Use of a small molecule drug, plx4032, in the preparation of a medicament for treating cerebral arteriovenous malformation disease

Abstract

This invention discloses the application of the small molecule drug PLX4032 in the preparation of a drug for the treatment of cerebral arteriovenous malformation. The small molecule drug PLX4032 of this invention can inhibit Braf... V600E Overactivation of the mutant protein Braf-MEK1 signaling pathway, thereby reducing Braf V600E The mutant protein's kinase activity, thereby preventing Braf from occurring in arteriovenous malformations of the brain. V600E The expression of mutant proteins can directly target diseased cells, resulting in good therapeutic effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to application of a small molecule drug PLX4032 in preparation of a drug for treating brain arteriovenous malformation disease. BACKGROUND

[0002] Brain arteriovenous malformation (bAVM) is one of the four major vascular malformations, the other three including venous malformation, cavernous malformation (CCM) and capillary telangiectasia. Among them, arteriovenous malformation is one of the most prominent phenotypes of vascular malformation types. bAVM was first described in 1895, and bAVM is characterized by arterial blood supply, high flow and low resistance shunt between arteriovenous. The malformation blood vessels are composed of dilated arteries converging from all directions and twisted and pulsating veins, without capillaries.

[0003] For the treatment of AVM disease, different grades of AVM have different treatment options. Generally, if diagnosed with intracranial AVM, the doctor will recommend to observe the development of the lesion first. If it is non-malignant, it can be controlled with drugs and does not need to be operated. Conversely, if the lesion is obvious, it can be surgically removed and embolization ("glue treatment") therapy can be considered. If the lesion is in an inaccessible position, Gamma Knife radiosurgery (GKRS) can be considered. In fact, GKRS is the preferred treatment for most arteriovenous malformation patients in the UK. In terms of therapeutic drugs, Bevacizumab has been reported to have reached clinical phase I. Bevacizumab is an anti-tumor drug also known as Avastin. It is a humanized monoclonal antibody that can target vascular endothelial growth factor (VEGF) in arteriovenous malformation, thereby inhibiting angiogenesis and growth in the lesion area. Since Avastin is a protein drug, it is difficult to effectively pass through the blood-brain barrier and act on the lesion brain area, so small molecule compounds represent a more attractive solution. How to target lesion cells and design and screen small molecule inhibitors based on BRAF-MEK1 signaling pathway kinases as targets has become the core direction of drug development. SUMMARY

[0004] In view of the problem of screening small molecule inhibitor drugs directly targeting lesion cells in drugs for treating brain arteriovenous malformation disease, the present application provides application of a small molecule drug PLX4032 in preparation of a drug for treating brain arteriovenous malformation disease. The drug is a small molecule inhibitor directly targeting lesion cells and based on BRAF-MEK1 signaling pathway kinases as targets.

[0005] Specifically, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a use of a small molecule drug PLX4032 in the preparation of a medicament for treating a cerebral arteriovenous malformation disease, wherein the small molecule drug PLX4032 has the following structure:

[0007]

[0008] Preferably, the use is characterized in that the cerebral arteriovenous malformation disease is manifested in Braf V600E mutant protein.

[0009] Preferably, the use is characterized in that the cerebral arteriovenous malformation disease is manifested in Braf V600E mutant protein.

[0010] Preferably, the use is characterized in that the cerebral arteriovenous malformation disease is manifested in over-activation of Braf-MEK1 signaling pathway.

[0011] Preferably, the use is characterized in that the small molecule drug PLX4032 is selected from one of a tablet, a solution, a suspension, an emulsion, a powder, a granule, a capsule, a microcapsule, a microsphere, and an injection.

[0012] Preferably, the use is characterized in that the small molecule drug PLX4032 further comprises a conventional carrier for pharmacy.

[0013] Preferably, the use is characterized in that the conventional carrier is selected from one or more than two of a filler, a binder, a humectant, an absorption promoter, and a cosolvent.

[0014] Advantages of the present application:

[0015] The small molecule drug PLX4032 of the present application can inhibit over-activation of Braf V600E mutant protein Braf-MEK1 signaling pathway, thereby reducing the kinase activity of Braf V600E mutant protein, and further avoiding expression of Braf V600E mutant protein in the cerebral arteriovenous malformation disease, and can directly target diseased cells, thus having a good treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1The expression data of Tek gene in human and mouse brain vasculature single cell and mixed cell transcriptome is shown, wherein a is the expression profile of Tek gene in human brain vasculature single cell transcriptome; wherein ART: artery; CAP: capillary; VEN: vein; aSMC: arterial vascular smooth muscle cell; aaSMC: arteriolar smooth muscle cell; T-PC: solute transport-pericyte; M-PC: extracellular matrix regulation-pericyte; P.FB: peripheral fibroblast; M.FB: meningeal fibroblast; TC: T cell; EPEN: ependymal cell; AST-HPc: hippocampal astrocyte; AST-Ctx: cortical astrocyte; PM: peripheral macrophage; MG: microglia; OL: oligodendrocyte; OPC: oligodendrocyte precursor cell; NEU: neuron; b is the expression profile of Tek gene in mouse brain vasculature single cell transcriptome; wherein PC: pericyte; vSMC: vein smooth muscle cell; aaSMC: arteriolar smooth muscle cell; aSMC: arterial smooth muscle cell; MG: microglia; FB1: fibroblast-like type 1; FB2: fibroblast-like type 2; OL: oligodendrocyte; EC1: endothelial cell type 1; EC2: endothelial cell type 2; EC3: endothelial cell type 3; vEC: vein endothelial cell; capilEC: capillary endothelial cell; aEC: artery endothelial cell; AC: astrocyte; c is the expression profile of Tek gene in mouse brain cell type-specific mixed cell transcriptome.

[0017] Figure 2 The Tek-Cre transgenic mouse can efficiently and specifically label the endothelial cells of each segment of the brain blood vessels, wherein a is the fluorescence microscopic imaging of the Tek-Cre; Ai47 transgenic mouse brain coronal section at 42 days after birth; b is the confocal fluorescence microscopic imaging of a single region of interest in the cortical region, HO: Hoechst 33342 nuclear dye, EGFP: reporter gene EGFP signal specifically expressed in the brain vascular endothelial cells of Tek-Cre positive mice, HO / EGFP: imaging result after merging the two channels of HO and EGFP, white arrow indicates brain vascular endothelial cells; c is the distribution of labeled vascular endothelial cells in the cerebral cortex, striatum and thalamus region of Tek-Cre; Ai47 double transgenic mice, white arrow indicates brain vascular endothelial cells.

[0018] Figure 3Fig. 1 shows the sequence alignment of the Tek gene promoter region in multiple species, wherein a is a DNA multiple sequence alignment analysis of the promoter region and 5' non-coding region (5' UTR) corresponding to the Tek gene in multiple species genomes including human, rhesus monkey, marmoset, pig, cow, rat and mouse; b is a phylogenetic tree analysis of sequences from each species, the promoter region and 5' non-coding region sequences corresponding to pig and cow, human and rhesus monkey and marmoset, and mouse and rat are more evolutionarily similar, respectively.

[0019] Figure 4 Fig. 2 shows the sequence alignment of the first intron sequence of the Tek gene in multiple species, wherein a is a DNA multiple sequence alignment analysis of the region corresponding to the first intron of the Tek gene in multiple species genomes including human, rhesus monkey, marmoset, pig, cow, rat and mouse; b is a phylogenetic tree analysis of sequences from each species, the first intron sequences corresponding to pig and cow, human and rhesus monkey and marmoset, and mouse and rat are more evolutionarily similar, respectively.

[0020] Figure 5 Fig. 3 shows the technical route adopted by the present application, a schematic diagram of the method process for separating a promoter from a cis-regulatory element functional segment and in vivo testing.

[0021] Figure 6 Fig. 4 shows the truncation of the Tek gene promoter segment and transduction efficiency analysis, wherein a is a schematic diagram of the truncation strategy of the Tek gene promoter segment; b is a column chart of the virus transduction efficiency mediated by each truncated version of the promoter in multiple brain regions; c is a schematic diagram of in vivo transduction fluorescence imaging of recombinant AAV virus mediated by each truncated version of the promoter, the scale bar is 100 μm.

[0022] Figure 7 Fig. 5 shows the truncation of the cis-regulatory element of the first intron segment of the Tek gene and transduction efficiency analysis, wherein a is a schematic diagram of the truncation strategy of the cis-regulatory element; b is the in vivo vascular endothelial cell transduction efficiency of recombinant AAV virus mediated by each truncated version; c is a schematic diagram of in vivo transduction fluorescence imaging of recombinant AAV virus mediated by each truncated version of the cis-regulatory element, the scale bar is 100 μm.

[0023] Figure 8(a) rAAV-miniBEND system can efficiently and specifically transduce and label brain vascular endothelial cells in whole brain, a is a sagittal section of mouse brain showing the representative image of rAAV-miniBEND system with best optimized mPro723-mCis700 truncated version transducing brain vascular endothelial cells in whole brain, transgenic fluorescent reporter mouse Ai14 (Rosa26-CAG-LSL-tdTomato), 80 μL virus AAV-PhP.eB-mPro723-Cre-mCis700 was injected via tail vein; (b) rAAV-miniBEND system co-localized with Glut1 immunofluorescence imaging in multiple brain regions, the experimental conditions of the cortex, hippocampus, thalamus group are the staining results of mouse brain sample sections after AAV-PhP.eB-miniBEND (mPro723)-Cre-mCis400 was injected into Ai47 transgenic reporter mice, the experimental conditions of the cerebellum group are the staining results of mouse brain sections after AAV-PhP.eB-miniBEND-Cre-mCis303 was injected into Ai47 transgenic reporter mice; EGFP signal indicates brain vascular endothelial cells labeled by rAAV-miniBEND system, Glut1 channel signal indicates the localization of Glut1 protein expression; HO: Hoechst 33342, a nuclear dye; Merge channel is the co-localization of Glut1 and EGFP signals.

