TPK expression virus capable of being administered through intravenous injection and use thereof in treating alzheimer's disease

Abstract

The present invention relates to a TPK expression virus capable of being administered through intravenous injection. The TPK expression virus is AAV-TPK, and a genome thereof comprises a neuron-specific promoter, a nucleotide sequence for coding TPK, and a WPRE. Also provided are an AAV-TPK construction method and a use thereof in the preparation of a drug for preventing or treating Alzheimer's disease. The AAV-TPK is serotype rAAV-CAP-B10. In clinical application, the AAV-TPK can be administrated through direction injection across a blood-brain barrier, or is administrated through peripheral intravenous injection, and is used for treating or preventing Alzheimer's disease.

Description

TPK-expressing virus capable of intravenous administration and its application in treating Alzheimer's disease Technical Field

[0001] The present invention relates to a TPK expression virus capable of intravenous administration and application thereof in treating Alzheimer's disease, belonging to the technical field of biomedicine. Background Art

[0002] Alzheimer's disease (AD) is a severe neurodegenerative disorder that is prevalent in the elderly. Its primary clinical manifestations include memory impairment, decreased language and spatial orientation, and mental abnormalities. Pathologically, it is often accompanied by massive neuronal death and subsequent brain atrophy. Currently, several leading hypotheses regarding the pathogenesis of AD include the β-amyloid (Aβ) cascade hypothesis, the phosphorylated tau protein hypothesis, the neuroinflammatory response hypothesis, and the oxidative stress hypothesis. Despite extensive research based on these hypotheses, effective treatment options remain elusive.

[0003] The brain accounts for approximately 2% of body weight but consumes approximately 20% of the body's total energy. Therefore, glucose metabolism is crucial for normal brain function. Previous studies have shown that abnormal brain glucose metabolism in Alzheimer's disease (AD) patients precedes the onset of clinical symptoms by 20 years and is closely associated with disease progression. Therefore, regulating glucose metabolism in brain cells has the potential to alleviate or halt the progression of AD.

[0004] Vitamin B1, also known as thiamine (TM), is converted into biologically active thiamine diphosphate (TDP) in the body by thiamine pyrophosphokinase (TPK). TDP is an essential coenzyme for transketolase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase in glucose metabolism. Previous studies have shown that transketolase activity decreases by over 45% and α-ketoglutarate dehydrogenase activity decreases by over 75% in the brain tissue of patients with AD. TDP levels in the whole blood of AD patients are significantly lower than normal levels and are highly correlated with decreased glucose metabolism in the brain of AD patients. Previous studies have found that TPK mRNA and protein expression levels in the brain tissue of AD patients are significantly lower than those in age- and sex-matched normal human brain tissue. In the brains of AD patients, TPK mRNA levels are negatively correlated with the Braak score, a measure of AD pathology, while in the brain tissue of cognitively normal individuals, TPK mRNA levels are positively correlated with the Braak score. The results suggest that TPK expression may be a protective factor in delaying the onset and progression of AD. Conditional knockout of the TPK gene in excitatory neurons in the mouse brain significantly led to abnormal brain glucose metabolism and cognitive impairment, brain atrophy caused by synaptic and neuronal loss, Aβ deposition and plaque formation, abnormal Tau phosphorylation and neurofibrillary tangle formation, microglia and astrocyte activation and neuroinflammation, microangiogenesis disorders, and peripheral glucose metabolism regulation disorders, all of which are important pathophysiological features of human AD. (See, for example, Sang et al., Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology, Biorxiv, 2020).

[0005] Adeno-associated virus (AAV) is the simplest, non-enveloped, single-stranded DNA virus discovered to date. Recombinant AAV is widely used as a gene therapy vector due to its high safety, low immunogenicity, broad host range, and stable expression. Currently, over 250 AAV gene therapy clinical programs are underway globally, and the US FDA has approved two AAV gene therapy approaches: Luxturna (subretinal injection) and Zolgensma (intravenous injection), for the treatment of Bernhardt's congenital amaurosis and spinal muscular atrophy, respectively.

[0006] New treatments for Alzheimer's disease are urgently needed. Gene therapy using recombinant adeno-associated virus (rAAV), which has gained popularity in recent years, primarily targets proteins involved in Aβ metabolism: APP, Bace1, γ-secretase, as well as Tau and ApoE. Gene therapy strategies that regulate energy metabolism have yet to be reported. Furthermore, current gene therapy approaches for AD often rely on intracerebral, intraventricular, or subarachnoid injections, which have drawbacks such as limited brain coverage, complex procedures, and the risk of brain injury.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a recombinant adeno-associated virus (rAAV) that can specifically target the brain to overexpress TPK through peripheral administration, which is convenient and avoids the potential side effects of peripheral overexpression of TPK, and its application in the treatment of Alzheimer's disease, in view of the shortcomings of existing gene therapy methods, such as the limited range of brain areas infected and the risk of brain injection damage. The recombinant adeno-associated virus comprises an expression cassette in its genome, and the expression cassette comprises a polynucleotide encoding thiamine pyrophosphokinase (TPK) that is operably linked to a promoter.

[0009] To achieve the above objectives, the present invention provides a TPK-expressing virus that can be administered intravenously. The TPK-expressing virus is a recombinant AAV virus, the genome of which contains a neuron-specific promoter, a nucleotide sequence encoding the TPK gene, and a WPRE element.

