3 in the S-SM3 group were produced in the cell lysate ( Figure 2 (The band corresponding to Nef-SM3-HA marked in A) was detected in the purified exosomes, and exosome markers such as CD9, CD63, and TSG101 were detected. The intracellular membrane component marker Calnexin was not detected. Furthermore, FLAG-tagged RVG-LAMP2b protein, as well as HA-tagged Nef-SM3 and Nef-Scramble proteins were detected simultaneously. Transmission electron microscopy (TEM) images in B show the vesicular structure and phospholipid bilayer structure of the exosomes. C shows that the particle diameters in the exosome samples are consistent and mainly around 110 nm. In summary, this example successfully constructed exosomes containing both the Nef-SM3 intracellular antibody and the RVG-LAMP2b protein, as well as exosomes containing both the Nef-Scramble protein and the 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 an in vitro HEK 293T cell uptake assay, and the control group was HEK 293T cells incubated with PBS.

[0073] The specific experimental steps are as follows.

[0074] (1) Exosome protein quantification: The exosomes from Example 4 were taken and the amount of exosome protein was determined using a commercially available BCA (Bicinchoninic Acid) protein concentration assay kit (performed according to the instructions);

[0075] (2) Preparation of dye working solution: Fluorescent dye PKH67 (Merck Life Sciences) stock diluent was diluted 10-fold 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 dye working solution to the exosomes according to the dosage in the instructions (add 50 μL of dye working solution to 10-200 μg of exosome protein). After adding the dye working solution, mix it by vortexing for 1 minute, and then incubate it for 10 minutes. Add 10 mL of 1× PBS to the incubated exosome-dye complex and mix it. Extract the exosomes again by ultra-high-speed centrifugation at 124,000 × g and 4°C for 135 minutes to remove excess dye, and finally resuspend the precipitate in 1× PBS. The precipitate is the fluorescently labeled exosomes;

[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 washed three times with PBS. The fluorescence enrichment was then observed under a confocal microscope.

[0078] The experimental results are as follows Figure 3 As shown, exosomes labeled with the green fluorescent dye PKH67 appeared near the cell nucleus labeled with the blue fluorescent dye DAPI, indicating that exosomes can be taken up into cells in vitro.

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

[0080] In vivo cellular uptake assays were performed on the N-SM3 exosomes prepared in Example 4. The control group consisted of C57 mice injected with PBS. The mice were purchased from Zhaoqing Ruisiyuan Biotechnology Co., Ltd. and housed in a standardized barrier environment at the Experimental Animal Management Center of Jinan University with a 12-hour light / dark cycle. All animal experimental procedures and animal husbandry strictly adhered to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Jinan University (approval number: IACUC20221117-03).

[0081] The specific steps are as follows.

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

[0083] (2) The exosomes obtained in step (1) were injected into the retroorbital venous plexus of mice (50 μL), 4.9×10 11particles / mL;

[0084] (3) 24 hours after injection, mice were anesthetized with isoflurane and perfused transcardially with 0.9% saline. The brains were then dissected and fixed with 10 mL of 4% paraformaldehyde for 24 hours and then dehydrated with 30% sucrose at 4°C for 48 hours.

[0085] (4) The brain dehydrated in (3) was embedded in a tissue cryoprotectant (OCT) and then cut into 20 μm slices using a cryostat (Leica CM1950) and mounted on a glass slide;

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

[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 and add NeuN antibody primary antibody solution (purchased from abcam, ab177487) diluted at a ratio of 1:1000 using the blocking solution, and incubate in a 4°C refrigerator overnight;

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

[0090] (9) Remove the fluorescent secondary antibody solution, wash with PBS three times, 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 with PBS three times, and then cover with a coverslip;

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

[0093] The experimental results are as follows Figure 4 As shown, exosomes labeled with the red fluorescent dye Dil can be seen appearing near the nuclei (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 Western immunoblotting experiment on the effect of exosome drugs on mHTT levels and aggregate clearance in the brain of Huntington's disease model mice

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

[0096] The HTT knock-in (140Q) mouse model used in this example was obtained 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 strictly adhered to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Jinan University (approval number: 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 intravenous injection. Each mouse was injected with 50 μL of the corresponding exosomes per day at a concentration of 4.9×10 11 particles / mL, and the drug was administered continuously for 7 days. After 7 days of injection, wait for 3 days for subsequent treatment effect evaluation.

[0099] (2) The mice prepared in step (1) were anesthetized with isoflurane, perfused transcardially with 0.9% saline, and then the brains were dissected.

[0100] (3) The mouse brain tissue obtained in (2) was ground using a Luka Grinding instrument (LUKYM-II) and then TM Protease inhibitor cocktail (Thermo Scientific), 50 mmol·L -1 The cells 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) containing NaF and PMSF.

[0101] (4) The tissue lysate was incubated with shaking at 4°C for 30 min. The sample was then ultrasonicated and centrifuged at 12,000 rpm for 10 min. The supernatant was used for subsequent experiments.

[0102] (5) The supernatant from (4) was loaded onto an SDS-PAGE gel and transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% milk / TBST (50 mM Tris-HCl, pH 7.4, containing 20 mM Tween 20) for 1 hour at room temperature. The primary antibody (purchased from Merck Bio) was diluted in 3% BSA / TBST and incubated with the NC membrane overnight at 4°C.

