Gene therapy for repairing phosphorus homeostasis in brain by targeting astrocytes

Abstract

The invention relates to an intracerebral phosphorus homeostasis accelerant and application thereof. Specifically, a gene compensation therapy for targeting glial cells, especially astrocytes, is developed on the basis of a molecular cytology mechanism of the intracerebral phosphorus homeostasis, and can be used for repairing the intracerebral phosphorus homeostasis imbalance condition, so that the occurrence of calcification is inhibited, or the further progress of intracerebral calcification is inhibited, and the intracerebral phosphorus homeostasis is inhibited. And clinical nervous system symptoms of related patients are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to gene compensation / gene replacement expression targeting astrocytes in the brain for hereditary brain calcification and its application. The method can effectively inhibit the occurrence of brain calcification and is expected to be used for clinical intervention of brain calcification in patients meeting the characteristics of the present invention. Background Art

[0002] Brain calcification is an age- and aging-related disease that is relatively common in clinical imaging. In cranial CT observations of the elderly population over 60 years old, more than 20% will have punctate calcifications, and more than 2-4% will have larger patchy calcifications. Some neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), have more than 20% of patients with brain calcification, and the lesions expand with age. Therefore, brain calcification is a neurodegenerative disease that needs to be paid attention to clinically and has treatment needs.

[0003] Hereditary brain calcification is a neurodegenerative disease caused by gene mutations, including many types, such as primary familial brain calcification (PFBC), Aicardi-Goutières syndrome (AGS), etc. Among them, PFBC is a more representative type, with an incidence of about 6 per 1,000. The pathological characteristics of PFBC are bilateral symmetrical calcification in the basal ganglia, thalamus and cerebellar dentate nucleus, and calcification is occasionally seen in the subcortical area of ​​some patients. As the disease progresses, the calcification foci can be connected into large sheets. High-resolution brain tissue pathological imaging studies have found that calcium salt particles are mainly deposited in the brain space around terminal arterioles, veins and capillaries. The clinical symptoms of hereditary brain calcification are varied, mainly manifested as movement disorders, mental disorders, cognitive disorders, epileptic seizures, dizziness, headaches, etc. In severe cases, personality and behavioral changes will occur, eventually leading to mental illness or dementia; some patients become disabled and cannot take care of themselves, and some patients have no obvious symptoms throughout their lives.

[0004] Hereditary brain calcification can be sporadic (mostly de novo mutations) or familial, with high genetic heterogeneity. In the past decade, human genetic studies have found that the pathogenic genes of PFBC include: SLC20A2, XPR1, PDGFB, PDGFRB, MYORG and JAM2. These genes suggest that the pathogenic mechanism of brain calcification may be mainly reflected in two aspects: first, the integrity of the blood-brain barrier (BBB) ​​structure is damaged or the pericyte coverage rate is reduced, resulting in increased BBB permeability. Patients related to this pathway may have mutations in genes such as PDGFRB, PDGFB, and JAM2, which are all expressed in cells that constitute the neurovascular unit.

[0005] The second pathway is the disorder of inorganic phosphate (Pi) transport in the brain and the resulting imbalance of brain phosphate homeostasis (BPH), and usually shows a significant increase in cerebrospinal fluid (CSF) Pi levels. Patients belonging to this pathway may have mutations in the SLC20A2, MYORG, or XPR1 genes, which encode the Na-Pi co-transporter PiT2 protein and the phosphate excretion transporter protein XPR1 and its regulator MYORG, respectively. Furthermore, statistical analysis of clinical calcification and symptoms showed that patients with Pi transport-related gene mutations had larger calcification foci and more severe clinical symptoms compared with patients with BBB pathway-related gene mutations. Moreover, among all reported familial calcification patients, SLC20A2 mutations accounted for 61% of all genetic factors, XPR1 accounted for 6% of all genetic factors, and MYORG mutations accounted for 13%.

[0006] Currently, the clinical treatment of PFBC is mainly symptomatic treatment, and there is no effective drug that can remove or reduce abnormal calcium deposition. In particular, targeting the imbalance mechanism of BPH may provide opportunities for the development of corresponding clinical treatment methods.

[0007] Therefore, there is an urgent need to develop a novel brain calcification intervention strategy that is convenient and effective and aims to restore phosphorus homeostasis. Summary of the invention

[0008] One of the purposes of the present invention is to provide a brain phosphorus homeostasis promoter and its application.

[0009] One of the purposes of the present invention is to provide a gene compensation therapy and application targeting astrocytes to reconstruct phosphorus homeostasis in the brain, including inhibiting the occurrence of brain calcification in individuals with PiT2 mutations, and delaying or curbing the progression of brain calcification in patients with PFBC or brain calcification associated with phosphorus homeostasis imbalance.