[0010] Furthermore, the AAV virus is serotype AAV.CAP-B10, the gene sequence of the neuron-specific promoter is shown in SEQ ID NO: 2, the nucleotide sequence encoding the TPK gene is shown in SEQ ID NO: 3, and the sequence of the WPRE element is shown in SEQ ID NO: 4.

[0011] Furthermore, the recombinant AAV virus sequence is shown in SEQ ID NO: 1.

[0012] The present invention provides a method for constructing a TPK expression virus that can be administered intravenously, comprising the following steps:

[0013] Step 1: obtain the TPK target gene by PCR amplification or restriction endonuclease digestion, and then assemble it with an adeno-associated virus vector by homologous recombination using the homologous recombinase Exnase or by T4 DNA ligation;

[0014] Step 2: Transform the viral vector assembled in step 1 into E. coli Stbl3 competent cells;

[0015] Step 3: Screen the transformants using colony PCR, send the positive clones for sequencing, and confirm the positive clones by sequence comparison;

[0016] Step 4: Co-transfect the screened and identified viral vector with pHelper plasmid and pAAV-R plasmid into AAV-293 cells to complete virus packaging and purification.

[0017] Furthermore, the primers for PCR amplification in step 1 or colony PCR in step 3 are 4423-BamHI-F primer and 4423-SalI-R primer, the sequence of the 4423-BamHI-F primer is shown in SEQ ID NO: 5, and the sequence of the 4423-SalI-R primer is shown in SEQ ID NO: 6.

[0018] Furthermore, the forward sequencing primer and reverse sequencing primer for transformant sequencing in step 3 are hSyn-CF primer and pEGFP-N-3' primer, respectively. The sequence of the hSyn-CF primer is shown in SEQ ID NO: 7, and the sequence of the pEGFP-N-3' primer is shown in SEQ ID NO: 8.

[0019] The present invention provides a kit for preparing a TPK-expressing virus that can be administered intravenously. The kit comprises a 4423-BamHI-F primer and a 4423-SalI-R primer for PCR amplification. The sequence of the 4423-BamHI-F primer is shown in SEQ ID NO: 5, and the sequence of the 4423-SalI-R primer is shown in SEQ ID NO: 6.

[0020] Furthermore, the kit also includes a forward sequencing primer and a reverse sequencing primer for detecting positive clones. The forward sequencing primer is hSyn-CF primer, the sequence of which is shown in SEQ ID NO: 7; the reverse sequencing primer is pEGFP-N-3' primer, the sequence of which is shown in SEQ ID NO: 8.

[0021] The present invention provides use of a TPK-expressing virus that can be administered intravenously in the preparation of a drug for preventing or treating Alzheimer's disease.

[0022] Furthermore, the present invention provides the use of a TPK-expressing virus that can be administered intravenously in the preparation of a drug for preventing or treating Alzheimer's-like neurodegenerative lesions, brain atrophy, Aβ and tau pathological changes, neuroinflammation, neurovascular disorders, or slowing the progression of Alzheimer's disease.

[0023] Furthermore, the administration method of the prepared drug is peripheral intravenous injection or trans-blood-brain barrier injection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. AD is a multi-region brain injury. The AAV.CAP-B10.hsyn-TPK of the present invention is a serotype rAAV that can specifically and efficiently cross the blood-brain barrier and act broadly across various brain regions. In clinical applications, the rAAV of the present invention can be directly administered via peripheral intravenous injection for the treatment or prevention of Alzheimer's disease. It is simple to use, has no significant side effects such as damage, and avoids the potential side effects of peripheral overexpression of TPK.

[0026] 2. Effectively avoid the shortcomings of other non-serotype AAV virus injections into the ventricle, subarachnoid space or brain tissue, such as complex operation and significant damage.

[0027] 3. The difference between the AAV.CAP-B10.hsyn-TPKV of the present invention and other gene therapies is that AD is treated by regulating brain energy metabolism through supplementing brain neuron TPK.

[0028] 4. The AAV.CAP-B10.hsyn-TPK of the present invention only exogenously mediates the compensatory expression of TPK, does not involve endogenous TPK, and does not involve the editing of other genes, which can greatly reduce other risks.

[0029] 5. The AAV.CAP-B10.hsyn-TPK of the present invention introduces highly active exogenous TPK, achieving beneficial therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a plasmid map of the recombinant adeno-associated virus genome before insertion of the TPK target gene in Example 1.

[0031] FIG2 is a plasmid map of the recombinant adeno-associated virus genome after insertion of the TPK target gene in Example 1.

[0032] FIG3 is a diagram showing the results of DNA gel electrophoresis detection of PCR amplification products in Example 2.

[0033] FIG4 is a diagram showing the results of DNA gel electrophoresis detection of the colony PCR amplification products in Example 2.

[0034] FIG5 is a diagram showing the comparison results of high-throughput sequencing of transformants in Example 2.

[0035] FIG6 is a graph showing the results of the Y-maze experiment in Example 3 to test the mice's round rate.

[0036] FIG7 is a graph showing the results of the rotating rod experiment for testing the terminal running speed of mice in Example 3.

[0037] FIG8 is a graph showing the nesting scores of mice 24 hours after the nesting experiment in Example 3.