[0024] Figure 9 (a) The continuous truncation of the 5' end sequence of the mouse Tek gene promoter region can reduce the non-specific labeling of miniBEND system in neurons in the cerebellum, where a is AAV-PhP.eB serotype used in each group, and the recombinant AAV vectors used are CAG-mScarlet, mPro973-Cre, mPro760-Cre, mPro723-Cre, respectively, and the Tek cis-regulatory elements used in the latter three groups are mCis700, and are injected into C57BL / 6J, Ai47, Ai47, Ai14 transgenic reporter mice, respectively. The virus titer information used in each group is shown in Appendix A (Table A.1), and the scale bar in the figure is 100 μm; (b) The proportion of non-specific labeling other than endothelial cells is evaluated by using the normalized labeling density (the number of labeled cells per 10 11 mm2of virus particles for in vivo transduction experiments in each mouse. 2

[0025] Figure 10 ​The truncation of human Tek gene promoter and cis-acting regulatory element and transduction efficiency analysis are shown in a, which is a schematic diagram of human Tek gene promoter truncation strategy; b is a schematic diagram of human Tek gene cis-acting regulatory element truncation strategy; c is the transduction efficiency evaluation in mice.

[0026] Figure 11 The conserved sequences of the corresponding cis-acting regulatory elements of Tek gene in different species can also mediate endothelial cell-specific gene transcription; Marmo-Cis700: truncated version of Tek gene cis-acting regulatory element Cis700 from Marmoset, Pig-Cis700: truncated version of Tek gene cis-acting regulatory element Cis700 from Pig, Rat-Cis737: truncated version of Tek gene cis-acting regulatory element Cis737 from Rat, and the promoter used in the recombinant vector is Rat-Pro1600.

[0027] Figure 12 The vector design and in vivo test results of the miniBEND overexpression system combined with tTA / TRE are shown in a, which is a schematic diagram of the vector design of the miniBEND overexpression system combined with tTA / TRE, double AAV vector strategy and single AAV vector strategy; b is the experimental mouse strain C57BL / 6J, double AAV strategy group: AAV-PhP.V1-mPro1576-tTA-mCis700+AAV-PhP.V1-TRE-EGFP-WPRE-pA, double virus co-transduction; single AAV vector strategy group: AAV-PhP.eB-TRE-EGFP-mPro723-tTA-mCis303-pA single virus transduction, virus titer information see Appendix A (Table A.1), and the scale bar in the figure is 100 μm.

[0028] Figure 13 The vector design and in vivo characterization of the overexpression system based on the miniBEND-globin fusion promoter are shown in a, which is a schematic diagram of the vector design of the overexpression system based on the miniBEND, and the chimeric intron includes two versions of glo566 and glo228, and GOI is the exogenous target gene in the figure; b is the fluorescence imaging result of the four overexpression systems packaged into AAV-PhP.eB serotype virus in C57BL / 6J mice in vivo; c is the statistical result of the corresponding cortical brain area in figure b, and the vertical coordinate represents the standardized endothelial cell marker density (n / mm2), and the standardization method is to standardize the endothelial cell marker density of each 10 11The density of endothelial cell markers corresponding to one viral particle. The number of mice used in each group and the number of statistical fields are as follows: miniBENDv1 group (4 mice, including 1 using AAV-PhP.vl, 3 using AAV-PhP.eB, 22 statistical fields); miniBENDv1 (glo566) group (3 mice, 18 statistical ROIs); miniBENDv2 (glo566) group (3 mice, 18 statistical fields); miniBENDv2 (glo228) group (3 mice, 18 statistical fields).

[0029] Figure 14 The method for constructing a focal bAVM disease model based on Braf-CA transgenic mice is shown; a is the working principle of Braf-CA transgenic mice (Dankort et al., 2007); b is an experimental schematic diagram; c is the mortality curve of the mouse model, indicating that the focal bAVM model has a certain mortality rate; a total of 12 successfully modeled mice (n = 12) were counted; d is the laser speckle blood flow imaging diagram of Braf-CA mice after bAVM modeling, which shows the representative result diagram of two model mice. Among them, Exon represents exon, Exon 1 represents the first exon of the gene, Exon 14, Exon 15, Exon 16 represent the 14th, 15th, and 16th exons.

[0030] Figure 15 The nuclear magnetic resonance imaging shows that the early stage of bAVM based on Braf somatic mutation has cerebral hemorrhage, and the later stage has tumor-like invasion and space-occupying effect, wherein a is a nuclear magnetic resonance imaging (MRI) schematic diagram, which is drawn using Biorender online software; b is a result diagram of MRI imaging of one of the successfully modeled mice at 5 days, 15 days, 22 days, and 41 days after virus injection, respectively, and the white arrow indicates the lesion area; c is the bAVM lesion growth curve, which is a total of 14 successfully modeled mice, AP Diameter (Diameter in Anterior-Posterior direction, diameter in anterior-posterior direction) represents the size of the position occupied by the lesion; PID: Post injection days (days after virus injection).

[0031] Figure 16 The Braf V600E Gene mutation can induce vascular malformations, wherein a is a representative diagram of vascular fluorescence imaging in normal brain regions and lesion regions after laminin antibody immunofluorescence experiment; b is a statistical box plot of blood vessel diameter; c is a statistical frequency distribution diagram of blood vessel diameter; the scale in the diagram is 50 μm.

[0032] Figure 17 The Braf-bAVM disease mouse model constructed is used for evaluating the therapeutic effect of the small molecule drug, wherein a is BRAF V600E The molecular structure diagram of the kinase selective inhibitor PLX4032 and the experimental process schematic diagram are shown, and the drug concentration is 75 mg / kg; the control group is injected intraperitoneally with an equal amount of DMSO (Vehicle, carrier); b is a columnar chart showing the lesion size comparison between the drug administration group and the control group, the vertical coordinate represents the bAVM lesion area in the anteroposterior direction (Nidus, AP diameter), the error bar represents the standard deviation of the mean value, the unpaired T test, ** represents p<0.01, the experimental group (n=9), and the control group (n=7); c is a representative diagram of the MRI T2 sequence scanning results of part of the experimental mice. Mouse numbers in the treatment group: #73, #78 and #85; mouse numbers in the control group: #68, #83 and #90. The Z-axis interval of the MRI scan is 0.5 mm.

[0033] Figure 18 The H&E histological staining results of the drug administration group and the control group of the Braf-bAVM disease mouse model constructed are shown. a is a representative image of the H&E staining of the cerebral cortex brain section of the Braf V600E-fl / fl mouse after local injection of AAV-miniBEND (mPro723-mCis700)-Cre, the left half of a shows the results of the control group (DMSO), and the right half of a shows the results of the drug administration group, a shows two representative areas of the control group and the treatment group; b shows the density of the malformed blood vessels in the control group and the drug administration group; c shows the diameter of the malformed blood vessels in the control group and the drug administration group. DETAILED DESCRIPTION

[0034] The present application relates to the application of a small molecule drug PLX4032 in the preparation of a drug for treating cerebral arteriovenous malformation disease, and provides a new use of the small molecule drug PLX4032. The present application will be further described in detail below in conjunction with the drawings and examples, and the following examples are only used to illustrate the present application and do not limit the scope of the present application. The experimental methods not specified in the examples are usually carried out under conventional conditions, such as the conditions described in Sambroom et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. All experimental data are statistically analyzed using GraphPad Prism8 software (GraphPad Software, USA), and all data are represented by error bars in the form of mean standard deviation, and analyzed by non-parametric intergroup t-test.

[0035] Experimental materials and methods

[0036] 1Tek gene

[0037] Tek gene encodes a receptor belonging to the protein tyrosine kinase Tie2 family. The encoded protein has a unique extracellular region containing two immunoglobulin-like domains, three epidermal growth factor (EGF)-like domains, and three fibronectin type III repeats. The ligand angiopoietin-1 binds to this receptor and mediates a signaling pathway that plays a role in embryonic vascular development. The gene is widely and specifically expressed in vascular endothelial cells.

[0038] There are two parts of core transcriptional regulatory sequences in Tek gene, in addition to the conventional promoter region (Tek Promoter), there is also the non-coding sequence of the first intron (Tek intron 1).

[0039] Inquiry GenBank database and literature to determine the promoter region and the first intron region of Tek gene.

[0040] 2 truncated sequence and recombinant plasmid construction

[0041] The recombinant plasmid constructed in the application is: AAV-miniTek-Cre-bGHpA-Cis-element, wherein miniTek is a Tek gene promoter region of different truncated sizes (Tek Promoter region), and Cis-element is a first intron region of different truncated sizes of Tek gene (Tek intron1 region). The SpeI and AgeI enzyme cutting sites are introduced at both ends of miniTek in the recombinant plasmid, and the KpnI and NotI enzyme cutting sites are introduced at both ends of Cis-element. The standardized plasmid backbone used in the recombinant plasmid is derived from the plasmid pAAV-CBA-cre-U6-sgRNA (Addgene #60229) purchased from the Addgene website, and the transgene segment on the Tek-Cre transgenic mouse genomic DNA includes a 2.1 kb sequence of the mouse-derived Tek gene promoter region and a 10 kb sequence of the first intron of the Tek gene, which can be used as a template (Schlaeger et al., 1997; Kisanuki et al., 2001). The 2.1 kb sequence of the Tek gene promoter region and the 10 kb sequence of the first intron segment of the Tek gene are used for truncation experiments, and different truncated lengths of the mouse-derived Tek promoter region (including mPro1576, mPro973, mPro723, mPro760, mPro583, and mPro471) are obtained by PCR using the genomic DNA of the Tek-Cre transgenic mouse as a template and designing primers upstream and downstream of the corresponding region. Different truncated lengths of the first intron region of the mouse (mCis1349, mCis700, mCis400, mCis303, mCis200, and mCis103) are obtained by PCR using the genomic DNA of the Tek-Cre transgenic mouse as a template and designing primers upstream and downstream of the corresponding region. The two ends of the PCR primers contain a 20 bp sequence homologous to the cohesive end of the recombinant AAV vector, and then the target recombinant plasmid can be obtained by homologous recombination. The detailed information of the primers is shown in Table 1.

[0042] The sequences of different truncated lengths of the Tek gene promoter region of human origin (including hPro1612, hPro762, found and determined by multi-species homologous sequence alignment) and the Tek intron region sequence (including hCis700, hCis400, hCis200, found and determined by multi-species homologous sequence alignment) were cloned by PCR reaction using the genomic DNA of the HEK293T cell line as the template. Primers were designed in the corresponding regions upstream and downstream, and obtained by PCR. The DNA materials of the Tek gene cis-regulatory elements (Cis-element) of rat, pig and marmoset origin were obtained by gene synthesis from Jinweizhi Company. The information of other plasmid vectors used in this paper is shown in Table 2.

