Cerebral microbleed animal model and preparation method therefor

Abstract

The present application relates to a cerebral microbleed animal model in which cerebral microbleeds are induced by Col4a1-targeting guide RNA (gRNA) packed with AAV-BR1.

Description

Animal model of cerebral microhemorrhage and method for producing the same

[0001] This paper relates to an animal model of cerebral microbleeds through cerebrovascular-specific COL4A1 gene editing.

[0002] Among the brain pathologies manifested by abnormalities in the cerebral small vessels, cerebral microbleeds (CMBs) are known to occur due to microbleeds and hemosiderin deposition caused by damage to the brain's small blood vessels. With the advancement of brain magnetic resonance imaging techniques, cerebral microbleeds smaller than a few millimeters are increasingly being found in the elderly population. Patients with CMBs are known to have a significantly worse prognosis when they develop a stroke, and the risk of developing intracerebral hemorrhage is also increased, and the possibility of developing intracerebral hemorrhage side effects from antithrombotic and anticoagulant treatment is also known to be higher. In addition, cerebral microhemorrhages are frequently found in cerebral amyloid angiopathy (CAA), a vascular abnormality found in Alzheimer's disease patients, and there are reports that the number of cerebral microhemorrhages is correlated with the degree of cognitive decline, raising the possibility that cerebral microhemorrhages may be a factor in cognitive decline due to dementia.

[0003] Therefore, there is a growing need for treatments that prevent or inhibit the progression of cerebral microhemorrhages. However, the molecular and cellular mechanisms by which cerebral microhemorrhages occur are currently unknown, and therefore, therapeutic targets for suppressing them are unclear.

[0004] In addition, although it is essential to establish an appropriate animal model to elucidate the pathophysiological mechanism of cerebral microhemorrhage, no animal model showing cerebral microhemorrhage similar to that observed in human MRI images has been reported to date.

[0005] Therefore, there is a need to develop an animal model of cerebral microhemorrhage to elucidate the pathophysiological mechanism of cerebral microhemorrhage and to develop therapeutic agents to prevent or treat cerebral microhemorrhage.

[0006] The background technology of this application, Republic of Korea Patent Publication No. 10-2023-0077849, relates to a method for detecting cerebral microhemorrhage and a learning method for detecting cerebral microhemorrhage.

[0007] The present invention aims to solve the problems of the above-mentioned conventional technology, and provides a cerebral microhemorrhage animal model that exhibits imaging and pathological findings very similar to human cerebral microhemorrhage by selectively deficient COL4A1 in cerebral microvascular endothelial cells.

[0008] In addition, a method for producing the above cerebral microhemorrhage animal model is provided.

[0009] In addition, a method for screening a cerebral microhemorrhage treatment agent using the cerebral microhemorrhage animal model is provided.

[0010] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0011] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a cerebral microhemorrhage animal model in which cerebral microhemorrhage is induced by a Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1.

[0012] According to one embodiment of the present invention, the expression of the COL4A1 gene may be suppressed by the Col4a1 targeting gRNA, but is not limited thereto.

[0013] According to one embodiment of the present invention, the elasticity of the blood-brain barrier may be reduced by suppressing the expression of the COL4A1 gene, but is not limited thereto.

[0014] According to one embodiment of the present invention, cerebral microhemorrhage may be induced by a decrease in the elasticity of the blood-brain barrier, but is not limited thereto.

[0015] According to one embodiment of the present invention, the Col4a1 targeting gRNA may be delivered specifically to cerebral vascular endothelial cells by the AAV-BR1, but is not limited thereto.

[0016] According to one embodiment of the present invention, the Col4a1 targeting gRNA (guide RNA) may include any one of the base sequences of SEQ ID NOs: 1 to 9, but is not limited thereto.

[0017] In addition, the second aspect of the present invention provides a method for producing a cerebral microhemorrhage animal model, comprising a step of inducing cerebral microhemorrhage by injecting a Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1 into a transgenic animal using CRISPR-Cas9.

[0018] According to one embodiment of the present invention, the expression of the COL4A1 gene may be suppressed by the Col4a1 targeting gRNA, but is not limited thereto.

[0019] According to one embodiment of the present invention, the elasticity of the blood-brain barrier may be reduced by suppressing the expression of the COL4A1 gene, but is not limited thereto.

[0020] According to one embodiment of the present invention, cerebral microhemorrhage may be induced by a decrease in the elasticity of the blood-brain barrier, but is not limited thereto.

[0021] According to one embodiment of the present invention, the Col4a1 targeting gRNA may be delivered specifically to cerebral vascular endothelial cells by the AAV-BR1, but is not limited thereto.

