A drug for Huntington's disease and its preparation method

Abstract

The present invention discloses a drug for Huntington's disease and a preparation method thereof. The method provided by the present invention prepares a gene drug for treating Huntington's disease by constructing a ssAAV-CMV-TRIM37-HA plasmid that overexpresses TRIM37. The drug for Huntington's disease acts on Huntington's disease by targeting TRIM37. The drug for Huntington's disease can mediate ubiquitin-specific targeting of mHTT protein, which is specifically low-expressed in the striatum of primates. When TRIM37 is over-expressed in the striatum, it can promote the degradation of mHTT aggregates, thereby alleviating the neurotoxicity of mHTT.

Description

Technical Field

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

[0002] Huntington's Disease (HD) is an autosomal, monogenic dominant neurodegenerative disease caused by abnormal polyglutamine (polyQ) expansion (>35) at the N-terminus of the huntingtin (HTT) protein. Mutant huntingtin (mHTT) is widely expressed in the brain, but the striatum of HD patients is the brain region most seriously affected by neuronal death. The mHTT protein misfolds and deposits in neurons in the brains of HD patients to form protein aggregates. mHTT aggregates are believed to be closely related to the neurotoxicity of HD, and reducing the deposition of mHTT aggregates may be a treatment for HD.

[0003] Although 30 years have passed since the discovery of the pathogenic gene, there is still no drug on the market that can effectively slow down the progression of HD. Meanwhile, drug treatment for HD mainly focuses on the treatment of chorea, cognitive impairment and mental disorders. Currently, Deutetrabenazine and Tetrabenazine are two drugs approved by the US Food and Drug Administration for symptomatic treatment. There is currently no drug that can effectively slow down the progression of HD. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a drug for Huntington's disease and a method for preparing the same. The method provided by the present invention prepares a gene drug for treating Huntington's disease by constructing a ssAAV-CMV-TRIM37-HA plasmid that overexpresses TRIM37. The drug for Huntington's disease acts on Huntington's disease by targeting TRIM37.

[0005] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0006] The present invention provides a drug for treating Huntington's disease, comprising TRIM37 protein.

[0007] The gene medicine for treating Huntington's disease provided by the present invention comprises a gene expressing TRIM37 protein.

[0008] Furthermore, the sequence of the gene expressing TRIM37 protein is shown in SEQ ID NO.4.

[0009] The present invention provides a method for preparing a gene drug for treating Huntington's disease, comprising the following steps:

[0010] RNA of human HEK293 cells was extracted and reverse transcribed to construct a cDNA library. The cDNA of TRIM37 protein was amplified by PCR (full-length TRIM37 cDNA was amplified using TRIM37-specific PCR primers). The cDNA of TRIM37 protein was ligated to a plasmid (ssAAV-CMV plasmid) by enzyme digestion (the full-length TRIM37 cDNA and plasmid were purified after double digestion with AgeI and EcoRI, and then ligated) to obtain a plasmid carrying the TRIM37 protein gene, which was then packaged by virus to obtain the gene drug for treating Huntington's disease.

[0011] Furthermore, in the PCR amplification, the upstream primer used is as shown in SEQ ID NO.1, specifically 5'-CCGACC GGT GCCACCATG GAT GAA CAGAGC GTG GAGAG-3';

[0012] Furthermore, in the PCR amplification, the downstream primer used is as shown in SEQ ID NO. 2, specifically 5'-CCGGAA TTC TTA AGC ATA ATC TGG AAC ATC ATA TGG ATA TCT TCCACTATT TTCATC T-3'.

[0013] Furthermore, the enzyme digestion includes: using AgeI restriction endonuclease and EcoRI restriction endonuclease to digest the plasmid and the cDNA of TRIM37 protein respectively.

[0014] Furthermore, the plasmid is a ssAAV-CMV plasmid, and the sequence is shown in SEQ ID NO.5.

[0015] Furthermore, the plasmid carrying the TRIM37 protein gene is a ssAAV-CMV-TRIM37-HA plasmid, and the sequence is shown in SEQ ID NO.3.