[0043] Table 1 Primer sequence list

[0044]

[0045]

[0046]

[0047]

[0048] Table 2 Related plasmid vector information

[0049]

[0050]

[0051]

[0052] 3Mice

[0053] The experimental animals (mice) used in this patent comply with the relevant regulations of the Chinese government on animal protection and use, and are approved by the animal management and use committee of the unit, in line with the management policy. Ai9 and Ai47 mice were from the laboratory of Dr. Zeng Hongkui of Allen Institute. Tek-Cre (catalog number 008863), NG2DsRed BACtg (catalog number 008241), and Ai14 (catalog number 007914) mice were purchased from Jackson Laboratory, USA. Braf-CA fl / fl , also known as Conditional activation Braf V600E Transgenic mice were purchased from Jackson Laboratory, USA, with the item number 017837. Wild-type mice C57BL / 6J (SPF level) were purchased from Vantianlihua, and some C57BL / 6J mice were purchased from the experimental animal platform of the unit. C57BL / 6J mice, Braf-CA fl / flTransgenic mice, Ai14 and Ai47 transgenic mice, etc. were used for AAV virus injection. The use and care of animals were completed under the guidance of the Animal Ethics Committee of Beijing Institute of Brain and Cognitive Sciences.

[0054] 4AAV virus packaging and purification

[0055] AAV virus packaging: (1) Cells in a 9cm cell culture dish were passaged to a 15cm cell culture dish. (2) Before transfection, the culture solution in the 15cm cell culture dish was aspirated with a liquid pump, and 20mL of complete culture medium (DMEM+10%FBS+PS / dual antibody) was added, and the culture dish was placed in the CO2 incubator for culture. After the expansion of culture, if the cells are to be passaged every other day, the culture solution (DMEM+10%FBS+PS / dual antibody) can be changed on the second day of expansion culture to make the cells grow better. (3) Prepare the transfection mixture (1 dish) according to Table 3.

[0056] Table 3 AAV virus packaging plasmid transfection system

[0057]

[0058] (4) After the mixed transfection reagent is shaken well on the shaker, it is placed in the biosafety cabinet for 30 minutes. (5) Add the mixture to the 15cm cell culture dish, cover the dish, and gently shake the dish on the operating table to evenly distribute the cells in the dish. (6) Place the cell culture dish in the CO2 incubator for culture, and after 8 hours, aspirate the culture solution and add 30mL of DMEM culture solution (without FBS, with PS / dual antibody). (7) Place the cell culture dish in the CO2 incubator again, and after 48-72 hours of transfection, collect the cells for virus purification.

[0059] Purification of AAV virus: (1) Collect cells: use 5 mL pipette to blow cells from the wall in the cell culture dish into a 50 mL centrifuge tube. (2) Add 1 / 10 volume of chloroform (3 mL). (3) Put into a shaker, 37°C, shake for 1 hour. (4) Add 1.928 g NaCl, put into a shaker, 25°C, shake for 20 minutes. (5) Centrifugation: 4°C, 12000 rcf, 20 minutes. (6) Transfer the supernatant to a new 50 mL centrifuge tube with a label. (7) Add 10% of the volume of PEG8000 to the supernatant. (8) Put into a shaker, 25°C, shake for 20 minutes. (9) Ice bath for 1 hour, if the experiment needs to be paused, it can be ice bathed overnight. (10) Centrifugation: 4°C, 12000 rcf, 30 minutes. (11) Gently discard the supernatant, and invert the tube on the absorbent paper. (12) Add 600 μL of filtered PBS, 1 μL of DNase, 1 μL of RNase, and fully dissolve the virus. (13) Put into a 37°C water bath or 37°C bacterial incubator, incubate for 30 minutes. (14) Transfer the incubated product to a new 1.5 mL centrifuge tube, add 600 μL of chloroform at a ratio of 1:1, shake and mix well. (15) Centrifugation: 4°C, 12000 rcf, 10 minutes. (16) Take the supernatant to a new 1.5 mL centrifuge tube, and concentrate it through a protein purification column. (Note: The packaging and purification of AAV virus in the present application are completed by the vector center of Beijing Institute for Brain and Cognition (CIBR).)

[0060] 5AAV virus injection

[0061] The in vivo transduction of AAV virus in mice is achieved by orbital venous plexus injection. First, a simple mouse anesthesia induction box is prepared by placing a liquid isoflurane-soaked cotton ball into a 50 ml centrifuge tube. Then, a 40U insulin needle is used to draw the diluted AAV virus of a specific titer. The mouse's head is inserted into the simple anesthesia induction box, and when the mouse's breathing rate slows down and the limbs become weak, the head is pinched to expose the right eyeball. The insulin needle containing the AAV virus (about 100 μl) is inserted into the orbital venous plexus and injected with the virus. After quickly pulling it out, the mouse is taken out of the induction box to wake up as soon as possible. If there is bleeding after pulling out the needle, a cotton swab should be used to wipe it off. The AAV virus and titer information in the laboratory can be found in the attached table (Table A.2).

[0062] 6Immunofluorescence staining experiment

[0063] Mice were perfused with sterile physiological saline through the heart and after the liver turned white, the perfusion was changed to 4% (w / v) paraformaldehyde (PFA). After the perfusion was complete, the mouse brain was dissected and transferred to a 15 mL centrifuge tube and placed in cold PFA (4°C) for 2 hours, washed 3 times in PBS, 10 minutes each on a shaker at room temperature. The last wash was changed to 30% sucrose and placed in a shaker at 4°C for dehydration, after 1-2 days, the mouse brain sank to the bottom, indicating that the dehydration was complete. The fixed brain was sectioned at 40-60 pm using a cryostat (CM3050S, Leica) or 60-70 pm using a vibratome (VT1000S, Leica). The sections were stained as previously described. Briefly, the sections were permeabilized with 0.25% Triton X-100 and then blocked with 5% BSA and 3% normal goat serum with 0.25% Triton X-100 for 2 hours. PDGFRbeta (1 :300, rat, eBioscience, 14-1402-81) or NG2 (1 :300, AB5320, rabbit, polyclonal, Millipore), Glutl (1 :300, rabbit, Thermofisher, RB-9052-P0) and CD31 (1 :50, rat, BD550274) primary antibodies were incubated with the brain sections at 4°C for 24-48 hours. After washing three times with PBS with Hoechst 33342 or DAPI (Life Technologies), the sections were incubated with Alexa 488, Alexa 546 or Alexa 633 / 647 (1 :500, Life Technologies) conjugated secondary antibodies at RT (22-25°C) for 2 hours. After washing three times with PBS, the sections were mounted with antifluorescence quenched mounting medium Vectorlab (or custom made (60% glycerol and Hoechst 33342)).

[0064] 7 Magnetic resonance imaging and three-dimensional reconstruction

[0065] Magnetic resonance imaging (MRI) was performed on a 7.0T scanner (Bruker Pharmascan 70 / 16, Bruker, Germany) equipped with a 23 mm surface coil and a 12 cm diameter self-shielded gradient system for small animal MR imaging of mice and rats. The operator interacted with the scanner via Paravision 5.1 software (Bruker BioSpin) and a Linux computer running Topspin 2.0. The procedure was as follows: the mouse was first anesthetized with a mixture of 5% isoflurane and 95% oxygen, and was transferred into the imaging chamber. The valve of the anesthetic machine was switched so that the mouse was continuously anesthetized with 2% isoflurane and 98% oxygen during the imaging procedure. The system was also equipped with a respiratory monitoring module, and a small respiratory frequency monitoring patch was placed on the abdomen of the mouse in the imaging chamber. The operator could adjust the anesthetic intensity by adjusting the valve of the anesthetic machine based on the feedback of the respiratory frequency of the mouse (which was generally controlled at 30-50 times / min). In this study, the mouse brain was imaged using T2-weighted (T2W) relaxation- enhanced (RARE) method, and the parameters used were as follows: repetition time (TR) / echo time (TE) was 3500 ms / 33 ms, 4RARE factor, 21 x 21 mm field of view (FOV), 256 x 256 matrix, 25 slices, and 0.5 mm slice thickness. The high signal regions in the images were brain edema and brain hemorrhage. The imaging data were processed using 3D slicer 4.11.20210226 software to reconstruct the three-dimensional images of the target lesions, and to quantify the volume of the lesion regions.

[0066] 8Fluorescence microscopy imaging

[0067] Widefield microscope imaging, brain slice fluorescence signal collection and photographing were performed using Olympus slide scanner VS120. The steps were as follows: first, select fluorescence scanning mode, second, load target slide, input target slide label information and data saving path, click preview, select 2X objective lens scanning in bright field mode, select 10X objective lens scanning in fluorescence mode, select scanning area, use multi-point focusing method, 5-point method covers the brain slice of target imaging. Next, set up illumination, FL Blue channel for Hoechst or DAPI stained samples, FL Green channel for EGFP protein expressing samples, FL Cy3 channel for DsRed, Tdtomato, mRuby3 and other red fluorescent protein expressing samples. Generally, it is FL Blue and FL Green dual channel imaging, FL Blue and FL Cy3 dual channel imaging, or FL Green and FL Cy3 dual channel imaging. Finally, check the multi-point focusing based on the top ranked channel, click next to start full area scanning.

[0068] Vital microscope imaging, vital microscope (Leica M205 FA), 1x objective lens. AAV locally injected mouse brain sample uses this microscope to determine the fluorescence expression of surface EGFP. The last step of the skull window surgery for two-photon imaging of mice uses this microscope to check the clarity of the skull window.

[0069] Confocal microscope imaging, use the flip scan function of the confocal microscope (Leica SP8) to take pictures of the whole brain slice image, with 10% overlap between adjacent fragments for automatic splicing. The imaging parameters of brain blood vessel samples are generally: 20x air lens, 1024x1024, z-axis step is 1 μm (many times using software optimized values); or 40x oil lens, 1024x1024, z-axis step is 0.5 μm, zoom factor is 1.25.