[0022] According to one embodiment of the present invention, the Col4a1 targeting gRNA may include any one of the base sequences of SEQ ID NOs: 1 to 9, but is not limited thereto.

[0023] In addition, the third aspect of the present invention provides a method for screening a cerebral microhemorrhage treatment agent, comprising the steps of administering a cerebral microhemorrhage treatment agent candidate to a cerebral microhemorrhage animal model according to the first aspect of the present invention; and comparing the animal model administered with the candidate substance with a control group not administered with the candidate substance, and determining that the animal model is a cerebral microhemorrhage treatment agent if the cerebral microhemorrhage is reduced by the candidate substance.

[0024] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0025] The cerebral microhemorrhage animal model according to the present invention packages a Col4a1-targeting gRNA that specifically targets the Col4a1 gene into an AAV-BR1 virus specifically directed to cerebral vascular endothelial cells and injects the same into an animal, thereby delivering the Col4a1-targeting gRNA only to cerebral blood vessels, thereby enabling cerebral blood vessel-specific editing of the Col4a1 gene, which weakens the elasticity of the cerebral blood vessels of the animal, thereby inducing cerebral microhemorrhage, and can thus be used as a cerebral microhemorrhage model.

[0026] In addition, the cerebral microhemorrhage animal model according to this invention can be used to develop treatments for various brain diseases accompanied by symptoms of cerebral microhemorrhage, and can be used to develop biomarkers that can detect cerebral microhemorrhage at an early stage.

[0027] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0028] Figure 1a shows the results of an indel experiment to confirm whether the Col4a1 gene was edited by delivering Col4a1-targeting gRNA sequences according to one experimental example of the present invention to mouse NIH3T3 cells, and Figure 1b shows the results of confirming the Col4a1 mRNA level.

[0029] Figure 2a shows the results of confirming the expression levels of Col4a1 mRNA and protein by real-time PCR through Col4a1-targeting gRNA treatment according to one experimental example of the present invention, and Figure 2b shows the results confirmed through a western blot experiment.

[0030] Figure 3a is a schematic diagram of an experiment to confirm Col4a1 gene editing and COL4A1 expression suppression in cerebrovascular endothelial cells by injecting Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention into a CRISPR Cas9 transgenic animal, Figure 3b is an Indel result, and Figure 3c is a result of measuring the out-of-frame ratio of the edited Col4a1 gene.

[0031] Figure 4a shows the results of real-time PCR to confirm whether Col4a1 mRNA and protein decrease in the cerebral blood vessels of animals 3 weeks after injecting a CRISPR Cas9 transgenic animal with a Col4a1-targeting gRNA packed with AAV-BR1 according to an experimental example of the present invention, and culturing primary cerebral vascular endothelial cells from the cerebral blood vessels of the animals. Figure 4b shows the results confirmed through a western blot experiment, and Figure 4c shows the results of staining brain tissue sections and observing them with a confocal microscope.

[0032] Figure 5 shows the results of magnetic resonance imaging of the brain of a CRISPR Cas9 transgenic animal injected with a Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention.

[0033] Figure 6a shows the results of cerebral microhemorrhage on the brain surface according to the injection concentration of Col4a1 targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention, Figure 6b shows the results of cerebral microhemorrhage in a brain section, and Figure 6c shows the results of measuring lesions due to cerebral microhemorrhage by region in a brain tissue section of an animal 3 months after injection.

[0034] Figure 7a shows the results of confirming cerebral microhemorrhages in brain tissue sections using the Prussian blue staining method 3 months after injecting animals with Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention at various concentrations, and Figure 7b shows the results of observing hemosiderin granules in the area stained with Prussian blue, and Figure 7c shows the results of performing an immunostaining method using an anti-Ferritin Light / Heavy chain antibody.

[0035] Figure 8a shows the results of measuring short-term memory using the Y-maze test from 0 to 6 months after injecting Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention into animals, and Figure 8b shows the results of measuring cognitive function and memory using the Novel objective recognition test, and Figure 8c shows the results of performing a neurobehavioral test to determine balance, limb coordination, muscle strength, etc. using the Rotarod test.

[0036] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0037] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0038] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0039] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0040] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0041] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0042] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0043] Hereinafter, the cerebral microhemorrhage animal model of this invention will be described in detail with reference to implementation examples, examples, and drawings. However, the invention is not limited to these implementation examples, examples, and drawings.

[0044]

[0045] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a cerebral microhemorrhage animal model in which cerebral microhemorrhage is induced by a Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1.