[0038] FIG9 is a graph showing the nesting scores of mice 48 hours after the nesting experiment in Example 3.

[0039] FIG10 is a graph showing the weighing results of the hemisphere, cortex, and hippocampus of mice in Example 4.

[0040] FIG11 is a graph showing the results of the brain surface area of ​​the left hemisphere of mice in Example 4.

[0041] FIG12 shows representative images and quantitative graphs of TPK protein detected by Western blotting in mouse cortical samples in Example 5.

[0042] FIG13 shows representative images and quantitative graphs of TPK protein detected by immunofluorescence staining in mouse cortical samples in Example 5.

[0043] FIG14 is an image of a mouse brain tissue section under a laser confocal microscope after AAV.CAP-B10 virus brain infection in Example 6.

[0044] Figure 15 is an image of the liver, heart, kidney, and testicle organ slices of mice after AAV.CAP-B10 virus infection under a laser confocal microscope in Example 6.

[0045] FIG16 is a graph showing the results of Western blot detection of APP, BACE1, Tau and p-Tau (S396) in Example 7.

[0046] FIG17 is a quantitative graph of Western blot detection of APP, BACE1, Tau and p-Tau (S396) in Example 7.

[0047] FIG18 shows representative images and quantitative graphs of immunofluorescence staining detection of NeuN antibody staining in Example 7.

[0048] FIG19 shows representative images and quantitative graphs of immunofluorescence staining detection using 4G8 and Iba1 antibodies in Example 7.

[0049] FIG20 shows representative images and quantitative graphs of immunofluorescence staining detection of GFAP antibody staining in Example 7. DETAILED DESCRIPTION

[0050] By the following examples, those skilled in the art will more clearly understand the present invention. It should be understood that the following examples are intended to illustrate embodiments of the present invention, rather than to limit the scope of the present invention. Unless otherwise specified, the methods used in the present invention are conventional methods in the art, and the experimental materials used are also commercially available.

[0051] Example 1. Preparation of AAV.CAP-B10.hsyn-TPK recombinant adeno-associated virus

[0052] The CRO company Wuhan Shumi Brain Science Technology Co., Ltd. was commissioned to produce laboratory-grade AAV encoding TPK (serotype AAV-CAP-B10), hereinafter referred to as AAV-TPK. Its genome contains: i) a neuron-specific promoter (SEQ ID NO: 2), ii) a nucleotide sequence encoding mouse TPK (SEQ ID NO: 3) and a WPRE element (SEQ ID NO: 4). Figures 1 and 2 show the plasmid maps (including restriction enzyme sites) of the recombinant adeno-associated virus genome before and after insertion of the TPK target gene, respectively.

[0053] 1. Experimental Methods

[0054] The construction method of the above-mentioned recombinant adeno-associated virus vector is as follows:

[0055] Step 1: obtain the TPK target gene by PCR amplification or restriction endonuclease digestion, and then assemble it with an adeno-associated virus vector by homologous recombination using the homologous recombinase Exnase or by T4 DNA ligation;

[0056] Step 2: Transform the viral vector assembled in step 1 into E. coli Stbl3 competent cells;

[0057] Step 3: Screen the transformants using colony PCR, send the positive clones for sequencing, and confirm the positive clones by sequence comparison.

[0058] The reagents, instruments, and vector information used in the construction method are as follows.

[0059] 1. Main Reagents

[0060] The main reagents used in the experiment of this embodiment are shown in Table 1.

[0061] Table 1

[0062] 2. Main instruments

[0063] The main instruments used in the experiment of this embodiment are shown in Table 2.

[0064] Table 2

[0065] 3. Plasmid Information

[0066] The vector plasmid information of this example is shown in Table 3.

[0067] Table 3

[0068] 4. PCR amplification primers

[0069] The sequences of the PCR primers used in step 1 of the construction method of this embodiment are shown in Table 4. The primers contain exchange pairing bases, restriction sites, and a partial sequence at the 5' end of the target gene for PCR fishing for the target gene.

[0070] Table 4

[0071] 5. PCR amplification reaction system

[0072] After DNA extraction, add the following components to an RNase-free PCR tube on ice, mix thoroughly, and centrifuge briefly:

[0073] After mixing, set up the PCR cycler. PCR cycle steps:

[0074] 2. Experimental Results

[0075] 1. Target gene PCR results

[0076] In step 1 of the above construction method, the TPK target gene was obtained by PCR amplification, and the PCR amplification product was detected by DNA gel electrophoresis. The result is shown in FIG3 .

[0077] In Figure 3, 1#: Plus II DNA Marker (bands from top to bottom are: 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp) recombinant plasmid construction; 2#: target fragment amplification band.

[0078] The results in Figure 3 show that the DNA molecular weight of the PCR amplification product is 775 bp, indicating that the target gene was amplified successfully.

[0079] After the target gene is successfully amplified by PCR, the linearized vector and the target gene fragment are mixed in proportion, and homologous recombination connection between the target gene fragment and the vector is achieved under the action of the homologous recombinase Exnase.

[0080] 2. Transformant PCR results

[0081] In step 3 of the above construction method, transformants were screened by colony PCR, and the PCR identification primer sequences are shown in Table 5. The colony PCR amplification products were detected by DNA gel electrophoresis, and the results are shown in Figure 4.

