Application of small molecule medicine PLX4032 in preparation of medicine for treating cerebral arteriovenous malformation diseases

Abstract

The invention discloses an application of a small molecule medicine PLX4032 in preparation of a medicine for treating cerebral arteriovenous malformation diseases, the small molecule medicine PLX4032 can inhibit excessive activation of a BrafV600E mutant protein Braf-MEK1 signal channel, so that the kinase activity of the BrafV600E mutant protein is reduced, the expression of the BrafV600E mutant protein in the cerebral arteriovenous malformation diseases is avoided, and the cerebral arteriovenous malformation diseases can be treated by using the small molecule medicine PLX4032 to treat the cerebral arteriovenous malformation diseases. Pathological cells can be directly targeted, and the treatment effect is good.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a small molecule drug PLX4032 in preparing a drug for treating cerebral arteriovenous malformation diseases. Background Art

[0002] Cerebral arteriovenous malformation (bAVM) is one of the four major vascular malformations, the other three being venous malformations, cavernous malformations (CCMs), and telangiectasias. Among them, arteriovenous malformations are one of the most phenotypically significant types of vascular malformations. First described in 1895, bAVMs are characterized by arterial blood supply and high-flow, low-resistance shunts between arteries and veins. The malformed vessels are composed of dilated arteries and tortuous, pulsating veins that converge in all directions, with no capillaries.

[0003] For the treatment of AVM disease, different grades of AVM have different treatment plans. Generally speaking, if you are diagnosed with intracranial AVM, the doctor will recommend waiting to see how the lesion develops. If it is non-malignant, you can wait and control it with drugs without surgery. On the contrary, if the lesion is more obvious, it can be surgically removed, and embolization ("glue therapy") therapy can be considered. If the lesion is in an inaccessible location, 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 Phase I clinical trials. Bevacizumab is an anti-tumor drug, also known as Avastin. It is a humanized monoclonal antibody that targets vascular endothelial growth factor (VEGF) in arteriovenous malformations, thereby inhibiting angiogenesis and growth in the lesion area. Since Avastin is a protein drug, it is difficult for it to effectively pass through the blood-brain barrier and act on the diseased brain area. Therefore, small molecule compound drugs represent a more attractive solution. How to target diseased cells and the design and screening of small molecule inhibitors based on the BRAF-MEK1 signaling pathway kinase protein as a target have become the core direction of drug development. Summary of the invention

[0004] The problem of screening small molecule inhibitor drugs that directly target diseased cells in drugs for treating brain arteriovenous malformations is addressed. The present invention provides an application of a small molecule drug PLX4032 in the preparation of drugs for treating brain arteriovenous malformations. The drug is a small molecule inhibitor that directly targets diseased cells and is based on the BRAF-MEK1 signaling pathway kinase protein as a target.

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

[0006] In a first aspect, the present invention provides a use of a small molecule drug PLX4032 in the preparation of a drug for treating cerebral arteriovenous malformation disease, wherein the structure of the small molecule drug PLX4032 is as follows:

[0007]

[0008] Preferably, the application is characterized in that the cerebral arteriovenous malformation disease is manifested in Braf V600E Expression of mutant proteins.

[0009] Preferably, Braf V600E The kinase activity of the mutant protein is increased.

[0010] Preferably, it is manifested in overactivation of the Braf-MEK1 signaling pathway.

[0011] Preferably, the application is characterized in that the small molecule drug PLX4032 is selected from one of tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres and injections.

[0012] Preferably, the application is characterized in that the small molecule drug PLX4032 also contains a conventional pharmaceutical carrier.

[0013] Preferably, the application is characterized in that the conventional carrier is selected from one or more of a filler, a binder, a wetting agent, an absorption promoter and a cosolvent.

[0014] Beneficial effects of the present invention:

[0015] The small molecule drug PLX4032 of the present invention can inhibit Braf V600E Overactivation of the Braf-MEK1 signaling pathway by the mutant protein reduces Braf V600E Kinase activity of mutant protein, thereby avoiding Braf in brain arteriovenous malformation disease V600E The expression of mutant proteins can directly target diseased cells and has a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The data show the expression of Tek gene in single cell and mixed cell transcriptomes of human and mouse cerebral vascular system, where a is the expression profile of Tek gene in single cell transcriptome of human cerebral vascular system; 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: periventricular fiber cell; M.FB: meningeal fiber cell; TC: T cell; EPEN: ependymal cell; AST-HPc: hippocampal astrocyte; AST-Ctx: cortical astrocyte; PM: periventricular macrophage; MG: microglia; OL: oligodendrocyte; OPC: oligodendrocyte precursor cell; NEU: neuron; b is the expression profile of Tek gene in the single-cell transcriptome of mouse brain vascular system; among them, PC: pericyte; vSMC: venous smooth muscle cell; aaSMC: arteriolar smooth muscle cell; aSMC: arterial smooth muscle cell; MG: microglia; FB1: type I fibroblast-like; FB2: type II fibroblast-like; OL: oligodendrocyte; EC1: type I endothelial cell; EC2: type II endothelial cell; EC3: type III endothelial cell; vEC: venous endothelial cell; capilEC: capillary endothelial cell; aEC: arterial endothelial cell; AC: astrocyte; c is the expression profile of Tek gene in the mouse brain cell type-specific mixed cell transcriptome.

[0017] Figure 2 The figure shows that Tek-Cre transgenic mice can efficiently and specifically mark endothelial cells in various segments of cerebral blood vessels, where a is a fluorescence microscopic image of a coronal brain section of a Tek-Cre; Ai47 transgenic mouse on the 42nd day after birth; b is a confocal fluorescence microscopic image of a single region of interest in the cortical area, HO: Hoechst 33342 nuclear dye, EGFP: indicates the reporter gene EGFP signal specifically expressed in brain vascular endothelial cells of Tek-Cre-positive mice, HO / EGFP: the imaging result after merging the HO and EGFP channels, and the white arrow indicates the brain vascular endothelial cells; c is the distribution of vascular endothelial cells marked in the cerebral cortex, striatum and thalamus of Tek-Cre; Ai47 double transgenic mice, and the white arrow indicates the brain vascular endothelial cells.

[0018] Figure 3The figure shows the region of sequence conservatism in the promoter of the Tek gene determined by multi-species sequence alignment, wherein a is a DNA multiple sequence alignment analysis of the promoter region and the 5' non-coding region (5'UTR) corresponding to the Tek gene on the genomes of multiple species including humans, rhesus monkeys, marmosets, pigs, cattle, rats, and mice; b is an evolutionary tree analysis of sequences from each species, and the promoter regions and 5' non-coding region sequences corresponding to pigs and cattle, humans and rhesus monkeys and marmosets, mice and rats are evolutionarily closer.

[0019] Figure 4 The figure shows the conserved region of the first intron sequence of the Tek gene determined by multi-species sequence alignment, wherein a is a DNA multiple sequence alignment analysis of the region corresponding to the first intron of the Tek gene on the genomes of multiple species including humans, rhesus monkeys, marmosets, pigs, cattle, rats, and mice; b is an evolutionary tree analysis of sequences from each species, and the first intron sequences corresponding to pigs and cattle, humans and rhesus monkeys and marmosets, mice and rats are respectively closer in evolution.

[0020] Figure 5 Shown is a technical route adopted by the present invention, a schematic flow chart of a method for separation and in vivo testing of functional segments of a promoter and a cis-regulatory element.

[0021] Figure 6 Shown is the analysis of truncation and transduction efficiency of the Tek gene promoter segment, where a is a schematic diagram of the truncation strategy of the Tek gene promoter segment; b is a statistical bar graph of the viral 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 viruses mediated by each truncated version of the promoter, with a scale bar of 100 μm.

[0022] Figure 7 Shown is the analysis of truncation and transduction efficiency of cis-regulatory elements in the first intron segment of the Tek gene, where a is a schematic diagram of the truncation strategy of cis-regulatory elements; b is the in vivo vascular endothelial cell transduction efficiency of recombinant AAV viruses mediated by various truncated versions; c is a schematic diagram of in vivo transduction fluorescence imaging of recombinant AAV viruses mediated by various truncated versions of cis-regulatory elements, and the scale bar is 100 μm.