[0046] The blood-brain barrier protects the brain from various pathogens and toxins of pathogens while only allowing selective permeation between the brain and blood vessels. It is composed of a structure that tightly surrounds blood vessels by cerebrovascular endothelial cells, cerebrovascular pericytes, and astrocytes, and is in the form of a basement membrane surrounding the outer surface of the endothelial cells, of which the COL4A1 protein, a subtype of collagen, is the main component.

[0047] The cerebral microhemorrhage animal model according to the present invention packages a Col4a1-targeting gRNA that specifically targets the Col4a1 gene into an AAV-BR1 virus specifically directed to cerebral vascular endothelial cells and injects the same into an animal, thereby delivering the Col4a1-targeting gRNA only to cerebral blood vessels, thereby enabling cerebral blood vessel-specific editing of the Col4a1 gene, which weakens the elasticity of the cerebral blood vessels of the animal, thereby inducing cerebral microhemorrhage, and can thus be used as a cerebral microhemorrhage model.

[0048] In addition, the cerebral microhemorrhage animal model according to this invention can be used to develop treatments for various brain diseases accompanied by symptoms of cerebral microhemorrhage, and can be used to develop biomarkers that can detect cerebral microhemorrhage at an early stage.

[0049] According to one embodiment of the present invention, the expression of the COL4A1 gene may be suppressed by the Col4a1 targeting gRNA, but is not limited thereto.

[0050] According to one embodiment of the present invention, the elasticity of the blood-brain barrier may be reduced by suppressing the expression of the COL4A1 gene, but is not limited thereto.

[0051] According to one embodiment of the present invention, cerebral microhemorrhage may be induced by a decrease in the elasticity of the blood-brain barrier, but is not limited thereto.

[0052] According to one embodiment of the present invention, the Col4a1 targeting gRNA may be delivered specifically to cerebral vascular endothelial cells by the AAV-BR1, but is not limited thereto.

[0053] The animal model according to the present invention can suppress the expression of the COL4A1 gene specifically in cerebral vascular endothelial cells of an animal by packaging a Col4a1-targeting gRNA into an AAV-BR1 virus specifically directed to cerebral vascular endothelial cells and injecting the same into the animal, thereby inducing cerebral microhemorrhage.

[0054] According to one embodiment of the present invention, the Col4a1 targeting gRNA (guide RNA) may include any one of the base sequences of SEQ ID NOs: 1 to 9, but is not limited thereto.

[0055] According to the present invention, an animal model can induce cerebral microhemorrhage by designing an sgRNA (single guide RNA) sequence that can specifically act on the mouse Col4a1 gene without acting on other genes and injecting the same into an animal, thereby suppressing the expression of the COL4A1 gene in cerebral vascular endothelial cells of the animal.

[0056]

[0057] In addition, the second aspect of the present invention provides a method for producing a cerebral microhemorrhage animal model, comprising a step of inducing cerebral microhemorrhage by injecting a Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1 into a transgenic animal using CRISPR-Cas9.

[0058] Regarding the method for producing a brain microhemorrhage animal model according to the second aspect of the present application, detailed descriptions of parts overlapping with the first aspect of the present application have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present application can be equally applied to the second aspect of the present application.

[0059] According to one embodiment of the present invention, the expression of the COL4A1 gene may be suppressed by the Col4a1 targeting gRNA, but is not limited thereto.

[0060] According to one embodiment of the present invention, the elasticity of the blood-brain barrier may be reduced by suppressing the expression of the COL4A1 gene, but is not limited thereto.

[0061] According to one embodiment of the present invention, cerebral microhemorrhage may be induced by a decrease in the elasticity of the blood-brain barrier, but is not limited thereto.

[0062] According to one embodiment of the present invention, the Col4a1 targeting gRNA may be delivered specifically to cerebral vascular endothelial cells by the AAV-BR1, but is not limited thereto.

[0063] According to one embodiment of the present invention, the Col4a1 targeting gRNA may include any one of the base sequences of SEQ ID NOs: 1 to 9, but is not limited thereto.

[0064]

[0065] In addition, the third aspect of the present invention provides a method for screening a cerebral microhemorrhage treatment agent, comprising the steps of administering a cerebral microhemorrhage treatment agent candidate to a cerebral microhemorrhage animal model according to the first aspect of the present invention; and comparing the animal model administered with the candidate substance with a control group not administered with the candidate substance, and determining that the animal model is a cerebral microhemorrhage treatment agent if the cerebral microhemorrhage is reduced by the candidate substance.

[0066] Regarding the screening method for a cerebral microhemorrhage treatment agent according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first aspect and / or the second aspect of the present invention are omitted, but even if the descriptions are omitted, the contents described in the first aspect and / or the second aspect of the present invention can be equally applied to the third aspect of the present invention.