[0010] The first aspect of the present invention provides a use of a brain phosphorus homeostasis promoter, characterized in that it is used to prepare a drug for preventing and / or treating brain calcification diseases.

[0011] In another preferred embodiment, the brain phosphorus homeostasis promoter is selected from the following group:

[0012] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0013] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0014] (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof;

[0015] (Z4) Any combination of the above Z1 to Z3.

[0016] In another preferred embodiment, the brain phosphorus homeostasis promoter includes an astrocyte phosphorus transport promoter.

[0017] In some embodiments, the prevention comprises arresting calcification before it occurs in a subject genetically identified to carry the mutation.

[0018] In some embodiments, the mutation comprises a mutation in PiT2, MYORG, XRP1, or a combination thereof.

[0019] In some embodiments, the calcification comprises brain calcification.

[0020] In some embodiments, the treatment includes arresting and / or delaying further progression of calcification in a subject.

[0021] In some embodiments, the treatment includes reversing and / or repairing imbalance of phosphorus homeostasis in the brain and / or brain calcification.

[0022] In another preferred embodiment, the expression vector comprises a viral vector.

[0023] In another preferred embodiment, the expression vector contains an expression cassette for expressing a foreign gene, and the foreign gene is selected from the following group: PiT2, MYORG, XRP1, or a combination thereof.

[0024] In another preferred embodiment, the exogenous gene is operably linked to an enhancer element selectively expressed in astrocytes and / or a promoter selectively expressed in astrocytes.

[0025] In another preferred embodiment, the promoter is gfaABC1D.

[0026] In another preferred embodiment, the promoter includes small nucleic acid drugs.

[0027] In another preferred embodiment, the promoter includes antisense oligonucleotides (ASOs).

[0028] In another preferred embodiment, the promoter includes: small molecule compounds, gene editing reagents, small nucleic acid drugs, or a combination thereof.

[0029] In another preferred embodiment, the gene editing reagent includes a base editing reagent.

[0030] In another preferred embodiment, the brain calcification disease includes brain calcification caused by abnormal phosphorus homeostasis in the brain.

[0031] In another preferred embodiment, the brain calcification disease includes brain calcification caused by upregulation of inorganic phosphate in cerebrospinal fluid.

[0032] In another preferred embodiment, the brain calcification disease includes primary familial brain calcification (PFBC).

[0033] As used herein, "abnormal phosphorus homeostasis," "imbalance in phosphorus homeostasis," "dysregulation of phosphorus homeostasis," and "deficient in phosphorus homeostasis" are used interchangeably.

[0034] As used herein, "phosphate transport disorder" and "phosphate transport defect" are used interchangeably.

[0035] In another preferred embodiment, the drug is administered to a subject selected from the following group: PFBC patients carrying PIT2, MYORG, XPR1 gene mutations, or a combination thereof, or patients with other types of brain calcification diseases associated with defects in brain phosphorus homeostasis.

[0036] In another preferred embodiment, the brain phosphorus homeostasis defect includes astrocyte phosphorus transport defect in the brain.

[0037] In another preferred embodiment, the brain calcification disease includes a brain calcification disease caused by an imbalance of phosphorus homeostasis in the brain.

[0038] In another preferred embodiment, for the subject (or patient) with brain calcification disease with imbalanced phosphorus homeostasis in the brain, the phosphorus transport capacity C1 of astrocytes is significantly lower than the phosphorus transport capacity C0 of astrocytes in a normal individual.

[0039] In another preferred embodiment, the “significantly lower than” refers to the ratio of the astrocyte phosphate transport capacity C1 to the astrocyte phosphate transport capacity C0 of a normal individual (C1 / C0) ≤ 2 / 3, preferably ≤ 1 / 2; more preferably ≤ 1 / 4.

[0040] In another preferred embodiment, the subject has a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphate transporters, or a combination thereof.

[0041] In another preferred embodiment, the genetic mutation includes exon mutation, intron mutation, 5'-UTR mutation, 3'-UTR mutation, or a combination thereof.

[0042] The second aspect of the present invention provides a preparation and a composition, characterized in that the preparation and the composition contain a promoter for promoting phosphorus homeostasis in the brain.

[0043] In another preferred embodiment, the preparation and composition contain (a) a therapeutically effective amount of a brain phosphorus homeostasis promoter, and (b) a pharmaceutically acceptable carrier or excipient.

[0044] In another preferred embodiment, the brain phosphorus homeostasis promoter is selected from the following group:

[0045] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0046] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0047] (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof;

[0048] (Z4) Any combination of the above Z1 to Z3.

[0049] In another preferred embodiment, the brain phosphorus homeostasis promoter includes an astrocyte phosphorus transport promoter.

[0050] In another preferred embodiment, the promoter includes small nucleic acid drugs.

[0051] In another preferred embodiment, the promoter includes antisense oligonucleotides (ASOs).