[0070] 9H&E staining

[0071] (1) Material: The mice were perfused with sterile normal saline through the heart, and after the liver turned white, they were replaced with 4% (w / v) paraformaldehyde (PFA) for perfusion. After perfusion, the mouse brain was dissected and transferred to a 15 mL centrifuge tube and placed in cold PFA (4°C) for fixation for more than 24 h. The tissue was taken out of the fixative and the target tissue was trimmed with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box. (2) Dehydration and wax immersion: Place the dehydration box in a hanging basket in the dehydration machine and dehydrate it in gradient alcohol. 75% alcohol for 4 h-85% alcohol for 2 h-90% alcohol for 2 h-95% alcohol for 1 h-anhydrous ethanol I for 30 min-anhydrous ethanol II for 30 min-alcohol benzene for 5-10 min-dimethylbenzene I for 5-10 min-dimethylbenzene II for 5-10 min-65° melting paraffin I for 1 h-65° melting paraffin II for 1 h-65° melting paraffin III for 1 h. (3) Embedding: The wax- immersed tissue is embedded in the embedding machine. First, put the melted wax into the embedding frame, and before the wax solidifies, take the tissue out of the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and paste the corresponding label. Cool on a -20°C freezing table, and after the wax solidifies, take the wax block out of the embedding frame and trim the wax block. (4) Sectioning: Place the trimmed wax block in a paraffin sectioning machine to section it at a thickness of 4 μm. Float the section on a 40°C water bath on the sectioning machine to flatten the tissue. Place the tissue on a glass slide and bake it in a 60°C oven. After the water-baked wax is roasted, take it out and store it at room temperature for future use. (5) Paraffin section dehydration to water: sequentially place the section in dimethylbenzene I for 15 min-dimethylbenzene II for 15 min-dimethylbenzene III for 15 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-95% alcohol for 5 min-85% alcohol for 5 min, and then wash with tap water. (6) Hematoxylin staining: place the section in hematoxylin staining solution for 1-2 min, wash with tap water, differentiate with differentiation solution, wash with tap water, return to blue with blue return solution, and rinse with running water. (7) Eosin staining: stain in eosin staining solution for 2-3 min. (8) Dehydration and mounting: sequentially place the section in anhydrous ethanol (5 min), anhydrous ethanol (5 min), anhydrous ethanol (5 min), dimethylbenzene (5 min), and dimethylbenzene (5 min) to make it transparent, and then mount it with neutral balsam. (9) Microscope examination, image acquisition and analysis.

[0072] Example 1

[0073] From the mouse and human corresponding brain cell type specific mixed cell transcriptome sequencing and single cell transcriptome sequencing data, we mined out those genes that are specifically highly expressed in brain vascular endothelial cells, and we determined that the Tek gene is specifically highly expressed in the endothelial cells of various segments of the brain blood vessels of both humans and mice, which meets our needs. (e.g. Figure 1Figure 1 shows the expression profile of Tek gene in human and mouse brain vasculature single cell transcriptome, where a is the expression profile of Tek gene in human brain vasculature single cell transcriptome; b is the expression profile of Tek gene in mouse brain vasculature single cell transcriptome; c is the expression profile of Tek gene in mouse brain cell type specific mixed cell transcriptome.

[0074] Based on the results of sections in Tek-Cre; Ai47 double transgenic mice, we found that Tek gene-labeled vascular endothelial cells were very comprehensive, basically covering 100% of the vascular endothelial cells, and had extensive distribution of arteriovenous capillary three segments (as shown in Figure 2 Figure 2 shows the fluorescence microimaging of Tek-Cre; Ai47 transgenic mouse brain coronal section at postnatal day 42, and the confocal fluorescence microimaging of a single region of interest in the cortical area, where a is the fluorescence microimaging of Tek-Cre; Ai47 transgenic mouse brain coronal section at postnatal day 42; b is the confocal fluorescence microimaging of a single region of interest in the cortical area, HO: Hoechst 33342 nuclear dye, EGFP: reporter gene EGFP signal specifically expressed in Tek-Cre positive mouse brain vascular endothelial cells, HO / EGFP: imaging results of HO and EGFP two channels combined, arrow indicates a single brain vascular endothelial cell; c is the distribution of Tek-Cre; Ai47 double transgenic mouse-labeled vascular endothelial cells in the cerebral cortex, striatum and thalamus region.

[0075] Since Tek gene is specifically expressed in mouse and human brain vascular endothelial cells, it indicates that the gene expression regulation mechanism in evolution is conservative, and there is a conservative core regulatory sequence in the genome. In order to lock the core sequence of transcription regulation, we performed multi-species sequence alignment analysis on the promoter region and the first intron region of the gene (species include human (Homo sapiens), macaque (Macaca mulatta), marmoset (Callithrix jacchus), pig (Sus domesticus), cow (Bos taurus), rat (Rattus norvegicus), mouse (Mus musculus)), and determined the conserved sequence between multiple species.

[0076] The results of multi-sequence alignment show that the Tek gene promoter region regulatory sequences of pig and cow have higher similarity, human and marmoset have higher similarity, and mouse and rat have higher similarity, as shown in Figure 3 Figure 3 shows the DNA multi-sequence alignment analysis of the promoter region and 5' non-coding region (5' UTR) corresponding to the Tek gene in the genomes of multiple species including human, rhesus, marmoset, pig, cow, rat, and mouse, and the phylogenetic tree analysis of sequences from each species, where a is the DNA multi-sequence alignment analysis of the promoter region and 5' non-coding region (5' UTR) corresponding to the Tek gene in the genomes of multiple species including human, rhesus, marmoset, pig, cow, rat, and mouse; b is the phylogenetic tree analysis of sequences from each species, the promoter region and 5' non-coding region sequences of pig and cow, human and rhesus and marmoset, and mouse and rat are more similar in evolution, respectively. The same is true for the multi-sequence alignment results in the intron region Figure 4), wherein a is DNA multiple sequence alignment analysis of the region corresponding to the first intron of Tek gene in multiple species genomes including human, rhesus monkey, marmoset, pig, cow, rat, mouse; b is phylogenetic tree analysis of sequences from each species, the first intron sequences of pig and cow, human and rhesus monkey and marmoset, mouse and rat are respectively more evolutionarily similar.

[0077] The conserved sequence of the core sequence regulating transcription between multiple species is as follows:

[0078] The promoter region is shown in SEQ ID NO. 1 (taking the sequence from mouse as an example, the letter "N" means that the base at this position has diversity between multiple species, and is not conserved):

[0079] 5'-AAGGNTGGTCNTCATCGCATACCATACATAGGTGGAGGGCTTGT TATTCNANTTNCNGCCTATGAGAGGATACCCCTATTGTTNCTGAAAATGCTGACCAGGNCCNACTTNAACAAANATNCCTCTNCCCCNANCNCAGNAANGCAGNAGCAGNAGCNANCNAGCANAGATAAGNTTNGATGAANGCAAGATGGATAGGGCTNGCNTGCCCCNAGCCCTGCTGATACCAANTGCCTTTAA-3'

[0080] The intron region is shown in SEQ ID NO. 15 (taking the sequence from mouse as an example, the letter "N" means that the base at this position has diversity between multiple species, and is not conserved):

[0081] 5'-GTGTGTGTGNGTGGTCACACCCATCTCAGCAGATCTGTCAGCTT TCCCGCTTTTGTTAGAGGGTGATATCATGCTTCCTGGGGGGAGCNCTGGAAGACAATGNCNGCCACTTTCCTCNAGATACAATAGGCGGAGTCAGGAAGGNAGTATTGACATTGCTGGGGCCNGGAGNACTCACTGCTCNGNGGCCGTCAGATGGTGAACCNGCNTAACCTTGGCACACAGNGCCTGGGNTGTNCAAGGCGTCTGGCTGCAGNGCCAAAGNGGACTCCACCCTNGGGACAGGAGTNCTTNAGACATCTGGGAATCTGGGATGGGTTNAAA-3'

[0082] Development and functional test of mini-promoters mediating endothelial cell-specific expression of brain vascular endothelial cells

[0083] To systematically analyze the function of various truncated versions of promoter sequences and cis-regulatory elements, we designed a standardized recombinant AAV expression vector (Figure 2) in which the upstream and downstream of the promoter were introduced with restriction sites SpeI and AgeI, respectively, and the Kozak sequence was followed by an expression frame of NLS-Cre-HA gene, followed by a polyA element bGHpA, and the downstream of the polyA element was the sequence of the cis-regulatory element to be inserted, which had single restriction sites KpnI and NotI at both ends for molecular cloning operations. Figure 5

[0084] First, Tek genomic sequences from multiple species from humans to mice were collected, and based on the BLAST analysis of multiple sequence alignment of the promoter, 5'-UTR region and first intron region, the homologous segments were obtained, and then the truncated cloning of the core promoter region and 5'-UTR region, and the bidirectional truncated cloning of the conserved cis-regulatory element in the first intron were performed. After obtaining the cloned fragments, they were subcloned into the recombinant AAV2 vector we had designed and standardized, and the AAV-PhP.eB capsid serotype was used for routine three-plasmid system for AAV virus packaging, and the downstream was based on the transgenic fluorescent reporter mouse Ai14 Rosa26 -CAG-LSL-Tdtomato or Ai47 Rosa26-CAG-LSL-3XGFP in vivo transduction experiments, and finally combined with dissection, sectioning, and fluorescence imaging analysis to obtain the transduction efficiency and specificity.