[0067] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0068]

[0069] [Experimental Method]

[0070] INDEL experiment

[0071] The target region was amplified from 30 ng of genomic DNA (gDNA) using Phusion High Fidelity DNA Polymerase PCR Polymerase (NEB). The amplified target region amplicon was further amplified using IDT for Illumina DNA / RNA UD Indexes (Illumina, San Diego, CA, USA) to generate a deep sequencing library. Paired-end sequencing was performed using the Illumina Nextseq system, and indel frequencies were calculated at 'http: / www.rgenome.net / '.

[0072] RNA extraction & real-time PCR

[0073] RNA was extracted from primary endothelial cells cultured from the brains of transgenic animals. The extraction method was as follows.

[0074] Primary endothelial cells are mixed with Trizol and chloroform in a 4:1 ratio, inverted, and left at room temperature for a while. When the layers are separated, centrifuge at 13,000 rpm for 15 minutes at 4°C to separate only the supernatant. Invert the separated supernatant with isopropanol in a 1:1 ratio, and leave at room temperature for 1 hour to precipitate RNA. After 1 hour, centrifuge at 13,000 rpm for 15 minutes at 4°C to completely remove the supernatant and leave only the pellet, wash with 75% ice-cold EtOH, dry the pellet well, and dissolve in 0.1% DEPC-DW.

[0075] RNA was synthesized into cDNA according to the protocol of cDNA synthesis mater mix (Cell safe, Korea). Col4a1 expression was analyzed by real-time PCR, and the primer sequences are as follows.

[0076] Sequence number: mouse Col4a1 [F] 5'-ATG GCT TGC CTG GAG AGA TAG G-3'

[0077] [R] 5'-TGG TTG CCC TTT GAG TCC TGG A-3'

[0078] Sequence number: mouse Gapdh [F] 5'-CAT CAC TGC CAC CCA GAA GAC TG-3'

[0079] [R] 5'-ATG CCA GTG AGC TTC CCG TTC AG-3'

[0080] Western Blot

[0081] Primary endothelial cells cultured from the brains of transgenic animals were dissociated in a lysis reagent for protein extraction. After centrifugation at 15,000 g for 5 minutes at 4°C, only the supernatant was separated, and the cells were incubated at 95°C for 5 minutes. The tissue samples were loaded onto Mini-PROTEAN® TGX™ Gel (Biorad, California, USA) and electrophoresed at 100 V for 1 hour and 30 minutes. After electrophoresis, the gel was transferred to NC membrane at 30 V for 18 hours at low temperature. After transfer, the NC membrane was incubated with anti-COL4A1 primary antibody (ab6586, Abcam, 1:1000) diluted in 5% skim milk at 4°C for 16 hours. Then, the cells were incubated with HRP conjugated anti-Rabbit igG (cell signaling, 1:5000) secondary antibody at room temperature for 50 minutes and detected with an X-ray film.

[0082] H&E staining & Prussian blue staining

[0083] Tissue samples for histological staining were prepared by fixing tissues by perfusion with PBS and 4% PFA after anesthetizing the experimental animals with ketamine. After perfusion, the tissues collected from the experimental animals were treated with 10% sucrose solution and 30% sucrose solution at 4°C. The sucrose-treated tissues were sectioned at 30 μm thickness using a frozen microtome. The sectioned tissues were then attached to tissue slides for tissue staining. Before staining, the tissue sections were dehydrated and rehydrated and then stained with Prussian blue staining solution or Hematoxylin & Eosin staining solution. The stained tissue slides were photographed with a slide scanner (Axioscan Z1, Zeiss, Jena, Germany) and then analyzed.

[0084] Tissue immunostaining (Ferritin Light & Heavy chain, COL4A1, Iba1 staining)

[0085] Tissue samples for tissue immunostaining were prepared by fixing tissues by perfusion with PBS and 4% PFA after anesthetizing experimental animals with ketamine. After perfusion, tissues collected from experimental animals were treated with 10% sucrose solution and 30% sucrose solution at 4℃. The sucrose-treated tissues were sectioned at 30 μm thickness using a frozen microtome. The sectioned tissues were attached to tissue slides and immunostained. Before immunostaining, the tissue slides were incubated for 30 minutes at room temperature in a solution containing 0.3% Triton X-100 and 10% NGS. The primary antibodies used for tissue immunostaining were anti-Ferritin Light chain (ab69090, Abcam, 1:250), anti-Ferritin Heavy chain (ab65080, Abcam, 1:100), and anti-COL4A1 (ab6586, Abcam, 1:100). The antibody labeling process was performed by incubating the primary antibodies diluted in 10% NGS on the tissue slides at 4℃ for 16 hours, followed by incubation with the secondary antibodies for 2 hours at room temperature. After antibody incubation, the tissue slides were fixed with mounting medium and photographed under a microscope. The tissue slides stained with the COL4A1 antibody were photographed using a Zeiss LSM900 (Jena, Germany) microscope. The tissue slides stained with the anti-Ferritin Light & Heavy chain antibodies were photographed using a slide scanner and then analyzed.