[0082] Table 5

[0083] In Figure 4, 1#: Markers from top to bottom are 8kb, 5kb, 3kb, 2kb, 1Kb, 750bp, 500bp, 250bp, and 100bp; 2-9#: transformants No. 1-8.

[0084] The results in FIG4 show that the DNA molecular weight of the colony PCR amplification product is 775 bp, indicating that the transformant contains the target gene fragment and is a positive clone.

[0085] 3. Transformant Sequencing Results

[0086] High-throughput sequencing was performed on the transformants of the above positive clones. The sequencing primer sequences are shown in Table 6. The sequencing alignment results are shown in Figure 5, where the color-marked parts are the target gene sequences.

[0087] Table 6

[0088] The comparison results in FIG5 show that among the transformants of the positive clones screened by colony PCR, the transformants containing the target gene fragment were determined to be positive clone transformants after sequence comparison.

[0089] Example 2: Packaging of recombinant adeno-associated virus

[0090] 1. Prepare Plasmid

[0091] The viral vector (virus carrying exogenous genes), pHelper (carrying adenovirus-derived genes), and pAAV-RC (carrying AAV replication and capsid genes) plasmids screened and identified in Example 1 were extracted in large quantities.

[0092] 2. AAV-293 Cell Culture

[0093] (1) AAV-293 cell recovery

[0094] Preheat the culture medium, then remove the cell cryopreservation tube from the liquid nitrogen tank and quickly place it in a water bath filled with 37°C water, shaking it occasionally to thaw it as quickly as possible; wipe and disinfect it with 70% alcohol, and move it to an ultra-clean workbench; aspirate the cell suspension and add it to 3 ml of culture medium, then add 5 ml of culture medium to dilute it, and gently blow it apart; centrifuge at 1000 rpm for 3 minutes to collect the cells; resuspend the cells in an appropriate amount of culture medium for inoculation.

[0095] (2) AAV-293 cell passage (10 cm dish as an example)

[0096] Discard the old culture medium, add 10 ml of sterile PBS, shake gently to wash the cell growth surface, and then discard the PBS; add 1 ml of trypsin digestion solution and digest for 1 to 2 minutes until the cells become round and begin to fall off; add 2 ml of complete culture medium to terminate the digestion and pipette the cells at the bottom of the dish into single cells; resuspend by centrifugation and count and inoculate as needed, or mix the cells and directly transfer them to a new culture flask for further culture.

[0097] (3) Cryopreservation of AAV-293 cells

[0098] Take the vigorously growing cells that have been cultured for 2 to 3 days, digest the cells and blow them into single cells according to the subculture procedure; collect the cells by centrifugation at 1000 rpm for 3 minutes; add an appropriate amount of PBS to resuspend the cells and count them; collect the cells by centrifugation at 1000 rpm for 3 minutes; resuspend the cells in freezing medium to 2×106 to 5×106 cells / ml, and then divide the cell suspension into 2 ml cryovials, 1 ml / tube; place the tubes in a programmed cooling box and then freeze the box at -80℃ overnight. The next day, transfer the tubes of frozen cells to liquid nitrogen for long-term storage.

[0099] 3. Cell Transfection

[0100] (1) Inoculation of AAV-293 cells

[0101] AAV-293 cells were seeded onto a 10 cm dish and used for transfection 48 hours later, when the confluency reached 70-80%. One hour before transfection, the cell culture plate was removed, the original cell culture medium was removed, and 10 ml of Opti-MEM medium was added.

[0102] Then prepare the complex of transfection reagent and plasmid as follows: dissolve 32 μg of viral vector plasmid to be transfected (pHelper: pAAV-RC: viral vector = 1:1:1) in Opti-MEM medium to a total volume of 500 μl, mix gently, and let stand for 5 minutes; dissolve the transfection reagent in Opti-MEM medium to a total volume of 500 μl, mix gently, and let stand for 5 minutes; add the transfection reagent dilution solution dropwise to the plasmid dilution solution, mix gently while adding, and let it stand at room temperature for 20 minutes to allow the DNA and transfection reagent to fully combine to form a stable transfection complex.

[0103] After preparing the complex of transfection reagent and plasmid, take out the cell dish and add the prepared DNA-transfection reagent complex to the cell culture plate.

[0104] (2) Change the medium after transfection

[0105] After 6 h, the culture medium was aspirated, the cells were washed once with PBS, and 10 ml of fresh complete culture medium was added for culture.

[0106] 4. Virus Harvest and Purification

[0107] (1) 60 hours after transfection, scrape the cells with a cell scraper and collect them into a centrifuge tube; centrifuge at 1500 rpm and 4°C for 5 minutes. Resuspend the cell pellet in 9 ml of lysis buffer and collect the culture medium into a 500 ml bottle; freeze the cell lysate in liquid nitrogen for 3 minutes, then freeze and thaw 3-4 times in a 37°C water bath; add 250 U of benzonase, mix well, and incubate at 37°C for 1 hour;

[0108] (2) Add NaCl to a final concentration of 150 mM, mix well, and incubate at 37°C for 30 min; centrifuge at 2500 g at room temperature for 10 min, take the supernatant and discard the precipitate; pour the supernatant after centrifugation into the supernatant collected by centrifugation in step (1), add PEG8000 and NaCl to final concentrations of 8% and 0.5 M, respectively, and incubate at 4°C overnight; centrifuge at 12000 rpm at 4°C for 1.5 h, discard the supernatant, and resuspend the precipitate in 10 ml PBS; add the resuspended virus stock solution to an ultracentrifuge tube containing iodixanol (the concentrations of iodixanol are 15%, 25%, 40%, and 60%, respectively), and centrifuge at 63000 rpm at 18°C ​​for 2 h;

[0109] (3) Use a 10 ml syringe to draw out the 40% layer of sample and place it into a dialysis bag. Dialyze at 4°C, changing the dialysate every 12-16 hours, using 2 L of buffer each time. Collect the dialyzed sample and concentrate it using an ultrafiltration tube.