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

[0024] Fig. 9 The figure shows that continuous truncation of the 5' end sequence of the mouse Tek gene promoter region can reduce the non-specific labeling of neurons in the cerebellum by the miniBEND system, where a is the virus used in each group is AAV-PhP.eB serotype, and the recombinant AAV vectors used are CAG-mScarlet, mPro973-Cre, mPro760-Cre, and mPro723-Cre, respectively. The Tek cis-regulatory element used in the recombinant vectors in the last three groups is mCis700, and they are 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), and the scale bar in the figure is 100μm; b is the proportion of non-specific labeling other than endothelial cells, with a standardized labeling density (per 10 11 Virus particles were used to transduce a mouse in vivo. 2 The number of labeled cells in the area was used as an indicator for evaluation. The number of experimental animals in each group was n=2. Five fields of view were selected from the slices of each mouse for cell counting and statistical analysis. The scale bar in the figure is 100 μm.

[0025] Fig.10Shown are the truncation and transduction efficiency analysis of the human Tek gene promoter and cis-regulatory elements, wherein a is a schematic diagram of the truncation strategy of the human Tek gene promoter; b is a schematic diagram of the truncation strategy of the human Tek gene cis-regulatory elements; and c is the transduction efficiency evaluation in mice.

[0026] Fig.11 Shown is the conserved sequence of the cis-regulatory element corresponding to the Tek gene in different species, which can also mediate endothelial cell-specific gene transcription; Marmo-Cis700: a truncated version of the cis-regulatory element Cis700 of the Tek gene from marmoset, Pig-Cis700: a truncated version of the cis-regulatory element Cis700 of the Tek gene from pig, Rat-Cis737: a truncated version of the cis-regulatory element Cis737 of the Tek gene from rat, and the promoter on the recombinant vector used in this group is Rat-Pro1600.

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

[0028] Fig.13 The figure shows the vector design and in vivo characterization of the overexpression system based on the miniBEND-globin fusion promoter, where a is a schematic diagram of the vector design of the overexpression system based on miniBEND, the chimeric introns include two versions of glo566 and glo228, and GOI in the figure is the exogenous target gene; b is the fluorescence imaging result of the four overexpression systems in Figure a packaged into AAV-PhP.eB serotype virus and tested in C57BL / 6J mice; c is the statistical result corresponding to the cortical brain area in Figure b, the ordinate represents the labeling density of endothelial cells after standardization (n / mm2), and the standardization method is every 10 11Endothelial cell labeling density corresponding to each viral particle. The number of mice used and the number of statistical fields in each group were: miniBENDv1 group (4 mice, including one using AAV-PhP.V1 and three 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] Fig.14 The figure shows the method of constructing a focal bAVM disease model based on Braf-CA transgenic mice; a is the working principle of Braf-CA transgenic mice (Dankort et al., 2007); b is the experimental schematic diagram; c is the death curve of the mouse model, indicating that the focal bAVM model has a certain mortality rate; a total of 12 mice with successful modeling were counted (n=12); d is the laser speckle blood flow imaging image of Braf-CA mice after bAVM modeling, which shows the representative results of two model mice. Among them, Exon means exon, Exon 1 means the first exon of the gene, Exon 14, Exon 15, and Exon 16 mean the 14th, 15th, and 16th exons.

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

[0031] Fig.16 Shown is Braf in brain endothelial cells V600E Gene mutations can induce vascular malformations, where a is a representative image of vascular fluorescence imaging in normal brain areas and lesion areas after Laminin antibody immunofluorescence experiment; b is a statistical box plot of vascular diameters; c is a statistical frequency distribution diagram of vascular diameters; the scale bar in the figure is 50 μm.

[0032] Fig.17 The Braf-bAVM disease mouse model constructed is used to evaluate the therapeutic effect of small molecule drugs, where a is BRAF V600E Molecular structure diagram of kinase selective inhibitor PLX4032 and schematic diagram of experimental flow, the drug concentration is 75 mg / kg; the control group was intraperitoneally injected with an equal amount of DMSO (Vehicle, carrier); b is a bar graph comparing the lesion size between the drug group and the control group, the ordinate represents the lesion diameter size (Nidus, AP diameter) of the bAVM lesion area in the anterior-posterior direction, the error bar represents the mean standard deviation, unpaired T test, ** indicates p < 0.01, experimental group (n = 9), control group (n = 7); c is a representative image of the MRI T2 sequence scan results of some experimental mice. The mice in the treatment group are numbered: #73, #78 and #85; the mice in the control group are numbered: #68, #83 and #90. The Z-axis interval of the MRI scan is 0.5 mm.

[0033] Fig.18 The results of H&E histological staining of the drug-treated group and the control group of the constructed Braf-bAVM disease mouse model are shown. a is a representative image of H&E staining of the cerebral cortex brain slice of 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), while the right half of a shows the results of the drug-treated group. a shows two representative areas of the control group and the treatment group; b shows the density of abnormal blood vessels in the control group and the drug-treated group; c shows the diameter of abnormal blood vessels in the control group and the drug-treated group. DETAILED DESCRIPTION

[0034] The present invention relates to the use of a small molecule drug PLX4032 in the preparation of a drug for the treatment of cerebral arteriovenous malformation diseases, and provides a new use of the small molecule drug PLX4032. The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are limited to illustrating the present invention and are not used to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the embodiments 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 according to the conditions recommended by the manufacturer. All experimental data were statistically analyzed using GraphPad Prism8 software (GraphPad Software, USA), and all data were expressed as mean standard deviations to represent error bars, and non-parametric inter-group t-test analysis was used.

[0035] Experimental Materials and Methods

[0036] 1Tek Gene

[0037] The 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 the receptor and mediates a signaling pathway that plays a role in embryonic vascular development. The gene has a wide range of specific expression in vascular endothelial cells.

[0038] The Tek gene has two core transcriptional regulatory sequences, including the conventional promoter region (TekPromoter) and the non-coding sequence of the first intron (Tek intron1).

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

[0040] 2 Truncated sequences and recombinant plasmid construction

[0041] The recombinant plasmid constructed by the present invention is: AAV-miniTek-Cre-bGHpA-Cis-element, wherein miniTek is a promoter region (Tek Promoter region) of different truncated sizes of the Tek gene, and Cis-element is a first intron region (Tek intron1 region) of different truncated sizes of the Tek gene. In the recombinant plasmid, SpeI and AgeI restriction sites are introduced at both ends of miniTek, and KpnI and NotI restriction sites are introduced at both ends of Cis-element. The standardized plasmid backbone source used by the recombinant plasmid is the plasmid pAAV-CBA-cre-U6-sgRNA (article number Addgene#60229) purchased from the Addgene website, and the transgenic segment on the genomic DNA of the Tek-Cre transgenic mouse includes a 2.1kb sequence of the promoter region of the mouse Tek gene and a 10kb 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 2.1kb sequence of the Tek gene promoter region and the 10kb sequence of the first intron segment of the Tek gene were used for truncation experiments. The Tek promoter regions of different truncated lengths of mouse origin (including mPro1576, mPro973, mPro723, mPro760, mPro583, and mPro471) were obtained by PCR using the genomic DNA of Tek-Cre transgenic mice as templates, and primers were designed upstream and downstream of the corresponding regions. The first intron regions of different truncated lengths of mouse origin (mCis1349, mCis700, mCis400, mCis303, mCis200, and mCis103) were obtained by PCR using the genomic DNA of Tek-Cre transgenic mice as templates, and primers were designed upstream and downstream of the corresponding regions. Both ends of the PCR primers contain 20bp sequences homologous to the sticky ends of the recombinant AAV vector, and the target recombinant plasmid can then be obtained by homologous recombination. The detailed information of the primers is shown in Table 1 below.