[0086] Animal magnetic resonance imaging

[0087] For imaging, experimental animals were anesthetized with 1.5% isoflurane. A high-field 9.4T Tesla animal MRI machine was used, and T2-weighted images were obtained according to a coronal T2-weighted RARE sequence (TR / TE = 3300 / 60 ms, echo train length = 4, FOV = 14 x 11 mm², matrix size = 256 x 200).

[0088] Y-maze test

[0089] The Y-maze test, which is an experiment to measure short-term memory and evaluate the ability to act sequentially, was conducted as follows. The measuring device consists of three branches, each 32.5 cm long, 7 cm wide, and 15 cm high, and the three branches can be folded at a 120° angle. The device is made of white polyvinyl plastic, and the three branches are designated A, B, and C, respectively, and the experiment is conducted. The experimental animal is placed in each branch for 10 minutes, and 1 point (actual alternation) is awarded for the number of times the animal's tail entered each branch and for each case in which the animal entered each branch sequentially. Alternation behavior is defined as entering all three branches without overlapping, and spontaneous alternation behavior was calculated using the following mathematical equation (1).

[0090] [Mathematical Formula 1]

[0091] Alternation triplet(%) = [Alternation / (Total number of entries - 2)] × 100

[0092] Novel Objective Recognition Test

[0093] Mice were placed in an open-field box (40 cm × 40 cm × 40 cm) made of acrylic for 10 minutes each to acclimate and stabilize them (habituation) to the box. On the second day (training day), two different objects (objects: A and B) made of acrylic were placed at regular intervals in the box, and the mice were trained to freely explore objects A and B for 10 minutes. After that, one of objects A and B was replaced with a new object (object: C), and the mice were allowed to explore again for 10 minutes. The exploration time for each object (A and C or B and C) was quantified, and the preference for the object was expressed as the ratio of exploration time using the following mathematical equation 2.

[0094] [Equation 2]

[0095] Preference(%) = [C / (A+C)]Х100

[0096] Rotarod test

[0097] The rotarod test involves placing a rat on a rotarod machine, starting at a speed of 4 rpm and gradually increasing to 40 rpm. The treadmill then begins to rotate, measuring the time (in seconds) it takes the rat to lose its balance and fall to the floor, as well as the average speed. A sensor is located on the floor to automatically measure the time when the rat touches the floor.

[0098]

[0099] [Example 1] Col4a1_E01-#01

[0100] To edit the gene sequence targeted by the gRNA delivered into the cell, mouse Col4a1-target sgRNA packaged with AAV-BR1 virus was intravenously injected through the orbital vein of transgenic animals (6J.129(Cg) -Gt(ROSA)26Sortm1.1 (CAG-cas9*,-EGFP) Fezh / J) expressing CRISPR Cas9 nuclease.

[0101] The sequence number of the injected Col4a1-target sgRNA is as follows.

[0102] Sequence number 1: 5'-GACGCTGAGCCGGGGCCCCA-3'

[0103] [Example 2] Col4a1_E01-#02

[0104] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 2 was used.

[0105] Sequence number 2: 5'-TGGGGCCCCGGCTCAGCGTC-3'

[0106] [Example 3] Col4a1_E01-#03

[0107] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 3 was used.

[0108] Sequence number 3: 5'-TGCGCTCCTCGTGGAGCAGA-3'

[0109] [Example 4] Col4a1_E01-#04

[0110] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 4 was used.

[0111] Sequence number 4: 5'-CGCCGCCCUUCUGCUCCACG-3'

[0112] [Example 5] Col4a1_E01-#05

[0113] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 5 was used.

[0114] Sequence number 5: 5'-CTGCTCGGCTGCGCTCCTCG-3'

[0115] [Example 6] Col4a1_E21-#01

[0116] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 6 was used.

[0117] Sequence number 6: 5'-GTTGCAGGTTTCCCTACTCC-3'

[0118] [Example 7] Col4a1_E21-#02

[0119] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 7 was used.

[0120] SEQ ID NO: 7: 5'-CACCAGCCTGGCCTGGAGTA-3'

[0121] [Example 8] Col4a1_E21-#03

[0122] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 8 was used.

[0123] SEQ ID NO: 8: 5'-GCACCAGCCTGGCCTGGAGT-3'

[0124] [Example 9] Col4a1_E21-#04

[0125] The same method as Example 1 was performed, but Col4a1-target sgRNA containing the base sequence of SEQ ID NO: 9 was used.