[0103] (6) The NC membrane from (5) was washed three times in TBST (10 minutes each time), and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (purchased from Boster) in 5% milk / TBST for 1 hour at room temperature.

[0104] (7) Western blot images were then developed using ECL and acquired using ChemiScop 6000 (CLiNqinxiang). The images were quantified using densitometry and analyzed using ImageJ software.

[0105] The experimental results are as follows Figure 5 As shown, compared with S-SM3 exosomes that have no clearance ability, N-SM3 exosomes showed higher mHTT levels and aggregates as a drug treatment for Huntington's disease model mice ( Figure 5 A) and the clearance effect had a significant effect on mHTT levels and aggregates ( Figure 5 B and C).

[0106] Example 8 Protein immunohistochemical staining experiment on the effect of exosome drugs on mHTT levels and aggregate clearance in the brain of Huntington's disease model mice

[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 in the brains of Huntington's disease model mice, with WT mice as a control.

[0108] The specific steps are as follows.

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

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

[0111] (3) Immerse the tissue sections obtained in (2) in 4% PFA precooled to 4°C and fix for 10 min;

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

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

[0114] (6) Soak 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 section and wash it with PBS three times, 10 min each time. Use an immunohistochemistry pen to draw a hydrophobic circle around the tissue section on the slide.

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

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

[0118] (10) Remove the primary antibody solution and wash with PBS three 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, and wash with PBS four times, 5 min each time;

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

[0121] (13) DAB Substrate and DAB Chromogen in the abcam DAB Substrate Kit were mixed at a ratio of 50:1 and added dropwise to the tissue sections. The sections were incubated at room temperature for 1–5 min until a specific signal was observed and the background was not too dark. PBS was added dropwise to terminate the reaction. The sections were washed with PBS 4 times for 10 min each.

[0122] (14) Tissue sections were dehydrated by immersing them in the corresponding liquids in the order of 70% ethanol, 80% ethanol, 95% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 5 min.

[0123] (15) After dehydration, the tissue sections were immersed in liquids in the order of xylene-xylene to clear the sections;

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

[0125] The results are as follows Figure 6 As shown in , after intravenous injection of S-SM3 exosomes without therapeutic ability into Huntington's disease model mice, the mHTT level and aggregates in the brain remained at a high level ( Figure 6 A), and compared with WT mice, there was a significant difference ( Figure 6 B), while the mHTT level and number of aggregates in the brain of mice treated with N-SM3 exosomes were reduced ( Figure 6 A), and significant ( Figure 6 (B) It can be seen that the Nef-SM3 fusion protein sequence prepared in the present application can significantly reduce the mHTT level and aggregates in the brain of Huntington's disease model mice, and the exosomes are modified to express RVG protein on the membrane surface, which makes them have good targeting.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An intracellular antibody, characterized in that The intracellular antibody is composed of a Nef protein sequence of HIV-1 and a sequence that targets and degrades mutant huntingtin protein; The sequence encoding the targeted degradation variant huntingtin protein has the sequence shown in SEQ ID NO:

5.

2. A nucleic acid molecule encoding the intracellular antibody according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule further includes a modified sequence; the modified sequence includes a sequence encoding an HA tag protein; Preferably, the nucleic acid molecule has the sequence shown in SEQ ID NO:

1.

4. A biomaterial comprising the intracellular antibody according to claim 1 or the nucleic acid molecule according to any one of claims 2 to 3, characterized in that: The biological material includes: (1) An expression cassette containing the nucleic acid molecule according to any one of claims 2 to 3; (2) a recombinant vector containing the nucleic acid molecule according to any one of claims 2 to 3, or a recombinant vector containing the expression cassette according to (1); (3) A recombinant cell containing the nucleic acid molecule according to any one of claims 2 to 3, or a recombinant cell containing the expression cassette according to (1), or a recombinant cell containing the recombinant vector according to (2), or a recombinant cell containing the intracellular antibody according to claim 1.

5. A method for preparing exosomes, characterized in that: The preparation method comprises the following steps: A plasmid is constructed based on the nucleic acid molecule according to claim 2 or 3 and / or a sequence encoding the rabies virus glycoprotein peptide RVG expressed on the exosome membrane, the plasmid is transfected into cells, and exosomes are collected after expression.

6. The preparation method according to claim 5, characterized in that The sequence encoding the rabies virus glycoprotein peptide segment RVG expressed on the exosome membrane is shown in SEQ ID NO:

2.

7. The preparation method according to claim 5, characterized in that The plasmid vector constructed based on the sequence encoding the rabies virus glycoprotein peptide segment RVG expressed on the exosome membrane includes a pcDNA3.1 plasmid.

8. The preparation method according to claim 5, characterized in that The cells include HEK 293T cells.

9. The exosomes prepared by the preparation method according to claim 5, characterized in that: The exosomes contain the intracellular antibody according to claim 1, and the membrane of the exosomes has the rabies virus glycoprotein peptide segment RVG.

10. Use of the intracellular antibody according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the biomaterial according to claim 4, or the exosome according to claim 9 in the preparation of a drug for treating Huntington's disease.

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