[0110] 5. Titer Determination

[0111] Currently, the titer of AAV is determined by quantitative PCR to detect the number of AAV vector genome copies in the genome to determine the number of AAV viral particles. The specific steps are as follows:

[0112] Prepare the standard: Use the plasmid standard, calculate the concentration of the standard, dilute to 1E+8 cells / μL, and then dilute to 1E+3 cells / μL, for a total of 6 gradients;

[0113] Prepare the sample: Alkaline lysis - Take 5 μL of concentrated virus sample, add NaOH to a final concentration of 1 M, incubate in a 55°C water bath for 30 minutes, then add HCl to neutralize, and dilute 10-fold with pure water or 1X PBS.

[0114] 18 μl of reaction solution was added to each reaction well, followed by 2 μl of template. The annealing temperature was set to 60°C, and the Ct value was obtained according to standard procedures to calculate the copy number in the AAV sample.

[0115] Example 3: Improvement of animal cognitive behavior in cKO mice by tail vein injection of AAV-TPK

[0116] The purpose of this example is to verify whether gene therapy can significantly improve the expression of TPK protein in the brain of CaMKII-CreERT2 / +; Tpkfl / fl mice and improve the cognitive function impairment of transgenic mice by injecting AAV virus into the mouse tail vein.

[0117] Three-month-old CaMKII-CreERT2 / +;Tpkfl / fl mice (cKO) and littermate control mice (WT) were induced with tamoxifen (30 mg / kg) and randomly divided into two groups (WT-Ctrl, n=9; WT-TPKovp, n=12; cko-Ctrl, n=9; cKO-TPKovp, n=11).

[0118] After 4 weeks of tamoxifen induction, mice were injected into each group through the tail vein, and a suspension of AAV-TPK (WT-TPKovp group and cKO-TPKovp) and control rAAV (same as TPK-AAV except for the absence of TPK coding sequence) with a titer of 2.5E+12vg / mL (prepared in PBS) was administered, 200 μL each. Specifically, the mice were fixed on a tail vein injection fixture, the mouse tail was wiped with an alcohol cotton ball for disinfection, and 200 μL of virus suspension was injected into the mouse tail vein using a 1 ml syringe. After the injection, the dry cotton ball pressed the injection site for 30 seconds to prevent the virus from overflowing. After the mice were injected with the virus and continued to be raised for 4 weeks, all mice were subjected to behavioral tests, biochemical assays and pathological examinations. The cognitive function behavioral evaluation experiment of this embodiment includes a Y maze experiment, a rotating rod experiment, and a nesting experiment, and the specific experimental methods are as follows.

[0119] 1. Experimental Methods

[0120] 1. Y-maze test

[0121] All mice were tested in a Y-maze experiment according to conventional methods. The results are shown in Figure 6.

[0122] 2. Rotarod Test

[0123] The diameter of the rotating rod is 3 cm and the channel width is about 8 cm. On the day of the test, the mice were acclimated to the test room for 30 minutes before the experiment. After 30 minutes, the mice were acclimated and placed in the runway. After the mice stood firmly, the rod rotated at a constant speed of 4 rpm. The acclimation time was 120 seconds. After a batch of mice had adapted, each runway was disinfected with 75% alcohol and wiped dry before the next batch of mice were acclimated. The final test was carried out at least half an hour after all mice had adapted. During the test phase, the runway was adjusted to an initial 4 rpm, with an acceleration time of 5 seconds and a duration of 10 seconds. After that, the rod was accelerated from 4 rpm to 35 rpm within 240 seconds, and 35 rpm was maintained for 60 seconds. The speed at which the mice fell from the rotarod was recorded. The results are shown in Figure 7.

[0124] 3. Nesting Experiment

[0125] Nesting behavior experiments are often used to evaluate the daily activities of mice with cognitive dysfunction. This experiment can evaluate the flexibility of fine movements, cognition and emotional state of mice while applying minimal pressure to them. The material used for nesting is cylindrical fiber cotton with a diameter of 3 cm and a length of 3 cm. Before the test, the mice were kept in single cages, and the materials that could be used for nesting in the cages were removed. The nesting materials were placed in the cages, and sufficient food and water were ensured. The nesting score was performed after 12 hours, and the scoring criteria are shown in Table 7. Among them, Figures 8 and 9 show the nesting scores of mice 24 hours and 48 hours after the start of the experiment, respectively.

[0126] Table 7

[0127] 2. Experimental Results

[0128] The results in Figure 6 showed that the Y-maze test showed that the bout rate of cKO mice was significantly reduced compared with that of the same littermate control mice, while the bout rate of cKO mice treated with TPK overexpression was significantly rescued.