[0126] Sequence number 9: 5'-AGGTTTCCCTACTCCAGGCC-3'

[0127] [Comparative Example 1] Rosa26-target sgRNA

[0128] The same method as Example 1 was used, but the mouse Rosa26-target sgRNA sequence was used.

[0129] The injected mouse Rosa26-target sgRNA sequence is as follows.

[0130] SEQ ID NO: 10: 5'-GGCGGUCCUCAGAAGCCAGG-3'

[0131]

[0132] [Experimental Example 1] Design and Selection of gRNA Sequences for Efficient Editing of the Col4a1 Gene

[0133] To efficiently edit the Col4a1 gene, CRISPR Cas9-gRNA sequences were designed and screened.

[0134] Figure 1a shows the results of an indel experiment to confirm whether the Col4a1 gene was edited by delivering Col4a1-targeting gRNA sequences according to one experimental example of the present invention to mouse NIH3T3 cells, and Figure 1b shows the results of confirming the Col4a1 mRNA level.

[0135] Referring to Fig. 1, it was confirmed that the gRNA (Col4a1_E01-#04) sequence used in Example 4 edited the Col4a1 gene most efficiently, and therefore, in the experimental examples described below, experiments were performed using the gRNA sequence of Example 4.

[0136] [Experimental Example 2] Observation of COL4A1 expression inhibition by Col4a1_E01-#04 selected in primary cerebral vascular endothelial cells.

[0137] Primary cerebral endothelial cells obtained from the cerebral blood vessels of CRISPR-Cas9 transgenic animals were cultured, and the Col4a1_E01-#04 sequence was packaged into lentivirus. These viruses were injected at a density of 5x10 5, 2.5x10 6 , 5x10 6 After delivery to cells at a concentration of TU / ml, the efficient reduction of Col4a1 mRNA and protein was confirmed through real-time PCR and western blot experiments.

[0138] Figure 2a shows the results of confirming the expression levels of Col4a1 mRNA and protein by real-time PCR through Col4a1-targeting gRNA treatment according to one experimental example of the present invention, and Figure 2b shows the results confirmed through a western blot experiment.

[0139] Referring to Fig. 2, a marked inhibition of COL4A1 expression in primary cerebral vascular endothelial cells was observed by treatment with Col4a1-targeting gRNA, and thus, in the experimental example described below, the gRNA sequence of Example 4 was used to package the AAV-BR1 virus and perform the experiment.

[0140] [Experimental Example 3] Confirmation of Col4a1 gene editing and COL4A1 expression suppression in actual cerebral vascular endothelial cells by AAV-BR1-Col4a1_E01-#04 virus injection in CRISPR Cas9 transgenic animals.

[0141] Col4a1_E01-#04 was packaged and produced as an AAV-BR1 virus using the cerebrovascular-specific AAV-BR1 system. This virus was delivered to the cerebral blood vessels by intravenous injection into the orbit at concentrations of 0.5E+11GC, 1E+11GC, and 2E+11GC. Three weeks after injection, primary cerebral vascular endothelial cells were cultured from the animal cerebral blood vessels, and whether the Col4a1 gene was actually edited in the cerebral vascular endothelial cells was confirmed through an Indel experiment.

[0142] Figure 3a is a schematic diagram of an experiment to confirm Col4a1 gene editing and COL4A1 expression suppression in cerebrovascular endothelial cells by injecting Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention into a CRISPR Cas9 transgenic animal, Figure 3b is an Indel result, and Figure 3c is a result of measuring the out-of-frame ratio of the edited Col4a1 gene.

[0143] Indel results confirmed that more than 95% of Col4a1 genes were edited when AAV-BR1-Col4a1_E01-#04 was injected at 1E+11GC scale or higher (Fig. 3b), and among the 95% of deleted genes, the out-of-frame ratio was confirmed to be more than 97%, confirming that almost all Col4a1 genes were perfectly edited (Fig. 3c).

[0144] Meanwhile, 3 weeks after virus injection, primary cerebrovascular endothelial cells were cultured from the cerebral blood vessels of animals, and the decrease in Col4a1 mRNA and protein in the cerebral blood vessels was confirmed through real-time PCR and western blot experiments. The decrease in COL4A1 expression was stained in brain tissue sections and observed using a confocal microscope.

[0145] Figure 4a shows the results of real-time PCR to confirm whether Col4a1 mRNA and protein decrease in the cerebral blood vessels of animals 3 weeks after injecting a CRISPR Cas9 transgenic animal with a Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention, and culturing primary cerebral vascular endothelial cells from the cerebral blood vessels of the animals. Figure 4b shows the results confirmed through a western blot experiment, and Figure 4c shows the results of staining brain tissue sections and observing them with a confocal microscope.