[0016] Furthermore, the virus is an adeno-associated virus.

[0017] Furthermore, the serotype of the adeno-associated virus (AAV) is AAV9.

[0018] The TRIM37 protein is an E3 ubiquitin ligase that can mediate ubiquitin-specific targeting of the mHTT protein, which is specifically low-expressed in the striatum of primates. When TRIM37 is overexpressed in the striatum, it can promote the degradation of mHTT aggregates.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] The present invention provides a drug for Huntington's disease and a preparation method thereof. The drug for Huntington's disease can mediate ubiquitin-specific targeting of mHTT protein, which is specifically low-expressed in the striatum of primates, and can promote the degradation of mHTT aggregates when TRIM37 is over-expressed in the striatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the result that ssAAV-CMV-TRIM37-HA can be stably expressed in HEK293 cells in the example.

[0022] Figure 2 The figure shows the result of TRIM37 regulating the ubiquitination level of mHTT.

[0023] Figure 3 This figure shows the results of TRIM37 regulating the homeostasis of mHTT protein.

[0024] Figure 4 The figure shows the result that overexpression of TRIM37 promotes the degradation of mHTT aggregates. DETAILED DESCRIPTION

[0025] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0026] Example 1

[0027] A method for preparing a gene drug for Huntington's disease comprises the following steps:

[0028] (1) Primer design: Design primers using primer 5 based on the base sequence of the target gene. After determining the primer sequence, add restriction sites (AgeI and EcoRI) and protective bases at both ends of the forward and reverse primers.

[0029] (2) After the primers are designed, the gene expressing TRIM37 protein is amplified in a PCR instrument using cDNA formed by reverse transcription of RNA extracted from HEK293 cells as a template (the amplification system is shown in Table 1). The sequence of the gene expressing TRIM37 protein is shown in SEQ ID NO.4.

[0030] Table 1

[0031]

[0032] In the PCR amplification, the upstream primer (Primer F) used is shown in SEQ ID NO.1, and the downstream primer (Primer R) used is shown in SEQ ID NO.2.

[0033] (3) After PCR, the target fragment was recovered using a PCR product recovery kit and the concentration was measured.

[0034] (4) The recovered target fragment and the plasmid to be connected (ssAAV-CMV) were digested with enzymes (AgeI and EcoRI) in a 37°C water bath overnight (overnight time is 16 hours).

[0035] (5) The next day, add 1 / 20 volume of calf intestinal alkaline phosphatase (CIP) to the EP tube containing the plasmid (ssAAV-CMV plasmid), vortex to mix, and continue to incubate at 37°C in a water bath for 10 min. After the water bath, incubate at 82°C for 2 min.

[0036] (6) After the water bath is completed, 1 wt% agarose gel electrophoresis is performed for identification and gel recovery, and the concentration is measured.

[0037] (7) Mix the target fragment and vector in a ratio of 3:1, add 5 μL of TaKaRa DNA Ligation Kit enzyme solution I (containing T4 DNA ligase), mix gently, and place at 16°C for 30 min.

[0038] (8) After ligation, add 50 μL of Trans1 blue (Disperse Blue 1) to the EP tube, mix gently, incubate on ice for 30 min, and then transfer to a 42°C dry incubator for heat shock treatment for 52 s.

[0039] (9) After the heat shock, incubate the EP tube on ice for 5 min. Then, add 800 μL of sterile LB culture medium to the EP tube and place it in a 37°C shaker for 1 h.

[0040] (10) Centrifuge at 500 × g for 5 min at room temperature, remove 700 μL of LB, mix gently, and drop the mixture onto the AMP-resistant plate. Use a spreading stick to evenly spread the bacterial solution on the plate. After the liquid is dried, place the plate in a 37°C incubator for overnight culture (16 h).

[0041] (11) Select monoclonal colonies and perform colony PCR. After identification by 1 wt% agarose gel electrophoresis, transfer the bacterial solution of the positive sample to a shake tube containing 6 mL LB (AMP resistance), cover the tube with a lid and place it in a 37°C shaker for overnight culture (overnight time is 16 hours).