[0052] In another preferred embodiment, the promoter includes: small molecule compounds, gene editing reagents, small nucleic acid drugs, or a combination thereof.

[0053] In another preferred embodiment, the gene editing reagent includes a base editing reagent.

[0054] In another preferred embodiment, the brain calcification disease includes brain calcification caused by abnormal phosphorus homeostasis in the brain.

[0055] In another preferred embodiment, the brain calcification disease includes primary familial brain calcification (PFBC).

[0056] The third aspect of the present invention provides a medicine kit for treating brain calcification diseases, characterized in that the medicine kit comprises:

[0057] (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect whether a genetic mutation exists in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and

[0058] (b) a second container, and a pharmaceutical composition in the second container, wherein the pharmaceutical composition comprises a pharmaceutical ingredient capable of regulating and repairing phosphorus homeostasis imbalance in the brain and a pharmaceutically acceptable carrier.

[0059] In another preferred embodiment, the pharmaceutical composition comprises a brain phosphorus homeostasis promoter.

[0060] In another preferred embodiment, the pharmaceutical ingredient comprises the preparation and composition as described in the second aspect of the present invention.

[0061] In another preferred embodiment, the medicine kit further comprises: (c) a third container, and a solution in the third container for assisting the dissolution or in vivo delivery of the drug in the second container.

[0062] In another preferred embodiment, optionally, the medicine kit further includes: (d) a fourth container, and a fourth detection reagent for detecting the phosphorus transport function of astrocytes located in the fourth container.

[0063] In another preferred embodiment, the brain phosphorus homeostasis promoter is selected from the following group:

[0064] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0065] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0066] (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof;

[0067] (Z4) Any combination of the above Z1 to Z3.

[0068] In another preferred embodiment, the expression vector comprises a viral vector.

[0069] In another preferred embodiment, the expression vector contains an expression cassette for expressing a foreign gene, and the foreign gene is selected from the following group: PiT2, MYORG, XRP1, or a combination thereof.

[0070] In another preferred embodiment, the exogenous gene is operably linked to an enhancer element selectively expressed in astrocytes and / or a promoter selectively expressed in astrocytes.

[0071] In another preferred embodiment, the promoter is gfaABC1D.

[0072] In another preferred embodiment, the promoter includes small nucleic acid drugs.

[0073] In another preferred embodiment, the promoter includes antisense oligonucleotides (ASOs).

[0074] In another preferred embodiment, the promoter includes: small molecule compounds, gene editing reagents, small nucleic acid drugs, or a combination thereof.

[0075] In another preferred embodiment, the gene editing reagent includes a base editing reagent.

[0076] In another preferred embodiment, the brain calcification disease includes brain calcification caused by abnormal phosphorus homeostasis in the brain.

[0077] In another preferred embodiment, the brain calcification disease includes primary familial brain calcification (PFBC).

[0078] As used herein, "abnormal phosphorus homeostasis," "imbalance in phosphorus homeostasis," and "deficient in phosphorus homeostasis" are used interchangeably.

[0079] In another preferred embodiment, the drug is administered to a subject selected from the following group: PFBC patients carrying PIT2, MYORG, XPR1 gene mutations, or a combination thereof, or patients with other types of brain calcification diseases associated with defects in brain phosphorus homeostasis.

[0080] In another preferred embodiment, the brain phosphorus homeostasis defect includes astrocyte phosphorus transport defect in the brain.

[0081] In another preferred embodiment, the brain calcification disease includes a brain calcification disease caused by an imbalance of phosphorus homeostasis in the brain.

[0082] In another preferred embodiment, for the subject (or patient) with brain calcification disease with imbalanced phosphorus homeostasis in the brain, the phosphorus transport capacity C1 of astrocytes is significantly lower than the phosphorus transport capacity C0 of astrocytes in a normal individual.

[0083] In another preferred embodiment, the “significantly lower than” refers to the ratio of the astrocyte phosphate transport capacity C1 to the astrocyte phosphate transport capacity C0 of a normal individual (C1 / C0) ≤ 2 / 3, preferably ≤ 1 / 2; more preferably ≤ 1 / 4.

[0084] In another preferred embodiment, the subject has a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphate transporters, or a combination thereof.

[0085] In another preferred embodiment, the genetic mutation includes exon mutation, intron mutation, 5'-UTR mutation, 3'-UTR mutation, copy number variations (CNVs), or a combination thereof.

[0086] In another preferred embodiment, the first detection reagent is used to detect genetic mutations of PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphate transporters, or a combination thereof in the desired subject. If the result of the detection indicates the presence of the mutation in the subject, the subject is considered to be suitable for treatment with the pharmaceutical composition in the kit.