[0146] Referring to Fig. 4, it was confirmed that COL4A1 expression was almost reduced when the virus was injected at a scale of 1E+11GC or higher in CRISPR Cas9 transgenic animals.

[0147] [Experimental Example 4] Magnetic resonance imaging of intracerebral microhemorrhages caused by AAV-BR1-Col4a1_E01-#04 virus injection in CRISPR Cas9 transgenic animals.

[0148] Three months after AAV-BR1-Col4a1_E01-#04 virus injection (2E+11GC scale), microhemorrhages in the animals' brains were confirmed through magnetic resonance imaging.

[0149] Figure 5 shows the results of magnetic resonance imaging of the brain of a CRISPR Cas9 transgenic animal injected with a Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention.

[0150] Referring to Fig. 5, microhemorrhage similar to microhemorrhage observed in humans was confirmed in CRISPR Cas9 transgenic animals injected with Col4a1-targeting gRNA packed with AAV-BR1, and thus, in the experimental examples described below, microhemorrhage observed in CRISPR Cas9 transgenic animals injected with Col4a1-targeting gRNA packed with AAV-BR1 was verified through various experiments.

[0151] [Experimental Example 5] Microhemorrhages observed on the brain surface and cross-sections following AAV-BR1-Col4a1_E01-#04 virus injection in CRISPR Cas9 transgenic animals.

[0152] Figure 6a shows the results of cerebral microhemorrhage on the brain surface according to the injection concentration of Col4a1 targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention, Figure 6b shows the results of cerebral microhemorrhage in a brain section, and Figure 6c shows the results of measuring lesions due to cerebral microhemorrhage by region in a brain tissue section of an animal 3 months after injection.

[0153] Referring to Fig. 6a, the AAV-BR1-Col4a1_E01-#04 virus was delivered to the cerebral blood vessels by intravenous injection into the orbit at concentrations of 1E+11GC and 2E+11GC scales, and traces of microhemorrhage were confirmed on the surface of the animal's brain 3 months after the virus injection.

[0154] Referring to Figure 6b, after intravenous injection of the AAV-BR1-Col4a1_E01-#04 virus into the orbit at concentrations of 0.5E+11GC, 1E+11GC, and 2E+11GC scale, microhemorrhages were observed in the brain cross-section 3 months later.

[0155] Referring to Fig. 6c, 3 months after the injection of the AAV-BR1-Col4a1_E01-#04 virus, lesions due to cerebral microhemorrhages were measured by area in the brain tissue sections of the animals through hematoxylin & eosin staining, and it was confirmed that the area where lesions appeared increased.

[0156] [Experimental Example 6] Tissue staining to confirm intracerebral microhemorrhage caused by AAV-BR1-Col4a1_E01-#04 virus injection in CRISPR Cas9 transgenic animals.

[0157] AAV-BR1-Col4a1_E01-#04 virus was intravenously injected into the orbit at concentrations of 0.5E+11GC, 1E+11GC, and 2E+11GC to deliver it to the cerebral blood vessels. Three months after the virus injection, Prussian blue staining was performed to confirm cerebral microhemorrhage in the animal brain sections. Three months after the AAV-BR1-Col4a1_E01-#04 virus injection (2E+11GC scale), hemosiderin granules were observed in the Prussian blue-stained area. In addition, immunostaining using anti-Ferritin Light / Heavy chain antibodies was performed to confirm cerebral microhemorrhage.

[0158] Figure 7a shows the results of confirming cerebral microhemorrhages in brain tissue sections using the Prussian blue staining method 3 months after injecting animals with Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention at various concentrations, and Figure 7b shows the results of observing hemosiderin granules in the area stained with Prussian blue, and Figure 7c shows the results of performing an immunostaining method using an anti-Ferritin Light / Heavy chain antibody.

[0159] Referring to Figure 7, microhemorrhages were confirmed through various staining methods in brain sections by AAV-BR1-Col4a1_E01-#04 virus injection in CRISPR Cas9 transgenic animals.

[0160] [Experimental Example 7] Observation of cognitive impairment and motor function decline in mice injected with AAV-BR1-mCol4a1-target gRNA.

[0161] To observe cognitive impairment and motor function decline in AAV-BR1-mCol4a1-target gRNA-injected mice, the Y-maze test, novel objective recognition test, and rotarod test were performed.