[0085] (1) Truncation analysis of Tek gene promoter segments

[0086] ​After we determined Tek as the target gene, we next performed a functional study of its promoter, aiming to identify the core sequence that regulates transcription of the Tek gene. First, we extracted the genomic DNA from the ear tissue of Tek-Cre transgenic mice, and, based on the results of our previous sequence alignment, we removed as much of the 5' distal sequence of the promoter as possible while retaining all of the conserved sequences. We did the same when selecting the sequence of the cis-regulatory element. In our preliminary experiments, we first constructed the longest version mPro1576+mCis1349 and the second truncated version mPro1576+mCis700 (the numbers represent the respective base lengths) of the cis-regulatory element, and performed in vivo functional analysis of the two. We found that the labeling efficiency of mCis700 and mCis1349 was consistent, indicating that they have the same effect on location-specific expression of brain vascular endothelial cells, and further indicating that the cis-regulatory element of the Tek gene can be truncated from mCis1349 to mCis700 without affecting its normal function. We then controlled the cis-regulatory element to the mCis700 version, and began to gradually truncate the sequence length of the promoter region. We further constructed mPro973 (truncated 5' distal sequence of the promoter region, retaining the entire 5' UTR region), mPro723-d224 (further truncated 5' distal sequence by 224 bp based on mPro973), mPro760 (truncated part of the 5' UTR sequence), mPro583 (5' UTR sequence retaining only the +1-+113 segment), and mPro471 (truncated all 5' UTR sequence) with the Tek gene promoter region. The schematic diagram of the truncation strategy of the Tek gene promoter region is shown in FIG. 10a. Figure 6

[0087] mPro723-d224 is truncated by 224 bp of the 5' distal sequence of the promoter based on mPro973, and the deleted region includes two transcription factor binding motifs (Octopus region I and Octopus region II) (Fadel et al., 1999). The results show that mPro723 can well maintain the effective activity of the promoter, and has a certain improvement in the promoter activity relative to mPro973 (FIG. 10b, c). Figure 6 mPro583 is a miniTek version truncated by part of the 5' UTR sequence, and the experimental results of mPro583 show that only sparse endothelial cells are labeled with fluorescence in the whole brain (FIG. 10d). Figure 6 ​c), indicating that truncation of the 5' UTR region affects the overall transcriptional activity of the promoter and that the mPro583 promoter loses its basic activity in most endothelial cells throughout the whole brain. mPro471 is a miniTek version that truncates all 5' UTR sequences, and the experimental results of mPro471 show that only sporadic endothelial cells express EGFP protein throughout the whole brain Figure 6 c), indicating that the mPro471 version loses more than 90% of its transcriptional activity. In summary, we believe that mPro723 (del 224bp, deletion of 224bp) is the optimal truncated version of miniBEND.

[0088] wherein the gene sequence of mPro1576 is shown in SEQ ID NO. 2:

[0089] cctcagtgtctgctcttgacctgttaacagctgagtcagggtctgccctcagctgtgcctgaggacagagctgagctat

[0090] ctacccctgcagattggaagcattacaggcactcaagatcagccctgaagtgataaaacctaaggcagaaatccacc

[0091] aagactagcagtgcctccgtgtctcttcctgtggctggtgggaaagagaggggcagtccttccttgatgcaaggtcgt

[0092] gtgtctagtggcacgcttccttcattcccagtgagagcaagtgatcacctgggtaaggaaggttcaggtgcctgagct

[0093] cgctggagaattcatcactcatccatcactctgctcctgtagacataatcacttctgttgggtctttatagagatgatttata

[0094] actttgttgtttatagtttttatgaatgtgtgtattcatttaggtcacatgggaggtacacattttcaggtgtctgtctttccatc

[0095] acacgggctttgaattaaactcagtcttggttttaccggctgagccatctcacctgcctgattatttaaaaatctccggagt

[0096] aatccaggagtgtggtttatgattgtagtatcaacactcgggaggctgagggagcatcgttatcatgagctccaggcta

[0097] gttccaggcttgcctaagctgtagagcaagtcactctcttaaaaagtgcctctcccatatttttgtatataatttgcatctga

[0098] aattctgtttgccaataactatgaaattattcacattactaaaatcttcctgtgccaagttctccaacgaattagatcacact

[0099] cagatgaaatgctaataaaaattaaagctgtagccagtagcatgcgtatatttgggctcagggccaacaggcaggcg

[0100] atctgggtgtaagaaaataggctaatggctgtggaatctggtctctagtggctccgctgagagctgacctcaaccacg

[0101] ctccctcaaattgattgccttccaggttatgatttctcatcacaggaaactttgttgcccaattcaaaccctgtgagtgaaa

[0102] acaaaaacaggagagcaagtgctgctccccgtgccccaaagccccttctgtcagggatcccaaatgcaccccaga

[0103] gaacagcttagcctgcaagggctggtcctcatcgcataccatacataggtggagggcttgttattcaattcctggcctat

[0104] gagaggatacccctattgttcctgaaaatgctgaccaggaccttacttgtaacaaagatccctctgccccacaatccag

[0105] ttaaggcaggagcaggagccggagcaggagcagaagataagccttggatgaagggcaagatggatagggctcg

[0106] ctctgccccaagccctgctgataccaagtgcctttaagatacagcctttcccatcctaatctgcaaaggaaacaggaaa

[0107] aaggaacttaaccctccctgtgctcagacagaaatgagactgttaccgcctgcttctgtggtgtttctccttgccgccaa

[0108] cttgtaaacaagagcgagtggaccatgcgagcgggaagtcgcaaagttgtgagttgttgaaagct

[0109] The gene sequence of mPro973 is shown as SEQ ID NO. 3:

[0110] ggaggctgagggagcatcgttatcatgagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctct

[0111] taaaaagtgcctctcccatatttttgtatataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaa

[0112] aatcttcctgtgccaagttctccaacgaattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagta

[0113] gcatgcgtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatct

[0114] ggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatca

[0115] caggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgcccc

[0116] aaagccccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcatac

[0117] catacataggtggagggcttgttattcaattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccagg

[0118] accttacttgtaacaaagatccctctgccccacaatccagttaaggcaggagcaggagccggagcaggagcagaa ​​​​​​​​​​​​​​​​gtatatttgg gctcagggcc aacaggcagg cgatctgggt gtaagaaaat aggctaatgg ctgtggaatct ggtctct

[0125] agtggctccg ctgagagctg acctcaacc acgctccct caaattgatt gccttccagg ttatgatttc tcacaggaa

[0126] actttgttgc ccaattcaaa ccctgtgagt gaaaacaaaa acaggagagc aagtgctgct cccgtgcccc aaagcc

[0127] ccttctgtca ggatcccaaa tgcaccccag agaacagctt agcctgcaag ggctggtcct catcgcatac catat

[0128] aggtggaggg cttgttattc aattcctggc ctatgagagg ataccctatt gttcctgaaa atgctgaccaggaccttac

[0129] ttgtaacaaa gatccctctg ccccacaatc cagttaagga ggagcaggag ccggagcagg agcagaagat aagc

[0130] cttggatgaa gggcaagatg gatagggctc gctctgccc aagccctgct gataccaagt gcctttaaga tacagcct

[0131] ttcccatcctaatctgcaaaggaaacaggaaaaaggaacttaaccctccctgtgctcagacagaaatgagactgttac

[0132] cgcctgcttc tgtggtgttt ctccttgccg ccaacttgta aacaagagcg agtggaccat gcgagcggga agtcgcaa

[0133] agttgtgagt tgttgaaagc

[0134] The gene sequence of mPro760 is shown as SEQ ID NO. 5:

[0135] gagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctcttaaaaagtgcctctcccatatttttgtat

[0136] ataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaaaatcttcctgtgccaagttctccaacg

[0137] aattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagtagcatgcgtatatttgggctcagggcc

[0138] aacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatctggtctctagtggctccgctgagagct

[0139] gacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatcacaggaaactttgttgcccaattcaaa

[0140] ccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgccccaaagccccttctgtcagggatccca

[0141] aatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcataccatacataggtggagggcttgttatt

[0142] caattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccaggaccttacttgtaacaaagatccctct

[0143] gccccacaatccagttaaggcaggagcaggagccggagcaggagcagaagataagccttggatgaagggcaag

[0144] atggatagggctcgctctgccccaagccctgctgataccaagtgcctttaa

[0145] The gene sequence of mPro583 is shown as SEQ ID NO. 6:

[0146] ggaggctgagggagcatcgttatcatgagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctct

[0147] taaaaagtgcctctcccatatttttgtatataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaa

[0148] aatcttcctgtgccaagttctccaacgaattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagta

[0149] gcatgcgtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatct

[0150] ggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatca

[0151] caggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgcccc

[0152] aaagccccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcatac

[0153] catacataggtggagggcttgttattc

[0154] The gene sequence of mPro471 is shown as SEQ ID NO. 7:

[0155] ggaggctgagggagcatcgttatcatgagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctct

[0156] taaaaagtgcctctcccatatttttgtatataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaa

[0157] aatcttcctgtgccaagttctccaacgaattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagta

[0158] gcatgcgtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatct

[0159] ggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatca

[0160] caggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccc

[0161] (2) Truncating analysis of cistropic regulatory elements in the first intron region of the Tek gene

[0162] To further explore the necessary functional sequences in cis-regulatory elements, we constructed recombinant AAV vectors containing four truncated versions of cis-regulatory elements: mCis400, mCis303, mCis200, and mCis103. To control for variables, these recombinant AAV vectors all contained the mPro1576 version sequence in the promoter region and were packaged into AAV viral particles with a PhP.eB capsid. First, specific primers were designed to clone different truncated versions of evolutionarily conserved segments in the intron-1 region of the Tek gene (e.g., mCis400, mCis303, mCis200, and mCis103). Figure 7a), mCis1349 contains the most complete regulatory elements, CACA repeat motif, GATA motif, Ets-1 transcription factor binding motif, etc. mCis700 has a deletion at the 5' end of a sequence containing a CACA repeat motif, and also has a small deletion at the 3' end. The mCis400 version is further truncated at both ends, with further deletion of the sequence containing the CACA repeat motif at the 5' end, and further deletion of the excess sequence adjacent to the Ets-1 motif at the 3' end. The mCis303 version is further truncated at the 5' end relative to the mCis400 version, with further deletion of the excess sequence adjacent to the GATA motif. The mCis200 version and the mCis103 version are designed to separately analyze the necessity of individual regulatory motifs by splitting the mCis303 version. The mCis200 retains the Ets-1 motif, and the mCis103 retains the CACA repeat motif and the GATA motif. Finally, the mCis0 version uses only the mPro1576 promoter sequence on the recombinant vector, and deletes all of the Intron 1 segment of the regulatory element sequence Figure 7 a). We found that the mCis200 and mCis103 versions were strongly inhibited in terms of transcriptional activity, indicating that the sequence loss of the regulatory sequence that activates normal transcription of the promoter segment. Among them, mCis200 retains partial transcriptional activity, indicating that the Ets-1 motif can only partially inhibit the transcriptional activity of the promoter region, and mCis103 almost loses all transcriptional activity, indicating that the GATA and CACA repeat motifs have a stronger inhibitory effect on the transcription of the promoter region Figure 7 b, c). The results of the mCis0 (Pro-only) group show that when there is only a promoter sequence, the Cre gene cannot be expressed in brain vascular endothelial cells, but is widely expressed in neurons, astrocytes, and brain vascular endothelial cells Figure 7 c), we believe that the role of the Intron 1 segment of the Tek gene in the cis-regulatory element is to limit the expression of the exogenous gene only in brain vascular endothelial cells by transcriptional inhibition, that is, in non-endothelial cells such as neurons and astrocytes, the mCis element has a transcriptional inhibitory effect on the promoter region. We can reasonably believe that there is a transcription factor that is specifically expressed in brain vascular endothelial cells that relieves the transcriptional inhibition of the mCis element on the Tek gene promoter region, and the downstream gene is thus expressed.