[0087] In another preferred embodiment, optionally, cells of the desired subject are collected, the cells are differentiated into astrocytes, and the phosphorus transport function of the astrocytes is detected using the fourth detection reagent. If the test result indicates that the phosphorus transport capacity C1 of the subject's astrocytes is significantly lower than the phosphorus transport capacity C0 of the astrocytes of a normal individual, the subject is considered to be suitable for treatment using the pharmaceutical composition in the kit.

[0088] In another preferred embodiment, the “significantly lower than” refers to the ratio of the phosphorus transport capacity C1 of the astrocytes derived from the subject to the phosphorus transport capacity C0 of the astrocytes of a normal individual (C1 / C0) ≤ 2 / 3, preferably ≤ 1 / 2; more preferably ≤ 1 / 4.

[0089] In another preferred embodiment, the subject includes an animal model.

[0090] In another preferred embodiment, the subject includes a mammal.

[0091] In another preferred embodiment, the subject includes a human.

[0092] In another preferred embodiment, the subject includes non-human mammals.

[0093] In another preferred embodiment, the subject is a non-human mammal.

[0094] In another preferred embodiment, the subject includes primates.

[0095] In another preferred embodiment, the subject includes a rodent.

[0096] In another preferred embodiment, the subject includes humans, mice, rats, monkeys, or a combination thereof.

[0097] In another preferred embodiment, the cells include primary cells and passaged cells.

[0098] In another preferred embodiment, the cells include somatic cells, germ cells, gametes, and stem cells.

[0099] In another preferred embodiment, the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells.

[0100] In another preferred embodiment, the stem cells are induced pluripotent stem cells (iPSCs).

[0101] In another preferred embodiment, the cells include: embryonic stem cells, adipose stem cells, neural stem cells, mesenchymal stem cells, oligodendrocyte progenitor cells, hematopoietic stem cells, immune cells (such as T cells, B cells, NK cells).

[0102] In another preferred embodiment, the cells include: healthy normal human cells and cancerous human cells.

[0103] In another preferred embodiment, the cells include: suspension cells and adherent cells.

[0104] In another preferred embodiment, the cells do not include embryonic stem cells.

[0105] In another preferred embodiment, the cells do not include human embryonic stem cells.

[0106] In another preferred embodiment, the preparation comprises a pharmaceutical composition.

[0107] In another preferred embodiment, the first container, the second container, the third container, and the fourth container are the same or different containers.

[0108] In another preferred embodiment, the drug in the second container includes a single and / or compound preparation containing a guide RNA or its expression vector, and / or a gene editing protein or its expression vector.

[0109] In another preferred embodiment, the gene editing protein includes a base editing protein.

[0110] In some embodiments, the single and / or compound preparations in the second container can be packaged in different drug containers due to drug transportation and storage requirements.

[0111] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines that are helpful in treating brain calcification diseases.

[0112] In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0113] In another preferred embodiment, the dosage form of the drug includes an injection dosage form, a transdermal adsorption dosage form, a nasal inhalation dosage form, or a combination thereof.

[0114] In another preferred embodiment, the medicine kit further contains instructions.

[0115] A fourth aspect of the present invention provides a method for promoting phosphorus transport in astrocytes, characterized in that it comprises the steps of:

[0116] Astrocytes are cultured in the presence of an astrocyte phosphate transport promoter, thereby promoting astrocyte phosphate transport.

[0117] In another preferred embodiment, the astrocyte phosphorus transport promoter is selected from the following group:

[0118] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0119] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0120] (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof;

[0121] (Z4) Any combination of the above Z1 to Z3.

[0122] In another preferred embodiment, the expression vector comprises a viral vector.

[0123] In another preferred embodiment, the expression vector contains an expression cassette for expressing a foreign gene, and the foreign gene is selected from the following group: PiT2, MYORG, XRP1, or a combination thereof.

[0124] In another preferred embodiment, the exogenous gene is operably linked to an enhancer element selectively expressed in astrocytes and / or a promoter selectively expressed in astrocytes.

[0125] In another preferred embodiment, the promoter is gfaABC1D.

[0126] In another preferred embodiment, the promoter includes small nucleic acid drugs.

[0127] In another preferred embodiment, the promoter includes antisense oligonucleotides (ASOs).

[0128] In another preferred embodiment, the promoter includes: small molecule compounds, gene editing reagents, small nucleic acid drugs, or a combination thereof.

[0129] In another preferred embodiment, the gene editing reagent includes a base editing reagent.

[0130] In another preferred embodiment, the astrocytes are in vitro cells.

[0131] A fifth aspect of the present invention provides a method for preventing and / or treating brain calcification diseases, comprising:

[0132] The enhancing agent of the first aspect of the present invention or the pharmaceutical kit of the third aspect of the present invention is administered to a subject in need thereof.

[0133] In another preferred embodiment, the subjects include: PFBC patients carrying PIT2, MYORG, XPR1 gene mutations, or a combination thereof, or patients with other types of brain calcification diseases accompanied by defects in brain phosphorus homeostasis.