[0042] The promoter region sequences of the human Tek gene of different truncated lengths (including hPro1612 and hPro762, found and confirmed by multi-species homologous sequence alignment) and the Tek intron region sequences (including hCis700, hCis400, and hCis200, found and confirmed by multi-species homologous sequence alignment) were cloned by PCR reaction using the genomic DNA of the HEK293T cell line as a template, and primers were designed upstream and downstream of the corresponding regions and obtained by PCR. The DNA materials of the cis-regulatory elements (Cis-element) of the Tek gene of rat, pig, and marmoset sources were obtained by gene synthesis from GENEWIZ. The information of other plasmid vectors used in this article 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] 3. Mice

[0053] The experimental animals (mice) used in this patent all comply with the relevant regulations of the Chinese government on animal protection and use, and have been approved by the Animal Care and Use Committee of this unit and are in compliance with management policies. Ai9 and Ai47 mice are from the laboratory of Dr. Zeng Hongkui of the Allen Institute. Tek-Cre (Cat. No. 008863), NG2DsRedBACtg (Cat. No. 008241), and Ai14 (Cat. No. 007914) mice were purchased from the Jackson Laboratory in the United States. Braf-CA fl / fl , also known as Conditional activation Braf V600E Transgenic mice were purchased from Jackson Laboratory, USA, with catalog number 017837. Wild-type mice C57BL / 6J (SPF grade) were purchased from Vital River, and some C57BL / 6J mice were purchased from the experimental animal platform of our 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 Science and Brain Induction.

[0054] 4AAV virus packaging and purification

[0055] AAV virus packaging: (1) Subculture the cells in the 9 cm cell culture dish to the 15 cm cell culture dish. (2) Before transfection, use a suction pump to remove the culture medium in the 15 cm cell culture dish, add 20 mL of complete culture medium (DMEM + 10% FBS + PS / antibody), and then return the culture dish to the CO2 incubator for culture. After expansion culture, if you want to subculture every other day, you can replace the culture medium (DMEM + 10% FBS + PS / antibody) 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 thoroughly shaken on a shaker to mix well, place it in a biosafety cabinet and let it stand for 30 minutes. (5) Add the mixed solution to a 15 cm cell culture dish, cover the dish with a lid, and gently shake the dish on the operating table to evenly distribute the cells in the dish. (6) Place the cell culture dish in a CO2 incubator for culture. After 8 hours, remove the culture medium and add 30 mL of DMEM culture medium (without FBS, with PS / double antibody). (7) Place the cell culture dish in a CO2 incubator again. After 48-72 hours of transfection, the cells can be collected for virus purification.

[0059] Purification of AAV virus: (1) Collect cells: Use a 5 mL pipette to blow the cells off the wall of the cell culture dish and collect them in a 50 mL centrifuge tube. (2) Add 1 / 10 volume of chloroform (3 mL). (3) Place in a shaker and shake at 37°C for 1 hour. (4) Add 1.928 g NaCl and place in a shaker and shake at 25°C for 20 minutes. (5) Centrifuge: 4°C, 12000 rcf, 20 minutes. (6) Transfer the supernatant to a labeled, new 50 mL centrifuge tube. (7) Add 10% of the supernatant volume of PEG8000. (8) Place in a shaker and shake at 25°C for 20 minutes. (9) Place in an ice bath for 1 hour. If the experiment needs to be paused, place in an ice bath overnight. (10) Centrifuge: 4°C, 12000 rcf, 30 minutes. (11) Gently discard the supernatant and turn the tube upside down on absorbent paper. (12) Add 600 μL of filtered PBS, 1 μL of DNase, and 1 μL of RNase to fully dissolve the virus. (13) Place in a 37°C water bath or a 37°C bacterial incubator and 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 thoroughly. (15) Centrifuge: 4°C, 12000 rcf, 10 minutes. (16) Take the supernatant to a new 1.5 mL centrifuge tube and concentrate it using a protein purification column. (Note: The packaging and purification experiments of the AAV virus in this invention were completed by the Vector Center of the Beijing Institute of Brain Science and Brain-Inspired Research (CIBR).

[0060] 5AAV virus injection

[0061] In vivo transduction of AAV virus in mice is achieved by injection into the orbital venous plexus. First, place a cotton ball soaked in liquid isoflurane into a 50ml centrifuge tube to prepare a simple mouse anesthesia induction box. Next, use a 40U insulin needle to absorb the diluted AAV virus of a specific titer, and insert the mouse's head into the simple anesthesia induction box. After the mouse's breathing rate slows down and its limbs become weak, pinch the head to expose the right eyeball, insert the insulin needle containing the AAV virus (about 100μl) into the orbital venous plexus and inject the virus. After quickly pulling it out, take the mouse out of the induction box and let it wake up as soon as possible. If there is bleeding after the needle is removed, it should be wiped off with a cotton swab. Please see the attached table (Table A.2) for information on AAV viruses and titers in this laboratory.

[0062] 6 Immunofluorescence staining experiment

[0063] Mice were perfused with sterile saline through the myocardium, and then replaced with 4% (w / v) paraformaldehyde (PFA) after the liver turned white. After perfusion, the mouse brain was dissected, transferred to a 15 mL centrifuge tube, and fixed in cold PFA (4°C) for 2 hours. It was washed three times in PBS, shaking on a shaker at room temperature for 10 minutes each time. Finally, it was replaced with 30% sucrose and placed on a shaker at 4°C for dehydration. After 1-2 days, the mouse brain sank to the bottom, indicating that dehydration was complete. The fixed brain was sliced ​​40-60 μm using a cryostat (CM3050S, Leica) or 60-70 mm using a vibrating microtome (VT1000S, Leica). The sections were stained as described previously. 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. Primary antibodies to PDGFRβ (1:300, rat, eBioscience, 14-1402-81) or NG2 (1:300, AB5320, rabbit, polyclonal, Millipore), Glut1 (1:300, rabbit, Thermofisher, RB-9052-P0) and CD31 (1:50, rat, BD550274) were incubated with brain slices at 4°C for 24-48 hours. After washing three times with Hoechst33342 or DAPI (Life Technologies) in PBS, the slices were incubated with secondary antibodies conjugated to Alexa 488, Alexa 546 or Alexa 633 / 647 (1:500, Life Technologies) at RT (22-25°C) for 2 hours. After washing three times in PBS, the slices were mounted with anti-fluorescence quenching mounting medium Vectorlab (or customized (60% glycerol and Hoechst33342)).

[0064] 7 Magnetic resonance imaging and 3D reconstruction

[0065] Magnetic resonance imaging (MRI) was performed by a 7.0T scanner (Bruker Pharmascan 70 / 16, Bruker, Germany), equipped with a 23mm surface coil and a 12cm diameter self-shielded gradient system, which can be used for small animal MRI scanning of mice and rats. The experimenter interacted with the scanner through Paravision 5.1 software (Bruker BioSpin) and a Linux computer running Topspin 2.0. The experimental operation steps are as follows: First, the mouse was anesthetized with a mixture of 5% isoflurane and 95% oxygen, and the mouse was transferred to the imaging chamber. At the same time, the valve of gas anesthesia was switched to continue to maintain anesthesia with 2% isoflurane and 98% oxygen during the imaging process. The system is also equipped with a respiratory detection module, with a small respiratory rate detection patch placed under the abdomen of the mouse in the imaging chamber. The experimenter can adjust the anesthesia machine switch valve and adjust the anesthesia intensity by observing the mouse's respiratory rate (generally controlled between 30 and 50 times / minute) feedback. In this study, the relaxation enhancement (RARE) method was used to perform T2-weighted (T2W) imaging of the mouse brain. The parameters used were as follows: repetition time (TR) / echo time (TE) was 3500ms / 33ms, 4RARE factor, 21×21mm scanning field of view (FOV), 256×256 matrix, 25 sections, and 0.5mm section thickness. The high signal areas in the imaging are brain edema lesions and cerebral hemorrhage lesions. The imaging data used 3D slicer4.11.20210226 software to perform three-dimensional reconstruction of the target lesions and quantify the volume of the lesion area.

[0066] 8 Fluorescence microscopy imaging

[0067] Wide-field microscope imaging, brain slice fluorescence signal collection and photography were performed using an Olympus slide scanner VS120. The steps are as follows: first select the fluorescence scanning mode, then load the target slide, enter the target slide's label information and data save path, click Preview, select 2X objective lens scanning for bright field mode, select 10X objective lens scanning for fluorescence mode, circle the scanning area, use multi-point focusing, and use the 5-point method to cover the target imaging brain slice. Next, set the illumination. Samples stained with Hoechst or DAPI use the FL Blue channel, samples expressing EGFP protein use the FL Green channel, and samples expressing red fluorescent proteins such as DsRed, Tdtomato, and mRuby3 use the FL Cy3 channel. Generally, FL Blue and FLGreen dual-channel imaging, FL Blue and FL Cy3 dual-channel imaging, or FL Green and FL Cy3 dual-channel imaging. Finally, use the top-ranked channel as the benchmark for multi-point focusing inspection, and click Next to start scanning the entire area.