[0042] (12) Take 200 μL of bacterial solution and add it to an EP tube containing 200 μL of 60% glycerol (for bacteria preservation treatment). Centrifuge the remaining bacterial solution at 6000 × g for 3 min.

[0043] (13) Use Magen's plasmid extraction kit to extract the plasmid.

[0044] (14) The extracted plasmid was sent to Shanghai Biotechnology Co., Ltd. for sequencing.

[0045] (15) The corresponding bacterial solution of the correctly sequenced plasmid was selected for expansion culture, and then the plasmid was extracted using Magen's extraction kit to obtain the plasmid ssAAV-CMV-TRIM37-HA plasmid (sequence shown in SEQ ID NO.3), and the concentration was measured.

[0046] (16) The plasmid ssAAV-CMV-TRIM37-HA was transfected into HEK293 cells using Invitrogen's liposome transfection reagent (Lipo3000). After 48 h, the cells were collected and total protein was extracted for Western blotting analysis. The results were referred to Figure 1 shown. Figure 1 This is a graph showing the result that ssAAV-CMV-TRIM37-HA can be stably expressed in HEK293 cells in the example. Figure 1 It can be clearly seen that a clear band can be detected at 130 kDa in both the anti-TRIM37 and anti-HA labeled TRIM37-HA lanes (the actual molecular weight of TRIM37 protein is at 130 kDa), while no corresponding signal was detected at the corresponding position in the control group (HEK293) lane, indicating that the ssAAV-CMV-TRIM37-HA plasmid was successfully constructed.

[0047] (17) The virus was sent to Guangzhou Paizhen Biotechnology Co., Ltd. for virus packaging to obtain the ssAAV-CMV-TRIM37-HA virus, that is, the gene drug for Huntington's disease.

[0048] Example 2

[0049] TRIM37 regulates the ubiquitination level of mHTT

[0050] (1) HEK293 (120Q) cells were cultured in a 10 cm dish containing DMEM medium (10% fetal bovine serum, 1% penicillin and streptomycin, 50 μg / μL HYB) and placed in an incubator at 37° C. and 5% CO 2 for culture.

[0051] (2) The next day, when the cells grew to 50% of the culture dish area, the plasmid (ssAAV-CMV-TRIM37-HA) and siRNA (TRIM37) were transfected into HEK293 (120Q) cells using Invitrogen's liposome transfection reagent and the cells were cultured in a 37°C, 5% CO2 incubator.

[0052] (3) 48 hours after transfection, add 10 μL of MG132 to the culture medium of the culture dish. After further culturing for 12 hours, digest the cells with 0.25% trypsin containing EDTA, collect the cells and transfer them to a 1.5 mL EP tube, centrifuge at 500×g for 5 minutes, wash twice with PBS, and store the cell pellet at -80°C for later use.

[0053] (4) Cells were lysed with a strong RIPA lysis buffer, and the protein concentration was determined by BCA. The protein sample solutions at different time points were adjusted to the same concentration (2 μg / μL), and 5×SDS protein loading buffer was added. After being thoroughly mixed in a vortex, the samples were centrifuged in a handheld centrifuge for 5 sec. The samples were boiled at 98°C in a dry thermostat for 10 min. The samples were placed on ice and ultrasonically treated after they were cooled.

[0054] (5) After ultrasound, Western blotting analysis was performed.