[0134] In another preferred embodiment, the subject has a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphate transporters, or a combination thereof.

[0135] In another preferred embodiment, the subject includes an animal model.

[0136] In another preferred embodiment, the subject includes a mammal.

[0137] In another preferred embodiment, the subject includes a human.

[0138] In another preferred embodiment, the subject includes non-human mammals.

[0139] In another preferred embodiment, the subject is a non-human mammal.

[0140] In another preferred embodiment, the subject includes primates.

[0141] In another preferred embodiment, the subject includes a rodent.

[0142] In another preferred embodiment, the subject includes humans, mice, rats, monkeys, or a combination thereof.

[0143] In another preferred embodiment, the administration includes intravenous injection, subcutaneous injection, head and neck lymphatic vessel injection, sinus administration, intracerebral ventricular injection, brain parenchyma injection, spinal lumbar puncture injection, or a combination thereof.

[0144] A sixth aspect of the present invention provides a method for screening candidate compounds for preventing and / or treating brain calcification diseases, the method comprising the steps of:

[0145] (a) In a test group, a test compound is added to the culture system of astrocytes or a cell line derived therefrom, and the phosphorus transport level (E1) and / or activity (A1) in the cells of the test group is observed; in a control group, no test compound is added to the culture system of the same cells, and the phosphorus transport level (E0) and / or activity (A0) in the cells of the control group is observed;

[0146] Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test compound has a promoting effect on the expression and / or activity of the phosphorus transport level and is a potential candidate compound that can be used to prevent and / or treat brain calcification diseases.

[0147] In another preferred embodiment, the method comprises step (b): administering the candidate compound determined in step (a) to a mammalian model to determine its effect on the mammal.

[0148] In another preferred embodiment, the influence includes the influence on the inorganic phosphate level in the brain of a mammal, and the effect on changing the occurrence or progression of calcification in the brain.

[0149] In another preferred embodiment, the mammal is a mammal suffering from brain calcification disease.

[0150] In another preferred embodiment, the “significantly higher than” refers to E1 / E0≥1.5, preferably ≥2.

[0151] In another preferred embodiment, the “significantly higher than” refers to A1 / A0≥1.5, preferably ≥2.

[0152] In another preferred embodiment, the cells include astrocytes.

[0153] In another preferred embodiment, the cells are cells cultured in vitro.

[0154] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0155] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 The effects of overall or conditional knockout of Pit2 (Slc20a2) on brain phosphorus homeostasis are shown, where Figure A shows that the average level of inorganic phosphate (Pi) in the cerebrospinal fluid (CSF) of PiT2 knockout (Pit2 KO) mice is ~1.8mM, which is significantly increased compared to the CSF Pi concentration in the brain of WT mice (~0.5mM). Figure B shows that there is no significant change in inorganic phosphate (Pi) in the serum of PiT2 knockout mice. Figure C shows the changes in the concentration of CSF Pi in the brain of Pit2 conditional knockout (Pit2 cKO) mice in different cell types, among which astrocytes conditionally knocked out (using Aldh1L1-CreER T2 The CSF Pi of mice induced by Tamoxifen (P1-CreER) increased most significantly (by about 170%), with an average level of about 1.35 mM. Other conditional knockout mice, including oligodendrocytes (Plp1-CreER T2 Figure D shows that there were no significant differences in serum Pi concentrations between the different conditional knockout mouse strains.

[0157] Figure 2 The selective expression of PiT2 in astrocytes in the brain mediated by intravenous injection of AAV virus is shown. Figure A shows the serotype of AAV (PHP.eB) and the structure of the expression vector, as well as a schematic diagram of intravenous injection of mice. Figure B shows sagittal brain slices prepared from mice fixed with PFA one month after intravenous injection. Based on immunofluorescence staining, the whole brain of the mouse shows widespread expression of PiT2-V5 signals (red) in astrocytes, and blue Hoechst indicates cell nuclei.

[0158] Figure 3 The results showed that selective compensatory expression of AAV in astrocytes inhibited calcification in the mouse brain. The images obtained by microCT scanning of the mouse brain after perfusion showed that the Pit2 KO mice treated with the control virus (expressing only GFP) showed obvious calcification foci in multiple brain regions in both the coronal and sagittal planes at 5 and 7 months of age. The dorsal midbrain, basal forebrain, thalamus, and hypothalamus showed significant large-scale calcification deposition in the mouse brain; the calcification coverage area of ​​7-month-old mice was larger than that of 5-month-old mice, and the calcification particles were larger. However, the calcification of the brain of Pit2 KO mice that successfully received therapeutic AAV injection (Replenished) was significantly suppressed at 5 and 7 months of age. DETAILED DESCRIPTION