[0068] Stereoscopic imaging, stereo microscope (Leica M205 FA), 1x objective. This microscope was used to determine the surface fluorescence expression of EGFP in the mouse brain sample after local injection of AAV. This microscope was used to check the clarity of the cranial window in the last step of cranial window surgery for two-photon imaging in mice.

[0069] Confocal microscopy imaging, using the flip scanning function of the confocal microscope (Leica SP8) to take whole brain slice images, adjacent segments have 10% overlap for automatic stitching. The imaging parameters of cerebrovascular samples are generally: 20x air objective, 1024×1024, z-axis step is 1μm (in many cases, the value optimized by software is used); or 40x oil objective, 1024×1024, z-axis step is 0.5μm, zoom factor is 1.25.

[0070] 9H&E staining

[0071] (1) Sampling: Sterile saline is perfused through the myocardium of mice, and after the liver turns white, the perfusion is replaced with 4% (w / v) paraformaldehyde (PFA). After the perfusion is completed, the mouse brain is dissected, transferred to a 15mL centrifuge tube, and placed in cold PFA (4°C) for fixation for more than 24 hours. The tissue is removed from the fixative and the target area is trimmed with a scalpel in a fume hood. The trimmed tissue and the corresponding label are placed in a dehydration box. (2) Dehydration and wax dipping: Place the dehydration box in a hanging basket and dehydrate it in a dehydrator with gradient alcohol. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h-absolute ethanol I 30min-absolute ethanol II 30min-alcohol benzene 5-10min-xylene I 5-10min-xylene II 5-10min-65° melted paraffin I1h-65° melted paraffin II 1h-65° melted paraffin III 1h. (3) Embedding: embed the wax-soaked tissue in the embedding machine. First put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20° freezer. 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 on a paraffin slicer and slice it with a thickness of 4μm. The slices were floated on 40℃ warm water on a slide spreader to flatten the tissues, and the tissues were picked up with a glass slide and baked in a 60℃ oven. After the wax was dried and melted, they were taken out and stored at room temperature for later use. (5) Dewaxing of paraffin slices to water: the slices were sequentially placed in xylene I for 15min-xylene II for 15min-xylene III for 15min-anhydrous ethanol I for 5min-anhydrous ethanol II for 5min-95% alcohol for 5min-85% alcohol for 5min, and washed with tap water. (6) Hematoxylin staining: the slices were dyed in hematoxylin solution for 1-2min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blueing solution, and rinsed with running water. (7) Eosin staining: the slices were dyed in eosin solution for 2-3min. (8) Dehydration and sealing: the slices were sequentially placed in anhydrous ethanol (5min), anhydrous ethanol (5min), anhydrous ethanol (5min), xylene (5min) to make them transparent, and sealed with neutral gum. (9) Microscopic examination, image acquisition and analysis.

[0072] Example 1

[0073] We mined genes that are highly expressed in brain vascular endothelial cells from mixed cell transcriptome sequencing and single cell transcriptome sequencing data of mouse and human brain cell types. We determined that the Tek gene is highly expressed in endothelial cells of all segments of brain vessels in both humans and mice, meeting our needs. (e.g. Figure 1As shown), where a is the expression profile of Tek gene in single-cell transcriptome of human cerebral vascular system; b is the expression profile of Tek gene in single-cell transcriptome of mouse cerebral vascular system; c is the expression profile of Tek gene in mixed cell transcriptome specific to mouse brain cell type.

[0074] Based on the slice results of Tek-Cre; Ai47 double transgenic mice, we found that the vascular endothelial cells marked by the Tek gene were very comprehensive, basically covering 100% of the vascular endothelial cells, and had a wide distribution in the three segments of arteriovenous capillaries (such as Figure 2 ). Among them, a is the fluorescence microscopic image of the coronal section of the brain of Tek-Cre; Ai47 transgenic mice on the 42nd day after birth. b is the confocal fluorescence microscopic image of a single region of interest in the cortex area, HO: Hoechst 33342 nuclear dye, EGFP: indicates the reporter gene EGFP signal specifically expressed in the brain vascular endothelial cells of Tek-Cre positive mice, HO / EGFP: the imaging result after the HO and EGFP channels are merged, and the arrow indicates a single brain vascular endothelial cell. c is the distribution of vascular endothelial cells marked in the cerebral cortex, striatum and thalamus of Tek-Cre; Ai47 double transgenic mice.

[0075] Since the Tek gene is specifically expressed in the cerebrovascular endothelial cells of mice and humans, it indicates that the gene expression regulation mechanism of the gene is conservative in evolution, and correspondingly, there are conservative core regulatory sequences in the genome. In order to lock the core sequence that regulates transcription, we conducted multi-species sequence comparison analysis on the promoter region and the first intron region of the gene (species include humans (Homo sapiens), macaques (Macaca mulatta), marmosets (Callithrix jacchus), pigs (Sus domesticus), cattle (Bos taurus), rats (Rattus norvegicus), mice (Mus musculus)), and determined the sequences that are conserved among multiple species.

[0076] The results of multiple sequence alignment showed that the regulatory sequences of the promoter regions of the Tek genes in pigs and cattle were more similar, those in humans and marmosets were more similar, and those in mice and rats were more similar. Figure 3 As shown, a is a DNA multiple sequence alignment analysis of the promoter region and 5' non-coding region (5'UTR) of the Tek gene in the genomes of multiple species including humans, rhesus monkeys, marmosets, pigs, cattle, rats, and mice; b is an evolutionary tree analysis of sequences from various species. The promoter region and 5' non-coding region sequences corresponding to pigs and cattle, humans and rhesus monkeys and marmosets, mice and rats are more similar in evolution. The multiple sequence alignment results in the intron region are also the same ( Figure 4), wherein a is a DNA multiple sequence alignment analysis of the region corresponding to the first intron of the Tek gene on the genomes of multiple species including humans, rhesus monkeys, marmosets, pigs, cattle, rats, and mice; b is an evolutionary tree analysis of sequences from each species, and the first intron sequences corresponding to pigs and cattle, humans and rhesus monkeys and marmosets, mice and rats are respectively more similar in evolution.

[0077] The conserved sequences of the core sequences regulating transcription among multiple species are as follows:

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

[0079] 5'-AAGGNTGGTCNTCATCGCATACCATACATAGGTGGAGGGCTTGT TATTCNANTTNCNGCCTATGAGAGGATACCCCTATTGTTNCTGAAAATGCTGACCAGGNCCNACTTNAACAAANATNCCTCTNCCCCNANCNCAGNAANGCAGNAGCAGNAGCNANCNAGCANAGATAAGNTTNGATGAANGCAAGATGGATAGGGCTNGCNTGCCCCNAGCCCTGCTGATACCAANTGCCTTTAA-3'

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

[0081] 5'-GTGTGTGTGNGTGGTCACACCCATCTCAGCAGATCTGTCAGCTT TCCCGCTTTTGTTAGAGGGTGATATCATGCTTCCTGGGGGGAGCNCTGGAAGACAATGNCNGCCACTTTCCTCNAGATACAATAGGCGGAGTCAGGAAGGNAGTATTGACATTGCTGGGGCCNGGAGNACTCACTGCTCNG NGGCCGTCAGATGGTGAACCNGCNTAACCTTGGCACACAGNGCCTGGGNTGTNCAAGGCGTCTGGCTGCAGNGCCAAAGNGGACTCCACCCTNGGGACAGGAGTNCTTNAGACATCTGGGAATCTGGGATGGGTTNAAA-3'

[0082] Example 2 Development and functional testing of mini-promoters and cis-regulatory elements that mediate gene-specific expression in cerebral vascular endothelial cells

[0083] In order to systematically analyze the functions of various truncated versions of promoter sequences and cis-regulatory elements, we designed a standardized recombinant AAV expression vector (such as Figure 5 ), wherein the restriction sites SpeI and AgeI were introduced into the upstream and downstream of the promoter respectively, the expression frame of the NLS-Cre-HA gene was connected after the Kozak sequence, followed by the polyA element bGHpA, and the cis-regulatory element sequence to be inserted was located downstream of the polyA element, which had single restriction sites KpnI and NotI at both ends to facilitate molecular cloning operations.