[0055] The results are as follows Figure 2 shown. Figure 2 Part A shows the result of knocking down TRIM37 protein, which leads to a decrease in mHTT ubiquitination level. Figure 2 From Part A, we can see that in the IP HTT group, knocking down TRIM37 protein resulted in a decrease in the ubiquitination level of mHTT, indicating that reducing the expression of TRIM37 protein can reduce the ubiquitination level of mHTT. Figure 2 Part B is a quantitative analysis of Part A. Figure 2As can be seen from part B, reducing the expression of TRIM37 protein has a significant effect on reducing the ubiquitination level of mHTT. Figure 2 Part C shows the result of increased mHTT ubiquitination level after overexpression of TRIM37 protein. Figure 2 From the C part, we can see that in the IP HTT group, when TRIM37 protein is overexpressed, the ubiquitination level of mHTT is increased. Figure 2 Part D is a quantitative analysis of Part C. Figure 2 As can be seen from part D, overexpression of TRIM37 protein has a significant effect on increasing the ubiquitination level of mHTT.

[0056] Example 3

[0057] TRIM37 regulates intracellular mHTT protein homeostasis

[0058] (1) HEK293 (120Q) cells were cultured in a 10 cm dish containing complete DMEM medium (10% fetal bovine serum, 1% penicillin and streptomycin, 50 μg / μL hygromycin (HYB)). When the cells grew to 80% of the dish area, they were digested with 0.25% trypsin containing EDTA, and the cell density was adjusted to plate in six-well plates and cultured in a 37°C, 5% CO2 incubator.

[0059] (2) The next day, when the cells grew to 50% of the culture dish area, the plasmid (ssAAV-CMV-TRIM37-HA) and siRNA (TRIM37) were transfected into HEK293 (120Q) cells using Invitrogen's liposome transfection reagent and the cells were cultured in a 37°C, 5% CO2 incubator.

[0060] (3) 48 hours after transfection, add 10 μL of cycloheximide to each well of cells, and collect cells at designated time points: 0h, 2h, 4h, 6h, 8h and 12h and transfer to 1.5mL EP tubes, centrifuge at 500×g for 5min, wash twice with PBS, and store the cell pellet at -80℃ for later use.

[0061] (4) Cells were lysed with RIPA lysis buffer, and protein concentration was determined by BCA. The protein sample solutions at different time points were adjusted to the same concentration (2 μg / μL), and 5×SDS protein loading buffer was added. After being thoroughly mixed in a vortex, the samples were centrifuged in a handheld centrifuge for 5 sec. The samples were boiled at 98°C in a dry thermostat for 10 min. The samples were placed on ice and ultrasonically treated after cooling.

[0062] (5) After ultrasound, Western blotting analysis was performed.

[0063] Results reference Figure 3 As shown, Figure 3 From part A, it can be seen that the mHTT protein in the siRNA group with TRIM37 knockdown has higher levels at different time points (2h, 4h, 6h, 8h and 12h) compared with the control group, indicating that reducing the expression of TRIM37 protein will enhance the homeostasis of mHTT protein. Figure 3 Part C is a quantitative analysis of Part A. Figure 3 As can be seen from part C, reducing the expression of TRIM37 protein has a significant difference in enhancing the homeostasis of mHTT protein. Figure 3 From part B, it can be seen that the mHTT protein in the TRIM37 protein overexpression group has lower levels at different time points (2h, 4h, 6h, 8h and 12h) compared with the GFP group, indicating that overexpression of TRIM37 protein will promote the degradation of mHTT protein. Figure 3 Part D is a quantitative analysis of Part B. Figure 3 As can be seen from part D, overexpression of TRIM37 protein has a significant effect on enhancing the degradation of mHTT protein.

[0064] Example 4

[0065] Overexpression of TRIM37 gene by stereotaxic injection of ssAAV-CMV-TRIM37-HA virus

[0066] (1) Experimental animals: 24-week-old HD140QKI mice (mice with Huntington's disease) were housed at the Experimental Animal Center of Jinan University.

[0067] (2) Experimental groups: A total of 9 HD140QKI mice were injected with 2 μL ssAAV-CMV-GFP virus on the right side as the control group, and 2 μL ssAAV-CMV-TRIM37-HA virus on the left side as the experimental group.