[0162] Figure 8a shows the results of measuring short-term memory using the Y-maze test from 0 to 6 months after injecting Col4a1-targeting gRNA packed with AAV-BR1 according to one experimental example of the present invention into animals, and Figure 8b shows the results of measuring cognitive function and memory using the Novel objective recognition test, and Figure 8c shows the results of performing a neurobehavioral test to determine balance, limb coordination, muscle strength, etc. using the Rotarod test.

[0163] Figure 8a The upper figure shows statistics by combining the number of male and female transgenic animals, the middle figure is a graph showing statistics derived from male transgenic animals under the same conditions, and the lower figure is a graph showing statistics derived from female transgenic animals under the same conditions. Through this, it was possible to confirm that short-term memory was reduced by comparing the number of times the animals entered three consecutive mazes in alternating order to the total number of entries into the Y maze in CRISPR Cas9 transgenic animals injected with Col4a1-targeting gRNA packed with AAV-BR1 at 6 months.

[0164] The upper figure in Fig. 8b shows statistics by combining the number of male and female transgenic animals, and shows a statistically significant decrease from 2 months after injection. The middle figure shows statistics derived from male transgenic animals under the same conditions, and shows a statistically significant decrease from 4 months after injection. The lower figure is a graph showing statistics derived from female transgenic animals under the same conditions, and shows a statistically significant decrease from 2 months after injection. Through this, we were able to confirm that cognitive function and memory ability were reduced by measuring the exploration time for a novel object in CRISPR Cas9 transgenic animals injected with Col4a1-targeting gRNA packed with AAV-BR1.

[0165] The upper figure in Fig. 8c shows statistics by combining the number of male and female transgenic animals, and shows a statistically significant decrease from 4 months after injection. The middle figure shows statistics derived from male transgenic animals under the same conditions, and shows a statistically significant decrease from 4 months after injection. The lower figure is a graph showing statistics derived from female transgenic animals under the same conditions, and shows a statistically significant decrease from 6 months after injection. Through this, it was possible to confirm that balance, limb coordination, and muscle strength were reduced by checking the fall time on the treadmill in CRISPR Cas9 transgenic animals injected with Col4a1-targeting gRNA packed with AAV-BR1.

[0166]

[0167] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0168] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Cerebral microhemorrhage induced by Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1, cerebral microhemorrhage animal model.

2. In paragraph 1, A cerebral microhemorrhage animal model in which the expression of the COL4A1 gene is suppressed by the above Col4a1-targeting gRNA.

3. In paragraph 2, A cerebral microhemorrhage animal model in which the elasticity of the blood-brain barrier is reduced by suppression of the expression of the COL4A1 gene.

4. In paragraph 3, A cerebral microhemorrhage animal model in which cerebral microhemorrhage is induced by a decrease in the elasticity of the blood-brain barrier.

5. In paragraph 1, A cerebral microhemorrhage animal model, wherein the Col4a1-targeting gRNA is specifically delivered to cerebral vascular endothelial cells by the AAV-BR1.

6. In paragraph 1, The above Col4a1 targeting gRNA (guide RNA) comprises any one of the base sequences of SEQ ID NOs: 1 to 9. Animal model of cerebral microhemorrhage.

7. A method for producing a cerebral microhemorrhage animal model, comprising the step of inducing cerebral microhemorrhage by injecting Col4a1-targeting gRNA (guide RNA) packed with AAV-BR1 into a transgenic animal using CRISPR-Cas9.

8. In paragraph 7, A method for producing a cerebral microhemorrhage animal model, wherein the expression of the COL4A1 gene is suppressed by the above Col4a1 targeting gRNA.

9. In paragraph 8, A method for producing an animal model of cerebral microhemorrhage, wherein the elasticity of the blood-brain barrier is reduced by suppressing the expression of the COL4A1 gene.

10. In paragraph 9, A method for producing an animal model of cerebral microhemorrhage, wherein cerebral microhemorrhage is induced by a decrease in the elasticity of the blood-brain barrier.

11. In paragraph 7, A method for producing a cerebral microhemorrhage animal model, wherein the Col4a1 targeting gRNA is specifically delivered to cerebral vascular endothelial cells by the AAV-BR1.

12. In paragraph 7, A method for producing a cerebral microhemorrhage animal model, wherein the Col4a1 targeting gRNA comprises any one of the base sequences of SEQ ID NOs: 1 to 9.

13. A step of administering a candidate substance for treating cerebral microhemorrhage to an animal model of cerebral microhemorrhage according to any one of clauses 1 to 6; and A step of comparing an animal model administered with the candidate substance with a control group that was not administered with the candidate substance, and determining that the candidate substance is a treatment for cerebral microhemorrhage if cerebral microhemorrhage is reduced by the candidate substance; Including, Screening methods for cerebral microhemorrhage treatment agents.

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