[0084] First, we collected Tek genome sequences from multiple species from humans to mice, analyzed the sequences of the promoter, 5'-UTR region and the first intron region based on multiple sequence alignment BLAST, and obtained the homologous segments. We then cloned the core promoter region and 5'-UTR region, and bidirectionally cloned the conserved cis-regulatory elements in the first intron. After obtaining the cloned fragments, we subcloned them into the recombinant AAV2 vector that we had designed and standardized, and used the AAV-PhP.eB capsid serotype for conventional three-plasmid system for AAV virus packaging. The downstream was based on the transgenic fluorescent reporter mouse Ai14 Rosa26 -CAG-LSL-Tdtomato or Ai47 Rosa26-CAG-LSL-3XGFP Perform virus transduction experiments in vivo, and finally combine dissection, sectioning, and fluorescence imaging analysis to determine the transduction efficiency and specificity.

[0085] (1) Analysis of truncation of the promoter region of the Tek gene

[0086] After determining the Tek gene as the target gene, our next step is to study the function of its promoter, with the goal of locking the core sequence that regulates the transcription of the Tek gene. First, we extracted the genomic DNA from the ear tissue of the Tek-Cre transgenic mouse. According to the results of our previous sequence alignment, we tried to cut off the non-essential promoter 5' distal sequence as much as possible while retaining all conservative sequences. The same is true for the sequence selection of cis-regulatory elements. In our preliminary experiment, we first constructed the longest version mPro1576+mCis1349 and the second truncated version of the cis-regulatory element mPro1576+mCis700 (where the numbers represent their respective base lengths), and performed in vivo functional analysis on the two. It was found that the labeling efficiency of the mCis700 and mCis1349 groups was consistent, indicating that they have the same role in the expression of specific genes in cerebrovascular endothelial cells, and further showed that the cis-regulatory element of the Tek gene can be further truncated from mCis1349 to mCis700 without affecting its normal function. Therefore, we controlled the cis-regulatory element to the mCis700 version and began to gradually shorten the sequence length of the promoter region. We further constructed mPro973 (truncated the 5' distal sequence of the promoter region and retained the complete 5'UTR region), mPro723-d224 (further shortened the 5' distal sequence by 224bp based on mPro973), mPro760 (truncated part of the 5'UTR sequence), mPro583 (only the +1-+113 segment of the 5'UTR sequence was retained), and mPro471 (truncated the entire 5'UTR sequence). The schematic diagram of the truncation strategy of the Tek gene promoter segment is shown in the figure. Figure 6 As shown in a.

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

[0088] 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 in SEQ ID NO.3:

[0110] ggaggctgagggagcatcgttatcatgagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctct

[0111] taaaaagtgcctctcccatatttttgtatataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaa

[0112] aatcttcctgtgccaagttctccaacgaattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagta

[0113] gcatgcgtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatct

[0114] ggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatca

[0115] caggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgcccc

[0116] aaagccccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcatac

[0117] catacataggtggagggcttgttattcaattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccagg

[0118] accttacttgtaacaaagatccctctgccccacaatccagttaaggcaggagcaggagccggagcaggagcagaa

[0119] gataagccttggatgaagggcaagatggatagggctcgctctgccccaagccctgctgataccaagtgcctttaaga

[0120] tacagcctttcccatcctaatctgcaaaggaaacaggaaaaaggaacttaaccctccctgtgctcagacagaaatgag

[0121] actgttaccgcctgcttctgtggtgtttctccttgccgccaacttgtaaacaagagcgagtggaccatgcgagcgggaa

[0122] gtcgcaaagttgtgagttgttgaaagct

[0123] The gene sequence of mPro723 is shown in SEQ ID NO.4:

[0124] gtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatctggtctct

[0125] agtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatcacaggaa

[0126] actttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgccccaaagcc

[0127] ccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcataccatacat

[0128] aggtggagggcttgttattcaattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccaggaccttac

[0129] ttgtaacaaagatccctctgccccacaatccagttaaggcaggagcaggagccggagcaggagcagaagataagc

[0130] cttggatgaagggcaagatggatagggctcgctctgccccaagccctgctgataccaagtgcctttaagatacagcct

[0131] ttcccatcctaatctgcaaaggaaacaggaaaaaggaacttaaccctccctgtgctcagacagaaatgagactgttac

[0132] cgcctgcttctgtggtgtttctccttgccgccaacttgtaaacaagagcgagtggaccatgcgagcgggaagtcgcaa

[0133] agttgtgagttgttgaaagct

[0134] The gene sequence of mPro760 is shown in 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 in 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 in SEQ ID NO.7:

[0155] ggaggctgagggagcatcgttatcatgagctccaggctagttccaggcttgcctaagctgtagagcaagtcactctct

[0156] taaaaagtgcctctcccatatttttgtatataatttgcatctgaaattctgtttgccaataactatgaaattattcacattactaa

[0157] aatcttcctgtgccaagttctccaacgaattagatcacactcagatgaaatgctaataaaaattaaagctgtagccagta

[0158] gcatgcgtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatct

[0159] ggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatca

[0160] caggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccc

[0161] (2) Analysis of truncation of cis-regulatory elements in the first intron of the Tek gene

[0162] In order to further explore the necessary functional sequences in cis-regulatory elements, we further constructed recombinant AAV vectors with four truncated length versions of cis-regulatory elements, namely mCis400, mCis303, mCis200, and mCis103. In order to control variables, these recombinant AAV vectors all have the sequence of mPro1576 version in the promoter region and are packaged into AAV virus particles with PhP.eB capsid. First, specific primers were designed to clone the sequence versions of different truncated lengths of the evolutionary conserved segment in the intron 1 region of the Tek gene (such as Figure 7a), mCis1349 contains the most complete regulatory elements, CACA repeat motif, GATA motif, Ets-1 transcription factor binding motif, etc. mCis700 deleted a sequence containing a CACA repeat motif at the 5' end, and also deleted a small sequence at the 3' end. The mCis400 version is also further truncated at both ends, the sequence containing the CACA repeat motif is further deleted at the 5' end, and the redundant sequence adjacent to the Ets-1 motif is further deleted at the 3' end. Compared with the mCis400 version, the mCis303 version further deletes the redundant sequence adjacent to the GATA motif at the 5' end. The design of the mCis200 version and the mCis103 version is to separate through the mCis303 version and analyze the necessity of a single regulatory motif separately. mCis200 retains the Ets-1 motif, and mCis103 retains the CACA repeat motif and the GATA motif. The final mCis0 version used only the mPro1576 promoter sequence on the recombinant vector and deleted all cis-regulatory element sequences in the Intron1 segment ( Figure 7 a). We found that compared with mCis1349, mCis700, mCis400, and mCis303, the transcriptional activity of mCis200 and mCis103 was strongly inhibited, indicating that the sequence had lost the regulatory sequence that could activate the normal transcription of the promoter region. Among them, mCis200 retained some transcriptional activity, indicating that the Ets-1 motif can only partially inhibit the transcriptional activity of the promoter region, while mCis103 almost lost all transcriptional activity, indicating that the GATA and CACA repeat motifs have a stronger transcriptional inhibitory effect on the promoter region ( Figure 7 b, c). The results of the mCis0 (Pro-only) group showed that when there was only a promoter sequence, the Cre gene could not be restricted to brain vascular endothelial cells, but was widely expressed in neurons, astrocytes, and brain vascular endothelial cells ( Figure 7 c), so we believe that the role of the cis-regulatory element of the Tek gene in the Intron1 segment is to restrict the expression of exogenous genes only in brain vascular endothelial cells through transcriptional inhibition, that is, in non-endothelial cells, such as neurons and astrocytes, the mCis element plays a transcriptional inhibitory role on the promoter region. We can reasonably believe that there is a transcription factor specifically expressed in brain vascular endothelial cells that relieves the transcriptional inhibition of the mCis element on the promoter region of the Tek gene, so that the downstream gene can be expressed.