[0129] The results in Figure 7 showed that compared with the control mice of the same littermate, the terminal running speed of cKO mice was significantly reduced using the rotarod test, while the terminal running speed of cKO mice treated with TPK overexpression was significantly rescued.

[0130] The results in Figures 8 and 9 show that compared with the same littermate control mice, the nesting test showed that the nesting scores of cKO mice were significantly reduced 24 hours and 48 hours after the start of the nesting experiment, while the nesting scores of cKO mice treated with TPK overexpression were significantly rescued.

[0131] Experimental conclusion: Tail vein injection of AAV.CAP-B10.hsyn-TPK can improve the cognitive function of cKO mice.

[0132] Example 4: Improvement of brain atrophy in cKO mice by tail vein injection of AAV.CAP-B10.hsyn-TPK

[0133] After the mouse behavioral experiment, the mice were euthanized and then perfused with pre-cooled PBS. After the blood was completely flushed out, the left hemibrain tissue of the mice was collected (the cerebellum, brainstem and olfactory bulb were removed, and the left and right brains were separated). The hemibrain, cortex and hippocampus of the mice were weighed respectively, and the results are shown in Figure 10. The left hemibrain was further perfused and fixed with pre-cooled 4% paraformaldehyde. After the perfusion, the hemibrain was taken out and photographed with a camera. Finally, ImageJ software was used to frame the cortical surface along the cortical surface and calculate the brain surface area of ​​the left hemibrain of the mouse. The results are shown in Figure 11.

[0134] The results in Figure 10 show that the weights of the cKO cortex, hippocampus and hemibrain of cKO mice were significantly reduced compared with control mice, while the weights of the cKO cortex, hippocampus and hemibrain of cKO mice treated with TPK overexpression were significantly rescued.

[0135] The results in FIG11 show that the left hemisphere surface area of ​​cKO mice was significantly reduced compared with control mice, while the left hemisphere surface area of ​​cKO mice treated with TPK overexpression was significantly rescued.

[0136] Experimental conclusion: Injection of AAV-TPK virus can significantly reduce neuronal loss in cKO mice and significantly increase the weight of the cKO cortex, hippocampus and hemibrain, thereby improving brain atrophy in cKO mice.

[0137] Example 5: Distribution of AAV-CAP-B10 in mice after tail vein injection and restoration of TPK protein expression in cKO mouse neuronal cells by AAV-TPK

[0138] 1. Experimental Materials

[0139] The specific sources of the primary and secondary antibodies used in the experiments of this example and the following examples are shown in Table 8.

[0140] Table 8

[0141] 2. Experimental Methods

[0142] 1. Western Blot Detection

[0143] Add 1x protease and phosphatase inhibitors to the tissue lysis buffer and pre-cool it on ice. In the experiment of Example 4, after the cortex of the left hemisphere of the mouse was weighed, pre-cooled tissue lysis buffer was added in proportion (about 100 μL for every 10 mg of tissue), and 2-3 grinding steel beads were added to the tube. The ep tube was placed in a grinding machine at 4°C and shaken for 100s. After shaking, it was placed on ice for 30 minutes to lyse. After thorough lysis, the homogenate was centrifuged at 12000 rpm in a high-speed centrifuge for 30 minutes. Then take the supernatant and transfer it to a new pre-cooled microcentrifuge tube and place it on ice. It is the protein sample, and the precipitate is discarded. The BCA method is used for protein quantification: bovine serum albumin (BSA) is used as the standard curve. Based on the biuret principle, proteins will Cu under alkaline conditions. 2+ Reduction to Cu 1+ , BCA (Bicinchoninic acid) chelated Cu 1+ As a color developer, it produces blue-purple and has an absorption peak at 562nm. 1+The specific experimental method of BCA method is as follows.

[0144] BCA Reagent A and Reagent B are mixed in a 50:1 ratio to create a working solution. Add 20 μL of standard samples at concentrations of 0, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL to a 96-well plate. Dilute the test sample to the appropriate concentration, bringing the total volume of the sample dilution to 20 μL. Add 200 μL of BCA working solution to each well. Oscillate the plate on a shaker and incubate at 37°C for 30 minutes. Then, measure the absorbance colorimetrically at 562 nm. Plot a standard curve with protein content (μg) on ​​the horizontal axis and absorbance on the vertical axis. Based on the absorbance value of the test sample, the corresponding protein content (μg) is calculated on the standard curve. Divide the calculated protein content by the total volume of the sample dilution (20 μL) and multiply by the sample dilution factor to obtain the actual sample concentration (μg / μL).

[0145] Based on the sample concentration determined by the BCA assay, all samples were adjusted to 1 μg / μL using lysis buffer and loaded. Mix thoroughly and heat in a 95°C water bath for 10 minutes. Protein sample preparation was complete. The precast gel was inserted into the electrophoresis tank, the electrophoresis buffer was added, and the comb was removed. A 10 μL marker was placed on both sides, and 10 μL of protein sample was added to each lane. The voltage was adjusted to 80 V, and the stacking gel was run for 30 minutes. The protein separation voltage was 120 V for 80 minutes. After the gel run, transfer to the membrane was performed. The sample clamps were installed in the following order: black plate (bottommost) → sponge pad → filter paper → gel → PVDF membrane (pre-soaked in methanol for 30 seconds) → filter paper → sponge pad → white plate → buckle (compacting each layer with a roller). The electrophoresis tank was then assembled (placed in ice, with an ice pack in the tank, with the clamps facing the black side and the red side facing the red side). The electrophoresis solution was added until the liquid level exceeded the PVDF membrane, and the flow was run at a constant current of 0.2 A for 60 minutes.