[0159] After extensive and in-depth research and a large amount of screening, the inventors have developed for the first time a gene compensation therapy targeting astrocytes that can effectively intervene in the progression of brain calcification. After in-depth experiments, the inventors have clarified for the first time the pathogenic mechanism of brain calcification caused by mutations and / or abnormalities of genes and / or molecules such as PiT2, MYORG, and XPR1. The mutations and / or abnormalities cause abnormalities in the phosphorus transport function of astrocytes, thereby leading to an imbalance in phosphorus homeostasis in the brain, which in turn leads to brain calcification. At the method level, the present invention provides a strategy for repairing phosphorus homeostasis in the brain and curbing the occurrence of calcification or blocking its progression by increasing the expression of PiT2, MYORG, or XPR1 genes in astrocytes in the brain.

[0160] Specifically, the present invention constructs a recombinant AAV virus that can selectively express PiT2 protein in astrocytes. Experimental results show that after intravenous injection of a recombinant AAV virus that can cross the blood-brain barrier, the expression module carrying the expression of PiT2 protein can selectively express the protein product PiT2 of the compensation gene carried by it in the astrocytes in the mouse brain. As a result, the astrocytes in the brain obtain the expression of compensated Pit2-KO mice to achieve the reconstruction of brain phosphorus homeostasis, thereby effectively curbing the occurrence and progression of brain calcification from the source. The strategy disclosed by the present invention also supports an alternative implementation form, that is, by delivering gene expression promoters, such as antisense nucleotide (ASO) drugs, or RNA splicing modification based on gene / base editing to astrocytes, thereby regulating the expression of PiT2, MYORG, or XPR1 in astrocytes, and the purpose of repairing brain phosphorus homeostasis and intervening in brain calcification can also be achieved. These data indicate that the gene compensation therapy of the present invention can be used in humans to repair brain phosphorus homeostasis, inhibit the occurrence of brain calcification or prevent the further development of brain calcification, and improve related neurological symptoms, and has very outstanding clinical translational value.

[0161] On this basis, the present invention has been completed.

[0162] the term

[0163] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0164] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0165] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0166] PiT2

[0167] SLC20A2 encodes type III sodium ion + )-The second transporter protein PiT2 in the SLC20 subfamily of inorganic phosphate (Pi) co-transporters. This transporter was initially discovered to be a receptor for mouse retrovirus (A-MuLV) to infect human cells, but it was soon confirmed that its actual function was a transporter located on the cell membrane that actively transports Pi, which can transport 2Na + The transport ratio of inorganic phosphate ions (mainly H2PO4 - ) transported into the cell.

[0168] In 2012, a genetic study based on a family with primary familial brain calcification (PFBC) found that SLC20A2 mutations caused defects in PiT2's Pi transport activity, leading to extracellular Pi accumulation and inducing the formation of brain calcification. Statistics show that mutations in the SLC20A2 / PIT2 gene account for nearly 40% of all PFBCs and 61% of all reported families with a clear diagnosis, making it the most important associated gene for PFBC.

[0169] Mice deficient in PiT2, such as Slc20a2 / Pit2 knockouts, or knock-in mice with mutations that mimic those in patients, develop extensive brain calcification and display an abnormal multisystem phenotype, including placental calcification, fetal growth restriction, developmental delay, lean body mass, reduced bone mass and strength, skeletal malformations, a high likelihood of eye defects, and impaired spatial learning, memory, and sensorimotor gating.

[0170] In addition, compared with wild-type mice, homozygous Slc20a2 knockout (Slc20a2 - / - ) mice had significantly increased CSF Pi levels, which is consistent with the increased CSF Pi levels in PFBC patients with SLC20A2 mutations. Considering that the CSF flowing along the paravascular space exchanges metabolites with the parenchymal ISF, it is reasonable to expect that the ISF Pi level also experienced an increase similar to that of the CSF Pi. In addition, PiT2 is widely and strongly expressed in neurons, astrocytes, oligodendrocytes, microglia, vascular smooth muscle cells (VSMC) and vascular fibroblast-like cells. Defects or mutations in PiT2 in these cells may impair Pi transport activity and contribute to the increase in extracellular ISF and CSF Pi. The applicant of the present invention further clarified that among the many cell types in the brain, astrocyte-mediated phosphate transport plays the most important regulatory role in maintaining phosphorus homeostasis in the brain.

[0171] In addition, several studies have shown that PiT2-deficient VSMCs were found to have high Pi-induced calcification and increased expression of Runx2 and osteopontin (OPN) in a high extracellular Pi environment. Therefore, the high extracellular Pi environment may induce VSMCs to transdifferentiate into osteoblast-like cells through a mineralization process similar to that during bone development, ultimately leading to the occurrence of brain calcification. However, this hypothesis still needs more research data to support it.