[0163] In order to finally determine whether the truncated version of the Tek gene promoter region mPro723 can maintain high transcriptional activity and cell type specificity when combined with the cis-regulatory element stage version of the Intron1 segment mCis700, we constructed the rAAV-mPro723-Cre-mCis700 recombinant plasmid and packaged it into AAV-PhP.eB virus. Rosa26-CAG-LSL-tdTomato In vivo transduction experiments were conducted in mice. The results of sagittal fluorescence imaging experiments showed that the best optimized truncated version of the rAAV-miniBEND system can efficiently and specifically transduce cerebral vascular endothelial cells throughout the brain (including olfactory bulb, cortex, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, etc.), and that many endothelial cells in large arteries and veins have labeled signals ( Figure 8 a, b).

[0164] In order to comprehensively evaluate the cell type specificity of rAAV-miniBEND system labeling, we conducted whole-brain interval slice experiments on mouse brains labeled with various truncated versions to ensure coverage of most brain regions. After carefully examining the fluorescent labeling signals in all brain regions, we found that the AAV-PhP.eB-CAG-mScarlet virus group had serious non-specific fluorescent labeling signals of neurons in the cerebellum, while the AAV-PhP.eB-mPro973-Cre group had non-specific fluorescent labeling signals in Purkinje cells in the cerebellum ( Fig. 9 ,in Fig. 9 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. The Tek cis-regulatory element used in the recombinant vectors in the last three groups was mCis700, and they were 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 in the figure is 100μm. Fig. 9 b The ratio of non-specific labeling other than endothelial cells was expressed as normalized labeling density - per 10 11 Virus particles were used to transduce a mouse in vivo. 2The number of labeled cells in the area was used as an indicator for evaluation. The number of experimental animals tested in each group was n = 2, and 5 ROIs were selected in the slices of each mouse for cell counting and statistical analysis. The scale bar in the figure is 100μm). This shows that although the miniBEND regulatory sequence can limit the expression of exogenous genes in most neurons, there is still leakage expression in Purkinje cells. We further compared the results of the mPro760-Cre group and the mPro723-Cre group and found that the density of non-specific labeling signals of Purkinje cells in these two groups was significantly reduced, especially in the mPro723-Cre group, whose non-specific labeling signal was close to zero. Only one non-specifically labeled cell could be found in multiple cerebellar slices throughout the 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 neurons in the cerebellum by the miniBEND system, and the optimal truncated version mPro723 we obtained has the highest cell type specificity.

[0165] The gene sequence of mCis1349 is shown in 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 in SEQ ID NO.11:

[0193] cccccaagattgtggaagctggactgaagatcacaatctgccagatgggcagaatctttactctttggcacatttgttgc

[0194] tgatggggagtgaatacccatggggacatggctgtcatggtgtggaagtgatagaaatgaaaacatgtatggatctgt

[0195] cacaggagctggtgaggctgatgggtgtgtgggtggccactgtttgctctctgcttgtcacagcctcttgttcagggctt

[0196] gatcagggaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagcagatctgtca

[0197] gctttcccgcttttgttagagggtgatatcatgcttcctggggggagctctggaagacaatgagcagccactttcctcta

[0198] ga

[0199] The gene sequence of mCis303 is shown in 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 Tek gene promoters and cis-acting elements in different species

[0212] Based on the conservation of the promoter and cis-acting elements of the Tek gene across species, and the fact that human promoters are an indispensable component of future clinical gene therapy programs, we conducted functional analysis experiments on subcloning and sequence truncation of the promoter and cis-acting elements of the human (Homo sapiens) Tek gene. We first subcloned the 1612bp sequence (hPro1612) of the human Tek gene promoter region including the 5' UTR, and then directly designed the human hPro762 version based on the results of the mouse miniBEND truncation experiment. This version truncated the sequence containing the non-essential elements Octopus region I and Octopus region II that we determined ( Fig.10 a). Fig.10 The schematic diagram of the truncation strategy of the cis-acting element of the human Tek gene is shown in b. The results of in vivo transduction experiments showed that the promoter of the human Tek gene combined with the cis-acting element (hPro1612-hCis700) can also mediate efficient brain vascular endothelial cell-specific gene expression ( Fig.10 c), this result proves our hypothesis that the human regulatory sequence also works in mouse somatic cells. Although the promoter and cis-acting elements have certain polymorphisms in DNA sequence, they are still conservative in function. In addition, the activity of the truncated version hPro762 is lower than that of hPro1612, but the specificity for brain endothelial cells is still very high. This shows that part of the Octopus region sequence is also needed in the human regulatory sequence to maintain the transcriptional activity of the promoter.

[0213] In order to test whether the cis-regulatory elements of Tek genes in other species can be used in combination with the Human promoter to mediate specific gene expression in brain vascular endothelial cells, we further synthesized and cloned the cis-regulatory elements corresponding to the first intron segment of the Tek gene in multiple other species (including marmosets (Callithrix jacchus), pigs (Sus scrofa) and rats (Rattus norvegicus)), and placed them one by one into the recombinant vectors we designed, packaged into AAV-PhP.eB capsid viruses for in vivo experimental tests in mice. The results showed that the cis-regulatory elements of Tek genes in other species (Marmo-Cis700 (marmoset), Pig-Cis700 (pig)) can be used in combination with the Human promoter to mediate specific gene expression in brain vascular endothelial cells ( Fig.11 ), in addition, the rat Tek gene promoter (Rat-Pro1600) and cis-regulatory element (Rat-Cis737) can also mediate the specific gene expression of brain vascular endothelial cells ( Fig.11 ).

[0214] The gene sequence of hPro1612 is shown in SEQ ID NO.47:

[0215]

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

[0217] ttgctctcatgagcaccatttttatcccaatctaatcctgtatgtttgtgtttttacacagattagtttttaaatgttatatataatttgcttctgaaacaccattgctcaatgactaccaaatctttctcattaccaaaatccttctatgccaacttcttcaagaaatttgatcacctttagatgaattgttaatgaaaattaaagctatagccggcaacatgggtatctttgggctaatggccaaccaacaggccatctgtgtgaaagaaaacaggctaacaattttggactctggtctcttggggctacattgagcattgacctcaccggtgctcactgaaattaattgcttttcaggttgtattttctcatcacggaaaccttcttctcccaattcaaaccatgtgggttaaaatgagaaaacaaaagccaaaacggcttcccacacccaaaagctccttctgtcagagatcccagtagccccgggagagctgttagaagtctgagaaggattggtcatcatcgcataccatacataggtggagggcttgttattctcagtttcccgcctatgagaggatacccctattgtttctgaaaatgctgaccgggacccacacttccaacaaaaattcctctgcccctacagcagcagcaaaagcagcagcagaagcaacagcaacagataagtgttttgatgaattgcgagatggatagggcttgagtgcccccagccctgctgataccaaatgcctttaa

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

[0219] ctttcaggaaagggaatgtctaaagttgcctcccttatttttggaattttgaactgataaccctattcttacccatctggccagacgattccttaactcgtgttacacctgcagaatgagttttagatctagctgtgacctcttcccccagccccacccccattgtccccttgtgtgccttcaggaatctgatcattcttctctcctgctccttcccaaaggctgcaggagcaggtgtgaagacgtggatgtgccagatgcagagtcctgacacttttcaacacatctgcatattagaggaagtacatacccattgcttggtggtttcatgtctaatgtggtatgagtgtgacaaagagagggagaaaatttggactagccaaagaagccagtcaggcgtggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcactcactgctctgcggccgtcagatggtgaaccagcttaaccttggc

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

[0221] aagtacatacccattgcttggtggtttcatgtctaatgtggtatgagtgtgacaaagagagggagaaaatttggactagccaaagaagccagtcaggcgtggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcactcactgctctgcggccgtcagatggtgaaccagcttaaccttggc

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

[0223] ggggtttgaagggcatcgtgggcggctgtcatttgctctctgcttgtcacagccccttgcccagggcttgaccagtgaggtgtatgtgctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactggaagacaatgctcggccactttcctccaga

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

[0225] Caggaaagtgaatgtctaaagttgtctcccttacttttggaattttaaactgataaccctattcttacccatctggccagactattccttaacttgcttcacacatgcagaatgagttttagatttagctatggctgcttccccctaagccccacccgcactttgcctccttttgtcccttcaggaatctgataattcttctctcatggttcttgccaaagattgcaggagcaggtgtgaagacgtggatatgccagatgggcagagtccttatgcttctcaacacatctgctgaatagagggagcgcatacccattccctgatggtttcatgtcttatgtggtgtgggtgtgacaaagagagggagaaatgtggactagccaaaggagccagtcaggcttggggttgagaggcatcgtgggcagctgccatttgctctgtgcttgtcacagccccttgcccaggtcttgaccagtgaggtgtgtgtcctggtcacacccatctcagcagatctgtcagctttcccgcttttgttaaagggtgatatcatgcttcctggggggagcactagaagacaatgctcggccactttcctccagatacaataggcggagtcaggaaggcagtattgacattgctggggctggggaggcgctcactgctctgcagccgtcagagggtgaaccagcttaaccttggc

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

[0227] Cagctggcatcactcccagccttcaggaaagggaatgtctaaagttgcctcccttatttctgaattttaaactgataacccaattcccaccggtctagccagtgtattccttacttgcattacacatgcagagcatttttcagaccgagccttgcctgcttgcctctacatccctcccccaaggcctcctttgtcctgaccatcttcctcctgctatcccttcccaaagactgcaggggcaggtgtggagacagggatctgccagatgagcagagtcctgactctttttaacacagctggtggtgagagcggggatattctcgccttgctgtgctgtgtctaccatggaacacatggagcaaagagaacaaacctaaacatgagccaaaggagcccatcaggcagcaacctgaggggcactgttggaggcactgttggctttctgctggtcacagccccctgcctggggcttgaccagtgaggtgtatatggcagtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggagcgctggaagacaatgccgggccacttgccttcagatacagtgggcggagtcaggaaggcagtattgacattgccgggccaggggggtactcactgctccagcggtcagatggtgaaccagcttaaccttggc

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

[0229]

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

[0231] ttgctgccctctagaccttcaaagacctattttataaaattatatttaccacccactcgtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtatgtgttatacatattggaagtcagagtttggttctcttcttccacaatgcggatcacagagcatcaggcttgtgagcaccttgctgcctgctttgtctcttcagcagtctgaccttgctccttctcgggggctgtggaagtaggactgaagttcagaatctgccagatgggcagaatctttactacttggtacatttgatgctgatagggaatgaattcccacagggacatggctgacacggtgtggaagtgacagaaatggggacgtttgtggatcaatcacatgagttggtgaggctgaggggtgttgtgggaggctgctgttggctccctgcttgttcagggattgaccagggagaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtcacacccatctcagcagatctgtcagctttcccgcttttgttagagggtgatatcatgcttcctggggggagctctggaagacaatgagcaggcactttcctctagatacaataggcggagtcaggaaggtagtattgacattgctggggcctaggagctactcactgctctgcggccgtcagatggtgaaccagcataaccttggcacacaggcct

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

[0233] In order to detect the strength of the miniBEND promoter obtained in Example 2 (1), 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 intravenous injection to transduce vascular endothelial cells throughout the brain. The slice results showed that its EGFP fluorescence intensity was much weaker than that expressed by the ordinary ef1a-EGFP virus ( Fig.13 ), which indicates that the promoter strength of the mPro1576 version is weaker than the conventional broad-spectrum medium-strength promoter ef1a, and is a weak promoter. Obviously, the intrinsic weak promoter characteristics of miniBENDv1 (a general term for the first version mPro1576, as distinguished from the truncated optimal version mPro723) limit its wide application in the application scenario of exogenous gene overexpression. Therefore, we need to further transform and optimize to improve the promoter strength of the miniBEND promoter. The optimization goal is to reach the promoter level of the ef1a promoter, that is, to meet the use requirements of in vivo overexpression.

[0234] The first thing we thought of was to introduce the Tet-off system (Loew et al., 2010) and achieve overexpression of the target gene through the transcriptional cascade amplification strategy of the tTA / TRE system. There are two specific implementation schemes for this strategy. The first scheme is a dual AAV vector, which requires the construction of plasmid vectors AAV-mPro1576-tTA-mCis700 and AAV-TRE-EGFP-WPRE-pA. This scheme allocates the expression space of the exogenous gene to another AAV vector, greatly increasing the restricted base length that can be inserted into the exogenous gene, which is approximately less than or equal to 4kb. The second scheme is a single AAV vector, which integrates the miniBEND promoter and tTA / TRE expression systems on the same rAAV vector, but the fluorescence expression intensity of this scheme is similar to that of the dual AAV vector group, and the fluorescent cell labeling density is low ( Fig.12 ). On the other hand, the vector capacity of this strategy is much smaller than that of the dual AAV vector, only 1.5 kb, which has certain limitations in the application of delivering many genes larger than 1.5 kb. Therefore, we also need a new single AAV vector strategy.

[0235] We increase the promoter strength by adding additional regulatory elements, or increase the stability of exogenous mRNA to increase the expression level of exogenous genes. We found that intronic regulatory sequences can increase the promoter strength, so we tried to introduce intronic regulatory sequences to construct a chimeric promoter to increase the promoter strength of the miniBEND promoter. However, there is a possible risk of failure in this scheme, that is, the new chimeric promoter may not maintain the cell type specificity of endothelial cells. We designed a set of experiments and constructed AAV-mPro1576-glo566-EGFP-pA-mCis700 plasmid, AAV-mPro723-glo566-EGFP-pA-mCis700 plasmid, AAV-mPro723-glo228-EGFP-pA-mCis700( Fig.13 a). It was found that the new chimeric promoter not only did not affect its endothelial cell-specific expression characteristics, the cis-regulatory elements could still work normally, and the promotion strength of the chimeric promoter was also enhanced, indicating that this method is feasible. Moreover, the truncated chimeric promoter (mPro723-glo566, mPro723-glo228) also worked equally well ( Fig.13 b, c), the length of the chimeric promoter of the mPro723-glo228 version is 978bp (less than 1kb), which leaves more space for exogenous genes.

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

[0237]

[0238] The gene sequence of mPro723-glo228 is shown in SEQ ID NO.55:

[0239] GtatatttgggctcagggccaacaggcaggcgatctgggtgtaagaaaataggctaatggctgtggaatctggtctctagtggctccgctgagagctgacctcaaccacgctccctcaaattgattgccttccaggttatgatttctcatcacaggaaactttgttgcccaattcaaaccctgtgagtgaaaacaaaaacaggagagcaagtgctgctccccgtgccccaaagccccttctgtcagggatcccaaatgcaccccagagaacagcttagcctgcaagggctggtcctcatcgcataccatacataggtggagggcttgttattcaattcctggcctatgagaggatacccctattgttcctgaaaatgctgaccaggaccttacttgtaacaaagatccctctgccccacaatccagttaaggcaggagcaggagccggagcaggagcagaagataagccttggatgaagggcaagatggatagggctcgctctgccccaagccctgctgataccaagtgcctttaagatacagcctttcccatcctaatctgcaaaggaaacaggaaaaaggaacttaaccctccctgtgctcagacagaaatgagactgttaccgcctgcttctgtggtgtttctccttgccgccaacttgtaaacaagagcgagtggaccatgcgagcgggaagtcgcaaagttgtgagttgttgaaagctagtaccggtAgTTCGTTTAGTGAACCGggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcag

[0240] The gene sequence of mPro1576-glo566 is shown in SEQ ID NO.56:

[0241]

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

[0243] Brain arteriovenous malformation (bAVM) is another major type of intracranial vascular malformation disease besides cavernous malformation (CCM). We investigated and analyzed the pathogenic genes of brain arteriovenous malformation and found that a recent research group reported that a bAVM patient had a somatic mutation of the 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 (Braf-CA) carrying Braf gene point mutations (Dankort et al., 2007), hoping to simulate the pathological phenotype of human bAVM in clinical practice in mice, and our research results can also prove whether the Braf gene is the real pathogenic gene.