[0146] After transfer, the membrane was blocked with 5% skim milk in TBST for 2 hours at room temperature. After blocking, the membrane was cut according to the size of the bands and incubated overnight at 4°C on a shaker with anti-TPK, anti-APP, anti-BACE1, anti-Tau, anti-pTau-S396, and anti-β-actin antibodies.

[0147] The next day, the primary antibody was aspirated, and the membrane was washed three times with TBST for 10 minutes each time. Then, the corresponding secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG) were prepared with 5% skim milk powder and incubated on a shaker at room temperature for 2 hours. The secondary antibody was aspirated, and the membrane was washed three times with TBST for 10 minutes each time. After drying slightly, it was incubated in ECL colorimetric solution (A solution: B solution is 1:1) in the dark, and then developed in a colorimeter to compare the relative content of protein in each sample. The results are shown in Figure 12, where Figure 12a and Figure 12b respectively show representative images and quantitative graphs of TPK protein detected by western blotting in cortical samples 4 weeks after virus infection.

[0148] The results in Figure 12 show that tail vein injection of AAV-TPK can significantly increase the TPK level in the cerebral cortex of WT mice and cKO mice.

[0149] 2. Immunofluorescence staining

[0150] The right hemisphere of the mouse was fixed with 4% paraformaldehyde (PFA) and then subjected to immunofluorescence staining as follows. After fixation with 4% PFA for 12-24 hours, the right brain was dehydrated and precipitated in 30% sucrose for 2 days. After precipitation, the right brain was embedded in cryostat (OCT) and stored at -80°C. The embedded right brain was sliced ​​into 30 μm thick slices using a cryostat, with the section angle being longitudinal (coronal) from the olfactory bulb to the cerebellum. After sectioning, the slices were washed three times with 1X PBS on a shaker for 10 minutes each time, the OCT was washed, and 0.5% Triton was added to permeabilize the membrane at room temperature for 30 minutes. Then, 3% BSA (bovine serum albumin) was replaced for blocking at room temperature for 1 hour. Anti-GFP antibody, anti-TPK1 antibody, and anti-NeuN antibody were added for co-staining and incubated at 4°C overnight; the primary antibody was recovered, and the slices were washed three times with 1X PBS for 5 minutes each time. The corresponding secondary antibodies (goat anti-chicken IgY H&L Alexa Fluor 488, donkey anti-rabbit IgG H&L Alexa Fluor 647, goat anti-mouse IgG H&L Alexa Fluor 555) were added and incubated at room temperature in the dark for 2 hours. Finally, the slices were attached to glass slides, slightly dried, and then mounting medium was added for sealing. The images were taken using a Nikon Ti2E inverted microscope. The results are shown in FIG13 , where FIG13 a and FIG13 b show representative images and quantitative graphs of TPK protein in cortical samples detected by immunofluorescence staining 4 weeks after AAV delivery, respectively.

[0151] The results in FIG13 showed that GFP was expressed in all groups, but TPK was strongly expressed only in the TPKovp group (including WT-TPKovp and cKOTPKovp groups), which was consistent with the changes in protein levels.

[0152] Experimental conclusion: Tail vein injection of AAV-TPK can significantly increase the TPK level in the cerebral cortex of cKO mice.

[0153] Example 6: Distribution of AAV-CAP-B10 in mice after tail vein injection

[0154] This example is used to explore the distribution of peripherally administered AAV.CAP-B10 virus in various organs in mice. Using the same method as the above example, after AAV-CAP-B10 was injected into the tail vein of mice, tissue sections of various organs in the mice were sliced ​​and fluorescent protein signals were detected. The results are shown in Figures 14 and 15. Among them, Figure 14 shows the imaging of mouse brain tissue sections under a laser confocal microscope after AAV.CAP-B10 virus brain infection; Figure 15 shows the imaging of mouse liver, heart, kidney, and testicular organ sections under a laser confocal microscope after AAV.CAP-B10 virus infection.

[0155] In Figure 14 , Figure 14A shows DAPI signals, Figure 14B shows virally expressed fluorescent protein signals, and Figure 14C shows a composite of Figures A and B, showing the region of viral expression. Scale bar: 500 μm.

[0156] Figure 15 : Figures 15A-C show images of liver tissue, where Panel A shows DAPI signal, Panel B shows virally expressed fluorescent protein signal, and Panel C is a composite of Panels A and B, showing the region of viral expression; Figures 15D-F show images of heart tissue; Figures 15G-I show images of kidney tissue; and Figures 15J-L show images of testicular tissue. The order of each row is the same as the first row. Scale bar: 100 μm.

[0157] The results in Figures 14 and 15 show that after tail vein injection of AAV-CAP-B10, the virus was expressed in the mouse brain tissue, but not in the mouse liver, heart, kidney, and testicle tissues.

[0158] Experimental conclusion: Peripheral administration of AAV.CAP-B10 virus can specifically target the brain without expressing in peripheral organs. This operation is convenient and avoids the potential side effects of peripheral overexpression of TPK.