[0172] Brain calcification

[0173] Brain calcification is a common neuropathological phenomenon in clinical practice. Its incidence rate increases from ~1% in young people to 20%-30% in the elderly (>60 years old), of which 2-4% are large patchy calcifications. Genetic defects, endocrine disorders (hypoparathyroidism and hypothyroidism), intracranial atherosclerosis, infection, brain tumors, neurotoxicity, physical injury and inflammation can promote or even cause brain calcification. In some neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), more than 20% of patients have brain calcification, and the lesions expand with age.

[0174] Primary familial brain calcification (PFBC), also known as Fahr's disease or idiopathic basal ganglion calcification (IBGC) because of the first pathological description of it by German doctor Fahr in the 1930s, is a hereditary disease characterized by progressive bilateral brain calcification, mainly distributed in the basal ganglia and / or ventral thalamus, cerebellar dentate gyrus, subcortical area and other brain regions. PFBC can serve as an ideal model for studying the pathogenesis of brain calcification and potential prevention and treatment. PFBC can cause a variety of clinical symptoms, ranging from occasional migraines to severe symptoms, including movement disorders (Parkinson's syndrome, tremor and dystonia), cognitive disorders (memory impairment, executive dysfunction symptoms and mental retardation) and neurological diseases (depression, affective disorders and psychosis), but nearly one-third of carriers of pathogenic gene mutations have brain calcification but no clinical symptoms.

[0175] Due to the high clinical heterogeneity, especially the large individual differences in clinical symptoms, the clinical diagnosis of PFBC mainly relies on the identification of morphological features of brain calcification through computed tomography (CT) imaging. The most typical feature in neuroimaging of patients with PFBC is symmetrical bilateral calcification of the basal ganglia, thalamus, frontal cortex or cerebellum. Moreover, compared with the hematological abnormalities of PTH and Pi in patients with brain calcification caused by abnormal parathyroid hormone, patients with PFBC have normal levels of calcium (Ca), phosphorus (P), alkaline phosphatase, parathyroid hormone and other serum biochemical indicators. The overall prevalence of PFBC is estimated to be 0.21%-0.66%. Since 2012, mutations in 6 genes have been associated with PFBC, including 4 autosomal dominant genes SLC20A2, PDGFRB, PDGFB and XPR1, and 2 autosomal recessive genes MYORG and JAM2. In the reported cases, the proportions of these genes are SLC20A2 (61%), PDGFB (12%), PDGFRB (5%), XPR1 (6%), MYORG (13%), and JAM2 (2%).

[0176] Gene inactivation

[0177] There are many methods for studying genes with unknown functions. The most important method is to manipulate the expression level or functional activity of the target gene, such as gene inactivation, and then observe the changes in related signal pathways and phenotypic studies. Methods for manipulating gene expression include gene knockdown, knockout and mutation knock-in. Gene inactivation can be achieved by gene knockout, gene interruption or gene insertion. The knockout strategy currently used in mice is to knock out (knockout, KO) the target gene in all cells of the body, or to rely on the Cre / loxP recombination system to drive the expression of Cre with the help of a gene promoter with expression selectivity to achieve conditional knockout (conditional knockout, cKO) of the target gene in a certain type of cell. After obtaining these KO or cKO mice of specific cell types, their molecular biochemical and behavioral phenotype changes are analyzed to study the functional information of the target gene. Gene knockout technology based on model animals or human cells is a very powerful means to study the function of human genes in the whole body. Another advantage of this research method is that it can associate gene function with disease, so that while obtaining gene function, we can also obtain information about the diseases that the gene can treat as a potential drug or drug target and animal models of the disease.

[0178] As used herein, the terms "gene inactivation" and "gene knockout" are used interchangeably and refer to a genetic operation such as interruption or knockout of a target gene, thereby significantly reducing or even completely losing the expression and / or activity of the target gene.

[0179] Adeno-associated virus

[0180] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependoviridae of the family Parvoviridae and is the simplest single-stranded DNA defective virus discovered so far. The wild-type AAV genome consists of a linear single-stranded DNA (ssDNA) of about 4.7 kb, flanked by inverted terminal repeats (ITRs), and the coding sequences of Rep and Cap proteins are included between the two ITR elements. Through the regulation of alternative promoters and splice variants at multiple locations, the Rep gene can express four different proteins that participate in viral genome replication, transcription, and integration. With the help of an important assembly activation protein AAP expressed by the open reading frame 2 (ORF2) of the Cap gene, the three structural capsid proteins VP1 / VP2 / VP3 expressed by the Cap gene are assembled into a 60-subunit icosahedral capsid at a ratio of 1:1:10. Adeno-associated virus (AAV) is considered to be an ideal delivery vector in gene therapy strategies because it is smaller than other viral vectors, has no or only weak immunogenicity, has an extremely low probability of genome integration, and can transfect both dividing and non-dividing cells.