[0163] To confirm the high transcriptional activity and cell type specificity of the combination of the truncated version mPro723 of the Tek gene promoter region and the stage version mCis700 of the cis-regulatory element of the Intron 1 region, we constructed the rAAV-mPro723-Cre-mCis700 recombinant plasmid and packaged it into the AAV-PhP.eB virus, and injected it into the Ai14 Rosa26-CAG-LSL-tdTomato In vivo transduction experiments were performed in mice. The results of sagittal section fluorescence imaging experiments showed that the rAAV-miniBEND system of the best optimized truncated version can efficiently and specifically transduce the endothelial cells of the blood vessels in the whole brain (including the olfactory bulb, cortex, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, etc.), and there are labeling signals in many large arteries and veins Figure 8 a, b) of FIG. 11.

[0164] To comprehensively evaluate the cell type specificity of the rAAV-miniBEND system, we performed whole-brain range interval section experiments on the brain of each truncated version labeled mouse to ensure coverage of most brain regions. After carefully checking the fluorescence labeling signals in all brain regions, we found that the AAV-PhP.eB-CAG-mScarlet virus group had serious non-specific fluorescence labeling signals in Purkinje cells in the cerebellum region, and the AAV-PhP.eB-mPro973-Cre group also had non-specific labeling fluorescence labeling signals in Purkinje cells in the cerebellum region Figure 9 wherein Figure 9 a The virus used in each group was AAV-PhP.eB serotype, and the recombinant AAV vectors used were CAG-mScarlet, mPro973-Cre, mPro760-Cre, and mPro723-Cre, respectively. In the latter three groups, the Tek cis-regulatory element used in the recombinant vector was mCis700, and was injected into C57BL / 6J, Ai47, Ai47, and Ai14 transgenic reporter mice, respectively. The virus titer information used in each group is shown in Appendix A Table A.1). The scale bar is 100 μm. Figure 9 b The proportion of non-specific labeling other than endothelial cells, with a standardized labeling density of 10 11 In vivo transduction experiments were performed in mice. The results of sagittal section fluorescence imaging experiments showed that the rAAV-miniBEND system of the best optimized truncated version can efficiently and specifically transduce the endothelial cells of the blood vessels in the whole brain (including the olfactory bulb, cortex, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, etc.), and there are labeling signals in many large arteries and veins 2Cell number of area-inlaid marker - For evaluation of the index, n = 2 of the test animals in each group were counted and statistically analyzed in 5 ROIs selected from each mouse section. The scale bar in the figure is 100 μm). It is shown that although the miniBEND regulatory sequence can limit the expression of exogenous genes in most neurons, there will be a phenomenon of leaky expression in Purkinje cells. We further compared the results of mPro760-Cre group and mPro723-Cre group, and found that the non-specific labeling signal density of Purkinje cells in these two groups was significantly reduced, especially in the mPro723-Cre group, the non-specific labeling signal was close to zero, and only 1 non-specific labeled cell was found in multiple cerebellum sections in the whole brain, and the signal was very weak. This shows that the continuous truncation of the 5' end sequence of the mouse Tek gene promoter region can reduce the non-specific labeling of miniBEND system in cerebellum neurons, and the optimal truncated version mPro723 we obtained has the highest cell type specificity.

[0165] wherein the gene sequence of mCis1349 is shown as SEQ ID NO. 8:

[0166] catagatatcttaatagtcaaggaatttttttttttttttttgaagagttagcagtcaggggatggtagaaactgcaaaacca

[0167] atccgtattctttcttgagatttttagacagttgatgctactagccacaaaaagagttttaagtgggaggagagtaagatg

[0168] caggcaccaaggtgacaggctccaggtctgtagcattagcttacagatgagattctttacagagagccaggcagctg

[0169] cattggctaaagcagatctgggagggggccaggagatcagctggcggcactcccagcctccaggaaaggcaacc

[0170] cttatttctggaattttaaactgataacccaattcccaccagcctggccaggctcttccttagctcacatcacaaacacag

[0171] aaggattgttttagatggagtcatgcttgattctttctatacctacttccaagaccaattttataaaagtttatttaccgccgt

[0172] gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcatggtatatatggatgtcagagtttggttctctccttc

[0173] tgcagtgtggctcttagagattgaactcagatcatgagcaagcaccttgctgcctgctatgtccctccagcagtctgac

[0174] catgttccttcccccaagattgtggaagctggactgaagatcacaatctgccagatgggcagaatctttactctttggca

[0175] catttgttgctgatggggagtgaatacccatggggacatggctgtcatggtgtggaagtgatagaaatgaaaacatgt

[0176] atggatctgtcacaggagctggtgaggctgatgggtgtgtgggtggccactgtttgctctctgcttgtcacagcctcttg

[0177] ttcagggcttgatcagggaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagca

[0178] gatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggagctctggaagacaatgagcagcca

[0179] ctttcctctagatacaataggcggagtcaggaaggtagtattgacattgctggggcctaggagctactcactgctcggt

[0180] ggccgtcagatggtgaaccggcgtaaccttggcacacaggcctgggctgtacaaggcgtctggctgcagggccaa

[0181] agaggactccaccctagggacaggagtacttcagacatctgggaatctgggatgggttttaaaattcagatcccaata

[0182] taaaaaaacaactcccaaacaaacagcagcaattaaaaaaaaaaaaaaaaaccagcctcccaagtaaaacaataatmCis700's gene sequence is shown in SEQ ID NO.10:

[0183] attttataaaagtttatttaccgccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcatggtatatatg

[0184] gatgtcagagtttggttctctccttctgcagtgtggctcttagagattgaactcagatcatgagcaagcaccttgctgcct

[0185] gctatgtccctccagcagtctgaccatgttccttcccccaagattgtggaagctggactgaagatcacaatctgccaga

[0186] tgggcagaatctttactctttggcacatttgttgctgatggggagtgaatacccatggggacatggctgtcatggtgtgg

[0187] aagtgatagaaatgaaaacatgtatggatctgtcacaggagctggtgaggctgatgggtgtgtgggtggccactgttt

[0188] gctctctgcttgtcacagcctcttgttcagggcttgatcagggaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg

[0189] tgtgtggtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggag

[0190] ctctggaagacaatgagcagccactttcctctagatacaataggcggagtcaggaaggtagtattgacattgctgggg

[0191] cctaggagctactcactgctcggtggccgtcagatggtgaaccggcgtaaccttggc

[0192] The gene sequence of mCis400 is shown as SEQ ID NO. 11:

[0193] cccccaagattgtggaagctggactgaagatcacaatctgccagatgggcagaatctttactctttggcacatttgttgc

[0194] tgatggggagtgaatacccatggggacatggctgtcatggtgtggaagtgatagaaatgaaaacatgtatggatctgt

[0195] cacaggagctggtgaggctgatgggtgtgtgggtggccactgtttgctctctgcttgtcacagcctcttgttcagggctt

[0196] gatcagggaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagcagatctgtca

[0197] gctttcccgcttttgttagagggtgatatcatgcttcctggggggagctctggaagacaatgagcagccactttcctcta

[0198] ga

[0199] The gene sequence of mCis303 is shown as SEQ ID NO. 12:

[0200] ccatggggacatggctgtcatggtgtggaagtgatagaaatgaaaaacatgtatggatctgtcacaggagctggtgag

[0201] gctgatgggtgtgtgggtggccactgtttgctctctgcttgtcacagcctcttgttcagggcttgatcagggaggtgtgt

[0202] gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagcagatctgtcagctttcccgcttttgttag

[0203] agggtgatatcatgcttcctggggggagctctggaagacaatgagcagccactttcctctaga

[0204] The gene sequence of mCis200 is shown in SEQ ID NO.13:

[0205] tttgctctctgcttgtcacagcctcttgttcagggcttgatcagggaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg

[0206] tgtgtgtggtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctgggggga

[0207] gctctggaagacaatgagcagccactttcctctaga

[0208] The gene sequence of mCis103 is shown in SEQ ID NO.14:

[0209] ccatggggacatggctgtcatggtgtggaagtgatagaaatgaaaaacatgtatggatctgtcacaggagctggtgag

[0210] gctgatgggtgtgtgggtggccactg

[0211] (3) Analysis of sequence polymorphism and functional conservation of the Tek gene promoter and cis-acting element across species.

[0212] Based on the conservation of the promoter and cis-acting elements of Tek gene among species, and the human promoter is an indispensable part of the future clinical gene therapy program, therefore we carried out subcloning and sequence truncation functional analysis experiments around the human (Homo sapiens) Tek gene promoter and cis-acting elements. We first subcloned the 1612bp sequence of the human Tek gene promoter region including the 5' UTR (hPro1612), and then designed the human hPro762 version according to the results of the mouse miniBEND truncation experiment, which truncated the sequence containing the non-essential elements Octopus region I and Octopus region II that we determined between them. Figure 10 a). Figure 10 b shows the truncation strategy of the cis-acting elements of the human Tek gene. The results of in vivo transduction experiments show that the promoter and cis-acting elements of the human Tek gene (hPro1612-hCis700) can also mediate efficient brain vascular endothelial cell-specific gene expression ( Figure 10 c), which proves our guess that the human regulatory sequence works in mouse somatic cells. Although the promoter and cis-acting elements have certain polymorphism in DNA sequence, they are still conserved in function. In addition, the activity of the truncated version hPro762 will be reduced relative to hPro1612, but the specificity of brain endothelial cells is still high. It shows that part of the Octopus region sequence is still needed in the human regulatory sequence to maintain the transcriptional activity of the promoter.