[0159] Example 7: Improvement of Multiple Pathological Changes in cKO Mice by Tail Vein Injection of AAV-TPK

[0160] To investigate neuronal loss and brain inflammatory activation, brain slices were incubated with anti-NeuN and anti-GFAP antibodies using the same methods as in Example 5. Separately, the slices were co-stained with anti-Iba1 and anti-4G8 antibodies. Western blotting and immunofluorescence staining were performed using the same methods as in Example 5. The results are shown in Figures 16-20.

[0161] 16 and 17 collectively show representative images and quantitative graphs of APP, BACE1, Tau, and p-Tau (S396) detected by Western blotting.

[0162] Figures 18 to 20 collectively show representative images and quantitative graphs of immunofluorescence staining using NeuN, 4G8, Iba1, and GFAP antibodies.

[0163] The results in Figures 16 and 17 show that compared with littermate control mice, the APP and BACE1 protein levels, p-Tau-S396, and p-Tau / Tau ratio in cKO mice were significantly increased, while the total Tau protein level was decreased. These changes can be significantly rescued by TPK overexpression treatment.

[0164] The results in Figures 18 to 20 show that compared with littermate control mice, cKO mice had significant neuronal loss, Aβ enhancement, and microglia and astrocyte activation, and these changes could be significantly rescued by TPK overexpression treatment.

[0165] Experimental conclusion: Tail vein injection of AAV-TPK can effectively improve the increase of APP and BAC1, the increase of p-Tau-S396 and the decrease of total Tau caused by the decrease of TPK levels, thereby effectively reversing these pathological changes; in addition, it can also effectively reverse the neuronal loss of cKO mice, and alleviate the activation of astrocytes and microglia caused by the decrease of TPK levels, and effectively reduce the deposition of Aβ plaques.

[0166] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that those skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be considered as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by those skilled in the art using the technical content disclosed above without departing from the spirit and scope of the present invention are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A TPK-expressing virus that can be administered intravenously, characterized in that: The TPK expression virus is a recombinant AAV virus, and its genome comprises a neuron-specific promoter, a nucleotide sequence encoding the TPK gene, and a WPRE element.

2. The intravenously administrable TPK expressing virus according to claim 1, characterized in that: The AAV virus is serotype AAV.CAP-B10, the gene sequence of the neuron-specific promoter is shown in SEQ ID NO:2, the nucleotide sequence encoding the TPK gene is shown in SEQ ID NO:3, and the sequence of the WPRE element is shown in SEQ ID NO:

4.

3. The intravenously administrable TPK expressing virus according to claim 1, characterized in that: The recombinant AAV virus sequence is shown in SEQ ID NO:

1.

4. The method for constructing a TPK expression virus that can be administered intravenously according to claim 1, characterized in that: The following steps are involved: Step 1, obtaining the TPK target gene by PCR amplification or restriction endonuclease digestion, and then assembling it with an adeno-associated virus vector by homologous recombination using a homologous recombinase Exnase or by T4 DNA ligation; Step 2, transforming the viral vector assembled in step 1 into E. coli Stbl3 competent cells; Step 3: Screen the transformants by colony PCR, send the positive clones for sequencing, and confirm the positive clones by sequence comparison; Step 4: Co-transfect AAV-293 cells with the screened and identified viral vector, pHelper plasmid, and pAAV-R plasmid to complete virus packaging and purification.

5. The method for constructing a TPK expression virus that can be administered intravenously according to claim 4, characterized in that: The primers for PCR amplification in step 1 or colony PCR in step 3 are 4423-BamHI-F primer and 4423-SalI-R primer, the sequence of the 4423-BamHI-F primer is shown in SEQ ID NO:5, and the sequence of the 4423-SalI-R primer is shown in SEQ ID NO:

6.

6. The method for constructing a TPK expression virus that can be administered intravenously according to claim 4, characterized in that: The forward sequencing primer and reverse sequencing primer for transformant sequencing in step 3 are hSyn-CF primer and pEGFP-N-3' primer respectively. The sequence of the hSyn-CF primer is shown in SEQ ID NO:7, and the sequence of the pEGFP-N-3' primer is shown in SEQ ID NO:

8.

7. A kit for preparing the TPK expression virus for intravenous administration according to claim 1, characterized in that: The kit includes 4423-BamHI-F primer and 4423-SalI-R primer for PCR amplification, the sequence of the 4423-BamHI-F primer is shown in SEQ ID NO:5, and the sequence of the 4423-SalI-R primer is shown in SEQ ID NO:

6.

8. A kit for preparing a TPK expressing virus for intravenous administration according to claim 7, characterized in that: The kit also includes a forward sequencing primer and a reverse sequencing primer for detecting positive clones. The forward sequencing primer is hSyn-CF primer, and its sequence is shown in SEQ ID NO:7; the reverse sequencing primer is pEGFP-N-3' primer, and its sequence is shown in SEQ ID NO:

8.

9. Use of the intravenously administrable TPK-expressing virus according to claim 1 in the preparation of a drug for preventing or treating Alzheimer's disease.

10. Use of the intravenously administrable TPK-expressing virus according to claim 9 in the preparation of a drug for preventing or treating Alzheimer's disease-like neurodegenerative lesions, brain atrophy, Aβ and tau pathological changes, neuroinflammation, neurovascular disorders, or slowing down the progression of Alzheimer's disease.

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