[0068] (3) Injection process: First check the amount of isoflurane in the anesthesia system, then open the anesthesia system correctly, adjust the anesthesia concentration and gas flow, and after the mouse is anesthetized, buckle the mouse's incisors into the mask device, adjust the mask device until the mouse's external auditory canal and the left and right ear bars of the locator are in a straight line, fix the mouse's head, and then use a medical cotton swab to apply 75% alcohol to the surgical site, and use scissors to cut the hair at the surgical site. Then use tweezers to gently lift the head skin and cut the scalp along the sagittal suture. Make an incision of about 1 cm, cut the meninges with scissors, and then use a cotton swab immersed in 3% H2O2 to wipe the incision until the Bregma is prominent; then use a drill to drill the left and right striatum (AP: +0.55mm, ML: +2.0mm or -2.0mm and DV-: 3.4mm)

[0069] Drill a hole; then place the virus on ice to thaw. After the virus thaws, use a microsyringe to first draw 1 μL of air and then draw 2 μL of ssAAV-CMV-TRIM37-HA or ssAAV-CMV-GFP virus. Correctly install the microsyringe on the mouse brain stereotaxic instrument and insert the needle according to the coordinate DV: -3.4 mm. When the injection needle is pushed to the specified coordinate, set the injection speed to 100 nL / min. After the injection of both sides of the striatum (left side: ssAAV-CMV-TRIM37-HA virus or right side ssAAV-CMV-GFP virus) is completed, stop the needle for 10 minutes, then slowly withdraw the needle and suture the wound with a suture needle. At the same time, apply antibiotics (penicillin and streptomycin) to the suture site, mark the ear with a puncher, remove the mouse from the fixator and put it in a mouse cage for warming care. After waking up, observe for 1 hour without obvious abnormalities and then put it back in the mouse cage for breeding.

[0070] (4) Four weeks after virus injection, samples were collected and analyzed (by Western blotting and immunofluorescence) for mHTT protein aggregates in the experimental and control groups. Figure 4 shown. Figure 4 The figure shows the result that overexpression of TRIM37 promotes the degradation of mHTT aggregates. Figure 4 Part A is the Western blotting result, which shows that the mHTT protein aggregates in the TRIM37 protein overexpression group are significantly reduced compared with the GFP group, indicating that overexpression of TRIM37 protein will reduce the formation of mHTT protein aggregates. Figure 4 Part B is a quantitative analysis of Part A. Figure 4 As can be seen from part B, overexpression of TRIM37 protein has a significant effect on reducing the formation of mHTT protein aggregates. Figure 4 Part C is the result of immunofluorescence staining. It can be seen that the aggregates of mHTT protein (red dots) in the TRIM37 protein overexpression group were significantly reduced compared with the GFP group, indicating that overexpression of TRIM37 protein will reduce the formation of mHTT protein aggregates.

[0071] Summary: The present invention clones the human TRIM37 gene to construct an overexpression ssAAV-CMV-TRIM37-HA plasmid and packages the virus. In the cell and mouse models of HD, the TRIM37 gene is overexpressed by transfection and brain stereotactic injection, respectively, to achieve the clearance of mutant huntingtin (mHTT) aggregates, thereby alleviating the neurotoxicity of mHTT.

[0072] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. Application of overexpressed TRIM37 protein in the preparation of drugs for the treatment of Huntington's disease.

2. Application of genes expressing TRIM37 protein in the preparation of drugs for the treatment of Huntington's disease.

3. The use according to claim 2, characterized in that: The sequence of the gene expressing TRIM37 protein is shown in SEQ ID NO.

4.

4. Use of a plasmid carrying the TRIM37 protein gene in the preparation of drugs for the treatment of Huntington's disease.

5. Use of virus-packaged plasmids carrying the TRIM37 protein gene in the preparation of drugs for the treatment of Huntington's disease.

6. The use according to claim 4 or 5, characterized in that: The plasmid carrying the TRIM37 protein gene is a ssAAV-CMV-TRIM37-HA plasmid, and the sequence is shown in SEQ ID NO.

3.

7. The use according to claim 5, characterized in that: The virus is an adeno-associated virus.

8. The use according to claim 7, characterized in that: The serotype of the adeno-associated virus is AAV9.

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