[0244] Braf-CA mice are a type of mouse with a human BRAF gene fragment knocked in. The design principle is based on the fact that the amino acid sequences of human BRAF kinase protein and mouse BRAF kinase protein are highly conserved. When designing the homologous recombination knock-in vector, in order to ensure that the normal expression of the endogenous Braf gene and the sequence of the normal Braf protein are not destroyed, a DNA fragment of Exon15-Exon18 of the human BRAF gene with double-end flanking LoxP sites is used in the middle of the knock-in vector, and the 3' end is downstream of the Poly A element and the Neo screening gene. Downstream of the second LxoP site on the knock-in vector is the T base at position 1799 in the coding region of Exon15 of the Braf locus of the mouse genome, which is mutated to an A base (c.1799T to A), and accordingly, the valine (V) at position 600 in the coding protein sequence will mutate to glutamic acid (E).

[0245] Under the action of Cre recombinase, the LoxP site on the Braf gene locus will be removed, and the human Exon15-Exon18 fragment after Exon14 of the mouse endogenous Braf gene will be removed. Correspondingly, the downstream fragment carrying the V600E point mutation can be transcribed and translated normally, and finally BrafV is expressed in the target cells. 600E Mutant protein ( Fig.14 We designed a set of experiments, selected homozygous Braf-CA mice, injected AAV-miniBEND-Cre (AAV-PhP.eB-mPro723-Cre-pA-mCis700) virus in situ into the unilateral cerebral cortex of mice aged P30-P50, and induced endothelial cells to overexpress Braf in local brain regions. V600E Mutant protein ( Fig.14b), homozygous Braf-CA mice raised normally are viable and can reproduce normally. We observed 12 mice after modeling for a long time and found that the focal bAVM model mice died one after another after the 20th day of virus injection, and the final survival rate was less than 50% ( Fig.14 c), the abnormal structure of blood vessels in the lesion area can be observed through the skull of the successfully modeled mice by laser speckle blood flow imaging ( Fig.14 d).

[0246] During this period, we used magnetic resonance imaging (MRI) to observe the development of intracranial bAVM lesions in mice ( Fig.15 The results showed that brain hemorrhage was found on the 15th day after virus injection (PID15), and the area of ​​intracranial edema was observed to expand on the 22nd day, indicating that the bAVM lesion gradually had an invasive effect similar to a tumor. By the 41st day, tissue necrosis and compression of the hippocampus in the bAVM lesion area had occurred, indicating that the bAVM lesion had a space-occupying effect similar to a tumor ( Fig.15 b, c). The focal bAVM disease model constructed based on Braf-CA transgenic mice is very similar to the clinical imaging phenotype of human intracranial arteriovenous malformations. At the same time, the phenotypes of hemiplegia and occasional epilepsy we observed in mice (with video records) are also consistent with the behavioral manifestations of clinical bAVM patients. In order to further confirm the changes in the morphological structure of cerebral blood vessels in the bAVM lesion area, we further used Laminin antibodies to perform immunofluorescence experiments on the samples, and performed statistical analysis on the vascular diameters of normal brain areas and lesion areas. We found that there were almost no capillary-sized microvessels in the bAVM lesion area, and they were all thickened malformed blood vessels ( Fig.16 The statistical results also showed that the diameter of blood vessels in the bAVM lesion area was significantly larger than that in the normal brain area ( Fig.16 b, c).

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

[0248] Based on the construction of a disease model of cerebral arteriovenous malformation based on Braf-CA transgenic mice in Example 4, we have been able to confirm that Braf V600E The mutant protein can induce malformation of blood vessels in the bAVM lesion area. V600EThe kinase activity of the mutant protein is more than 500 times higher than that of the wild-type Braf protein (references as follows: 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 speculate that the main cause of vascular malformations is the overactivation of the Braf-MEK1 signaling pathway. Next, we further explored the signaling pathway regulatory mechanism of bAVM occurrence and development. After consulting the literature, we found that PLX4032 (Vemurafenib) is a small molecule drug of Braf. V600E The mutant protein-specific kinase activity inhibitor can effectively inhibit Braf in living mice V600E The kinase activity of the mutant protein was inhibited (Bollag et al., 2010). V600E The kinase activity of the mutant protein can hinder the development of bAVM lesions, which can prove that Braf V600E The high kinase activity of the mutant protein and the activation of downstream signaling pathways are sufficient for the development of bAVM disease. In order to further confirm the molecular mechanism of bAVM disease and test our hypothesis, we designed a set of pharmacological inhibition experiments and used the anterior and posterior diameters of the lesions after MRI as detection indicators. Drug administration began on the 7th day after injection of the modeling virus, and the control group was given an equal amount of the drug solvent carrier DMSO. MRI began on the 22nd day ( Fig.17 The experimental results showed that the anterior-posterior diameter of the lesion area in the drug-treated experimental group was significantly smaller than that in the control group ( Fig.17 b and c), indicating that Braf V600E The specific inhibitor PLX4032 (Vemurafenib) can hinder the development of bAVM disease. Further H&E histological staining results showed that the number of abnormal blood vessels in the lesion area was significantly reduced after treatment with the small molecule drug PLX4032, and the area of ​​the lesion area in the experimental group was significantly smaller than that in the control group. Fig.18As shown, the left half of a shows the results of the control group (DMSO), while the right half of a shows the results of the drug-treated group; b shows the density of abnormal blood vessels in the control group and the drug-treated group; c shows the diameter of abnormal blood vessels in the control group and the drug-treated group.

[0249] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

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

[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 summarized and recorded below:

[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.

[0259] 2.Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual[M]. Cold spring harbor laboratory press, 1989.

[0260] 3.Kisanuki Y Y,Hammer R E,Miyazaki J I,et al.Tie2-Cre transgenicmice:a new model for endothelial cell-lineage analysis in vivo[J].Developmental Biology,2001,230(2):230-42.

[0261] 4.Fadel B M,Boutet S C,Quertermous T.Octamer-dependent in vivoexpression of the endothelial cell-specific TIE2 gene[J].Journal ofBiological Chemistry,1999,274(29):20376-83.

[0262] 5.Loew R,Heinz N,Hampf M,et al.Improved Tet-responsive promoters withminimized background expression[J].BMC Biotechnology,2010,10(1):81.

[0263] 6.Albright B H,Storey C M,Murlidharan G,et al.Mapping the StructuralDeterminants Required for AAVrh.10 Transport across the Blood-Brain Barrier[J].Mol Ther,2018,26(2):510-23.

[0264] 7.Hong T,Xiao X,Ren J,et al.Somatic MAP3K3 and PIK3CA mutations insporadic cerebral and spinal cord cavernous malformations[J].Brain,2021,144(9):2648-58.

[0265] 8.Hong T,Yan Y,Li J,et al.High prevalence of KRAS / BRAF somaticmutations in brain and spinal cord arteriovenous malformations[J].Brain,2019,142(1):23-34.

[0266] 9.Huo R,Yang Y,Sun Y,et al.Endothelial hyperactivation of mutantMAP3K3induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation[J].Angiogenesis,2023.

[0267] 10.Bollag G,Hirth P,Tsai J,et al.Clinical efficacy of a RAF inhibitorneeds broad target blockade in BRAF-mutant melanoma[J].Nature,2010,467(7315):596-9.

[0268] 11.Dankort D,Filenova E,Collado M,et al.A new mouse model to explorethe initiation,progression,and therapy of BRAFV600E-induced lung tumors[J].Genes Dev,2007,21(4):379-84.

Claims

1. Use of a small molecule drug PLX4032 in the preparation of a drug for treating cerebral arteriovenous malformation, wherein: The structure of the small molecule drug PLX4032 is as follows:

2. The use according to claim 1, characterized in that: The cerebral arteriovenous malformation disease is manifested in Braf V600E Expression of mutant proteins.

3. The use according to claim 1 or 2, characterized in that: The cerebral arteriovenous malformation disease is manifested in Braf V600E The kinase activity of the mutant protein is increased.

4. The use according to any one of claims 1 to 3, characterized in that: The brain arteriovenous malformation disease is manifested by excessive activation of the Braf-MEK1 signaling pathway.

5. The use according to any one of claims 1 to 4, characterized in that: The small molecule drug PLX4032 is selected from one of tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres and injections.

6. The use according to any one of claims 1 to 5, characterized in that: The small molecule drug PLX4032 further comprises a conventional pharmaceutical carrier.

7. The use according to claim 6, characterized in that: The conventional carrier is selected from one or more of a filler, a binder, a wetting agent, an absorption promoter and a cosolvent.

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