[0213] To test whether the cis-regulatory elements of Tek genes in other species can be used with the Human promoter and mediate brain vascular endothelial cell-specific gene expression, we further synthesized and cloned the cis-regulatory elements of the first intron segment of the Tek gene in other species (including Callithrix jacchus, Sus scrofa, and Rattus norvegicus), and put them one by one into the recombinant vector we designed, packaged into AAV-PhP.eB capsid virus for in vivo testing in mice. The results show that the Tek cis-regulatory elements of other species (Marmo-Cis700 (Callithrix jacchus), Pig-Cis700 (Sus scrofa)) can be used with the Human promoter and mediate brain vascular endothelial cell-specific gene expression ( Figure 11 ), in addition, the promoter (Rat-Pro1600) and cis-regulatory elements (Rat-Cis737) of the rat Tek gene can also mediate brain vascular endothelial cell-specific gene expression ( Figure 11 ).

[0214] wherein the gene sequence of hPro1612 is shown as SEQ ID NO. 47:

[0215]

[0216] The gene sequence of hPro762 is shown as SEQ ID NO. 48:

[0217] ttgctctcatgagcaccatttttatcccaatctaatcctgtatgtttgtgtttttacacagattagtttttaaatgttatatataatttgcttctgaaacaccattgctcaatgactaccaaatctttctcattaccaaaatccttctatgccaacttcttcaagaaatttgatcacctttagatgaattgttaatgaaaattaaagctatagccggcaacatgggtatctttgggctaatggccaaccaacaggccatctgtgtgaaagaaaacaggctaacaattttggactctggtctcttggggctacattgagcattgacctcaccggtgctcactgaaattaattgcttttcaggttgtattttctcatcacggaaaccttcttctcccaattcaaaccatgtgggttaaaatgagaaaacaaaagccaaaacggcttcccacacccaaaagctccttctgtcagagatcccagtagccccgggagagctgttagaagtctgagaaggattggtcatcatcgcataccatacataggtggagggcttgttattctcagtttcccgcctatgagaggatacccctattgtttctgaaaatgctgaccgggacccacacttccaacaaaaattcctctgcccctacagcagcagcaaaagcagcagcagaagcaacagcaacagataagtgttttgatgaattgcgagatggatagggcttgagtgcccccagccctgctgataccaaatgcctttaa

[0218] The gene sequence of hCis700 is shown as SEQ ID NO. 49:

[0219] ttttcaggaa agggaatgtc taaagttgcc ctcttatttt tggaattttg aactgataac cctattctta cccatctggc cagacgattc cttaactcgt gttacacctg cagaatgagt tttagatcta gctgtgacct cttcccccag cccacccccattgtccccttgtgtgccttcaggaatctgatcattcttctctcctgctccttcccaaaggctgcaggagcaggtgtgaagacgtggatgtgccagatgcagagtcctgacacttttcaacacatctgcatattagaggaagtacatacccattgcttggtggtttcatgtctaatgtggtatgagtgtgacaaagagagggagaaaatttggactagccaaagaagccagtcaggcgtggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcactcactgctctgcggccgtcagatggtgaaccagcttaaccttggc

[0220] The gene sequence of hCis400 is shown as SEQ ID NO. 50:

[0221] aagtacatacccattgcttggtggtttcatgtctaatgtggtatgagtgtgacaaagagagggagaaaatttggactagccaaagaagccagtcaggcgtggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcactcactgctctgcggccgtcagatggtgaaccagcttaaccttggc

[0222] The gene sequence of hCis200 is shown as SEQ ID NO. 51:

[0223] aagtacatacccattgcttggtggtttcatgtctaatgtggtatgagtgtgacaaagagagggagaaaatttggactagccaaagaagccagtcaggcgtggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccaga

[0224] The gene sequence of Marmo-Cis700 is shown as SEQ ID NO. 52:

[0225] Caggaaagtgaatgtctaaagttgtctcccttacttttggaattttaaactgataaccctattcttacccatctggccagactattccttaacttgcttcacacatgcagaatgagttttagatttagctatggctgcttccccctaagccccacccgcactttgcctccttttgtcccttcaggaatctgataattcttctctcatggttcttgccaaagattgcaggagcaggtgtgaagacgtggatatgccagatgggcagagtccttatgcttctcaacacatctgctgaatagagggagcgcatacccattccctgatggtttcatgtcttatgtggtgtgggtgtgacaaagagagggagaaatgtggactagccaaaggagccagtcaggcttggggttgagaggcatcgtgggcagctgccatttgctctgtgcttgtcacagccccttgcccaggtcttgaccagtgaggtgtgtgtcctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactagaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcgctcactgctctgcagccgtcagagggtgaaccagcttaaccttggc

[0226] The gene sequence of Pig-Cis700 is shown as SEQ ID NO. 53:

[0227] Cagctggcatcactcccagccttcaggaaagggaatgtctaaagttgcctcccttatttctgaattttaaactgataacccaattcccaccggtctagccagtgtattccttacttgcattacacatgcagagcatttttcagaccgagccttgcctgcttgcctctacatccctcccccaaggcctcctttgtcctgaccatcttcctcctgctatcccttcccaaagactgcaggggcaggtgtggagacagggatctgccagatgagcagagtcctgactctttttaacacagctggtggtgagagcggggatattctcgccttgctgtgctgtgtctaccatggaacacatggagcaaagagaacaaacctaaacatgagccaaaggagcccatcaggcagcaacctgaggggcactgttggaggcactgttggctttctgctggtcacagccccctgcctggggcttgaccagtgaggtgtatatggcagtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggagcgctggaagacaatgccgggccacttgccttcagatacagtgggcggagtcaggaaggcagtattgacattgccgggccaggggggtactcactgctccagcggtcagatggtgaaccagcttaaccttggc

[0228] The gene sequence of Rat-Pro1600 is shown as SEQ ID NO. 54:

[0229]

[0230] The gene sequence of Rat-Cis737 is shown as SEQ ID NO. 9:

[0231] ttgctgccctctagaccttcaaagacctattttataaaattatatttaccacccactcgtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtatgtgttatacatattggaagtcagagtttggttctcttcttccacaatgcggatcacagagcatcaggcttgtgagcaccttgctgcctgctttgtctcttcagcagtctgaccttgctccttctcgggggctgtggaagtaggactgaagttcagaatctgccagatgggcagaatctttactacttggtacatttgatgctgatagggaatgaattcccacagggacatggctgacacggtgtggaagtgacagaaatggggacgtttgtggatcaatcacatgagttggtgaggctgaggggtgttgtgggaggctgctgttggctccctgcttgttcagggattgaccagggagaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggagctctggaagacaatgagcaggcactttcctctagatacaataggcggagtcaggaaggtagtattgacattgctggggcctaggagctactcactgctctgcggccgtcagatggtgaaccagcataaccttggcacacaggcct

[0232] Example 3 Optimization of the overexpression system based on miniBEND

[0233] To test the strength of the miniBEND promoter obtained in (1) of Example 2, we first constructed the AAV-mPro1576-EGFP-mCis700 plasmid, packaged it into AAV-PhP.eB, and injected the virus into the blood circulation of C57BL / 6J wild-type mice by orbital vein injection to transduce the vascular endothelial cells throughout the brain, and the results showed that the EGFP fluorescence intensity of the section was much weaker than that of the ordinary ef1a-EGFP virus expression ( Figure 13 ), which indicates that the mPro1576 version of the promoter has a weaker strength than the conventional broad-spectrum medium-strength promoter ef1a, and belongs to a weak promoter. Obviously, the inherent weak promoter characteristics of miniBENDv1 (the general term for the first version of mPro1576, distinguished from the truncated optimal version mPro723) limit its wide application in the application scenarios of overexpression of exogenous genes, so we need to further optimize and improve the strength of the miniBEND promoter. The optimization goal is to reach the starting level of the ef1a promoter, that is, to meet the use requirements of in vivo overexpression.

[0234] We first thought of introducing the Tet-off system (Loew et al., 2010) to achieve the overexpression of the target gene through the transcription cascade amplification strategy of the tTA / TRE system. This strategy has two specific implementation schemes. First, the first scheme is a double AAV vector, which requires the construction of plasmid vectors AAV-mPro1576-tTA-mCis700 and AAV-TRE-EGFP-WPRE-pA. This scheme divides the expression space of the exogenous gene into another AAV vector, greatly increasing the limit of the length of the insertable exogenous gene, which is less than or equal to 4 kb. The second scheme is a single AAV vector, which integrates the miniBEND promoter and the tTA / TRE two expression systems into the same rAAV vector, but the fluorescence expression intensity of this scheme is similar to that of the double AAV vector group, and the fluorescence cell labeling density is low ( Figure 12 ). On the other hand, the vector capacity of this strategy is much smaller than that of the double AAV vector, only 1.5 kb, and has certain limitations in the delivery of many genes greater than 1.5 kb. Therefore, we also need new single AAV vector strategies.

[0235] We tried to increase the expression level of foreign genes by adding extra regulatory elements to increase the strength of the promoter or to increase the stability of the foreign mRNA. We found that intron regulatory sequences could increase the strength of the promoter, so we tried to introduce intron regulatory sequences to construct chimeric promoters to increase the strength of the miniBEND promoter. However, there was a risk that the new chimeric promoter might not maintain the cell type specificity of endothelial cells. We designed a series of experiments to construct AAV-mPro1576-glo566-EGFP-pA-mCis700 plasmids, AAV-mPro723-glo566-EGFP-pA-mCis700 plasmids, AAV-mPro723-glo228-EGFP-pA-mCis700 plasmids, and AAV-mPro723-glo566-EGFP-pA-mCis700 plasmids (a) of Example 1. Figure 13 We found that the new chimeric promoter not only did not affect its endothelial cell-specific expression characteristics, but also the cis-regulatory elements worked normally, and the strength of the chimeric promoter was also enhanced, indicating that this method was feasible. Moreover, the truncated version of the chimeric promoter (mPro723-glo566, mPro723-glo228) also worked (b, c) of Example 1, and the length of the mPro723-glo228 version of the chimeric promoter was 978 bp (less than 1 kb), which gave more space to the foreign gene. Figure 13

[0236] The gene sequence of mPro723-glo566 is shown in SEQ ID NO. 46:

[0237] ​

[0238] wherein the gene sequence of mPro723-glo228 is set forth in SEQ ID NO. 55:

[0239] GtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatctggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatcacaggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgccccaaagccccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcataccatacataggtggagggcttgttattcaattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccaggaccttacttgtaacaaagatccctctgccccacaatccagttaaggcaggagcaggagccggagcaggagcagaagataagccttggatgaagggcaagatggatagggctcgctctgccccaagccctgctgataccaagtgcctttaagatacagcctttcccatcctaatctgcaaaggaaacaggaaaaaggaacttaaccctccctgtgctcagacagaaatgagactgttaccgcctgcttctgtggtgtttctccttgccgccaacttgtaaacaagagcgagtggaccatgcgagcgggaagtcgcaaagttgtgagttgttgaaagctagtaccggtAgTTCGTTTAGTGAACCGggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcag

[0240] wherein the gene sequence of mPro1576-glo566 is shown as SEQ ID NO. 56:

[0241]

[0242] Example 4 Construction of a mouse model of focal pathology of brain arteriovenous malformation (bAVM)

[0243] Brain arteriovenous malformation (bAVM) is another major category of intracranial vascular malformation disease in addition to cavernous malformation (CCM). We investigated the pathogenic genes of brain arteriovenous malformation and found that a recent study reported a case of bAVM patient with somatic mutation of Braf gene (Hong et al., 2019), indicating that Braf gene mutation is closely related to the occurrence and development of bAVM disease. Therefore, we purchased conditional knock-in mice carrying Braf gene point mutation (Braf-CA) (Dankort et al., 2007) in the hope that we can simulate the pathological phenotype of human bAVM in mice, and our research results can also prove whether Braf gene is a real pathogenic gene.

[0244] Braf-CA mice are a kind of mice with human BRAF gene fragment knock-in. The design principle is based on the high conservation of amino acid sequences between human BRAF kinase protein and mouse BRAF kinase protein. In the design of homologous recombination knock-in vector, in order to ensure the normal expression of endogenous Braf gene and the sequence of normal Braf protein, the DNA fragment of Exon15-Exon18 of human BRAF gene with double-end flanking LoxP site is used in the middle of the knock-in vector, and the Poly A element and Neo selection gene are downstream of the 3' end. The T base at position 1799 of the coding region on the Exon15 of the mouse genomic Braf locus downstream of the second LxoP site in the knock-in vector is mutated to A base (c.1799T to A), and the valine (V) at position 600 of the corresponding protein sequence is mutated to glutamic acid (E).

[0245] Under the action of Cre recombinase, the LoxP site on the Braf locus will be removed, and the Exon15-Exon18 fragment of human origin after Exon14 of the mouse endogenous Braf gene will be removed, and the corresponding downstream fragment carrying the V600E point mutation will be normally transcribed and translated, and finally the BrafV 600E mutant protein Figure 14 a). We designed a set of experiments, selected homozygous Braf-CA mice, and in the P30-P50 age group, the mice were injected with AAV-miniBEND-Cre (AAV-PhP.eB-mPro723-Cre-pA-mCis700) virus in situ in the unilateral cerebral cortex, to induce endothelial cells to overexpress Braf V600E mutant protein Figure 14b), the homozygous Braf-CA mice raised normally and were viable and fertile. We observed 12 mice after modeling for a long time, and found that the mice with focal bAVM model died one after another after 20 days of virus injection, and the final survival rate was less than 50% (Fig. 1 Figure 14 c), the successfully modeled mice could be observed by laser speckle blood flow imaging instrument to observe the abnormal structure of blood vessels in the lesion area through the skull (Fig. 1 Figure 14 d).

[0246] During the observation, we used magnetic resonance imaging (MRI) to observe the development of intracranial bAVM lesions in mice (Fig. 1 Figure 15 a). The results showed that brain hemorrhage began to be found at 15 days after virus injection (PID15), and at 22 days, the area of intracranial edema in mice was observed to expand, indicating that the bAVM lesion gradually had a tumor-like invasion effect. By the 41st day, the bAVM lesion area had tissue necrosis and compression of the hippocampus, indicating that the bAVM lesion had a tumor-like space-occupying effect (Fig. 1 Figure 15 b, c). The focal bAVM disease model based on the Braf-CA transgenic mouse is similar to the clinical intracranial arteriovenous malformation in the imaging phenotype, and we also observed the mouse hemiplegia and occasional epilepsy phenotype (with video recording) consistent with the behavior of clinical bAVM patients. In order to further confirm the changes in the morphological structure of brain blood vessels in the bAVM lesion area, we further used Laminin antibody to perform immunofluorescence experiments on the samples, and statistically analyzed the blood vessel diameters in the normal brain area and the lesion area. We found that there were almost no capillary-sized microvessels in the bAVM lesion area, but only thickened malformation vessels (Fig. 1 Figure 16 a), and the statistical results also showed that the blood vessel diameter in the bAVM lesion area was significantly larger than that in the normal brain area (Fig. 1 Figure 16 b, c).

[0247] Example 5 PLX4032 small molecule compound can effectively inhibit the development of brain arteriovenous malformation disease model in mice

[0248] Based on the brain vascular arteriovenous malformation disease model constructed based on the Braf-CA transgenic mouse in Example 4, we have been able to confirm that the Braf V600E mutant protein can induce the formation of malformation vessels in the bAVM lesion area, and since the Braf V600EThe kinase activity of the mutant protein is more than 500 times higher than that of the wild-type Braf protein (see Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R; Cancer Genome Project. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004 Mar 19;116(6):855-67. doi: 10.1016 / s0092-8674(04)00215-6). We suspect that the main cause of vascular malformation is the over-activation of the Braf-MEK1 signaling pathway. Next, we further explore the signal pathway regulation mechanism of the occurrence and development of bAVM. After consulting the literature, we find that the PLX4032 (Vemurafenib) small molecule drug is a specific inhibitor of the kinase activity of the Braf V600E mutant protein, which can effectively inhibit the Braf V600E mutant protein kinase activity (Bollag et al., 2010) in living mice. If the Braf V600E mutant protein kinase activity is inhibited in the bAVM model mice, it can hinder the development of bAVM lesions, which can prove that the high kinase activity of the Braf V600E mutant protein and the activation of the downstream signaling pathway are sufficient for the development of bAVM disease. In order to further confirm the molecular mechanism of the occurrence of bAVM disease and test our guess, we designed a group of pharmacological inhibition experiments, and took the diameter of the lesion before and after the nuclear magnetic resonance imaging as the detection index. The drug was administered from the 7th day after the modeling virus injection, and the control group was given the same amount of drug solvent carrier DMSO. Nuclear magnetic resonance imaging was performed from the 22nd day (a) of the experiment. The results show that the front and back diameters of the lesion area of the experimental group are significantly smaller than the measured values of the control group (b and c of Figure 17 Figure 17 , indicating that the specific inhibitor PLX4032 (Vemurafenib) of the Braf V600E mutant protein can hinder the development of bAVM disease. Further H&E histological staining results show that the number of abnormal blood vessels in the lesion area after treatment with the PLX4032 small molecule drug is significantly reduced, and the area of the lesion area in the experimental group is significantly reduced compared with the control group, as shown in Figure 18 ​Figures 6a and 6b show the results of the histological analysis of the blood vessels of the mice treated with the AAV virus. In particular, Figure 6a shows the results of the control group (DMSO) while Figure 6b shows the results of the administration group; b shows the density of the malformed vessels in the control and administration groups; c shows the diameter of the malformed vessels in the control and administration groups.

[0249] Although the above describes the specific embodiments of the present application, it is not intended to limit the scope of protection of the present application. Various modifications or variations made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the scope of protection of the present application.

[0250] Appendix A AAV virus information used in this specification

[0251] Table A.1 AAV virus and titer information

[0252]

[0253]

[0254] Table A.2 AAV virus and titer information

[0255]

[0256] Appendix B References

[0257] The specific sources of the above documents described by the author's name appearing in the description of this patent specification are recorded as follows:

[0258] 1. Schlaeger, Thorsten M, Bartunkova, et al. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic [J]. Proceedings of the National Academy of Sciences of the United States of America, 1997.

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Claims

1. Use of a small molecule drug, PLX4032, for the manufacture of a medicament for treating a cerebral arteriovenous malformation disease, wherein, The structure of the small molecule drug PLX4032 is as follows: 。 2. Use according to claim 1, characterized in that, The cerebral arteriovenous malformation disease is manifested in Braf V600E Expression of mutant proteins.

3. Use according to claim 1, characterized in that, The cerebral arteriovenous malformation disease is manifested in Braf V600E The kinase activity of the mutant protein is enhanced.

4. Use according to claim 2, characterized in that, The brain arteriovenous malformation disease is manifested in Braf V600E The kinase activity of the mutant protein is enhanced.

5. The use according to claim 1, characterized in that, The cerebral arteriovenous malformation disease is manifested in over-activation of Braf-MEK1 signal pathway.

6. Use according to claim 2, characterized in that, The cerebral arteriovenous malformation disease is manifested in over-activation of Braf-MEK1 signal pathway.

7. Use according to claim 3, characterized in that, The cerebral arteriovenous malformation disease is manifested in over-activation of Braf-MEK1 signal pathway.

8. Use according to claim 4, characterized in that, The cerebral arteriovenous malformation disease is manifested in over-activation of Braf-MEK1 signal pathway.

9. Use according to any one of claims 1 to 8, characterized in that, The small molecule drug PLX4032 is selected from one of tablet, solution, suspension, emulsion, powder, granule, capsule, microcapsule, microsphere and injection.

10. Use according to any one of claims 1 to 8, characterized in that, The small molecule drug PLX4032 further comprises a conventional carrier for pharmacy.

11. Use according to claim 10, characterized in that, The conventional carrier is selected from one or more than two of filler, binder, humectant, absorption promoter and cosolvent.

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