[0181] The ITR elements of the virus contain cis-acting sequences required for genome replication and packaging. Therefore, AAV can be transformed into a viral delivery vector by removing all virally encoded gene sequences between the ITR elements and replacing them with exogenous DNA sequences encoding the gene of interest. These exogenous DNA sequences can encode conventional expression cassettes to express selected target genes in target cells. These engineered recombinant adeno-associated virus vectors (rAAV) are considered one of the most promising gene transfer vectors due to their good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long expression of exogenous genes in vivo. They are widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated viruses have been deeply understood, especially in terms of their application effects in various cells, tissues and in vivo experiments. In medical research, rAAV has been used in the study of gene therapy for a variety of diseases (including in vivo and in vitro experiments).

[0182] In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can be integrated into the genome of the cells they infect in a stable and site-specific manner, such as the AAVS1 site. The recombinant AAV genome has been sequenced, and the impact of its viral genome on the human genome has been widely studied and evaluated. A large number of animal and human cells, as well as human studies have shown that they can infect a range of cell types without significantly affecting cell growth, morphology or differentiation. These studies show that AAV is currently one of the most reliable vectors for gene delivery.

[0183] AAV vectors can be prepared using standard methods in the art. The production, purification and use of adeno-associated viruses of various serotypes have been tested in multiple laboratories and animal and human experiments. Methods for purifying vectors can be found in various technical books and biotechnology method articles, and can also be found in, for example, U.S. Patent Nos. 6566118, 6989264 and 6995006, the disclosures of which are incorporated herein by reference as a whole. The preparation of recombinant AAV vectors is described in, for example, PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference as a whole. There are existing literature and patent descriptions of embodiments of using recombinant AAV vectors to deliver target genes in vitro and in vivo (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference as a whole). These patents describe engineered AAV plasmids from various sources (strains, serotypes, mutant optimized strains, etc.) that have been removed from the original rep and / or cap genes in the AAV genome, which are used to load exogenous target genes for the production of recombinant AAV viruses and for in vitro (into cultured cells) or in vivo (directly into organisms) delivery of target genes. Based on safety considerations, AAV viruses used for therapeutic purposes must be replication-deficient, and the production of these recombinant AAVs is generally prepared by co-transfecting two plasmids into a stable cell line infected with a human helper virus (such as adenovirus): plasmid A is a plasmid loaded with a specific promoter and the target expression gene coding sequence with inverted terminal repeat sequences (ITR) at both ends, and plasmid B is a plasmid carrying the AAV genome replication gene (rep) and capsid protein coding gene (cap). In some production systems, based on higher safety requirements, the replication gene (rep) and capsid protein coding gene (cap) can also be constructed separately on different plasmids.

[0184] In some embodiments, in order to obtain better delivery and expression effects for targeted tissues and cell types, the expression vector of the recombinant AAV virus can be combined with viral capsid protein encoding plasmids of different serotypes, and these serotypes or capsid protein encoding genes are derived from different wild-type serotypes (including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV capsid proteins), or these capsid proteins can be derived coding sequences that have been modified by mutation screening or molecular biology directed evolution. Therefore, the present disclosure may use recombinant AAV viruses including any serotype described herein, or AAV serotypes with better effects developed in the future. The use of certain new serotypes may be protected by patents, but the basic methods for developing, screening and producing such viral capsid protein serotypes are known in the art and are described in published academic literature and U.S. Patent No. 6,596,535.

[0185] Expression vectors and host cells

[0186] The present invention also provides a plasmid construction and design ideas for expressing PiT2 protein in astrocytes, which includes a promoter for selectively driving transcription of a target gene in astrocytes and an open expression frame sequence encoding PiT2 protein. Through the provided sequence information, technicians can use public molecular cloning technology to construct a nucleic acid sequence or expression plasmid suitable for transduction into target cells.

[0187] Preferably, the vector contains a nucleic acid sequence encoding the PiT2 protein and is constructed in the form of an expression cassette well known to those skilled in the art, which can be used to introduce into astrocytes of the human or primate brain and express the target gene, thereby being used as a therapeutic vector. The vector can be viral or non-viral (e.g., a plasmid, or an mRNA sequence packaged in LNP). Viral vectors include viral vectors derived from the following types: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus and SV40. Preferably, the viral vector is replication defective, although it is envisioned that it may be replication deficient, capable of replication or conditional replication. Viral vectors can generally maintain an extrachromosomal state without integrating into the genome of the host cell.

[0188] The preferred viral vector for delivering the nucleic acid sequence encoding the PiT2 protein to astrocytes is an AAV vector, such as an AAV serotype that can be injected intravenously and penetrate the blood-brain barrier, such as PHP.eB, or PHP.AX, which has been proven to be effective in mice. Efficient viral delivery to the whole brain can be achieved by intravenous injection and then penetrating the blood-brain barrier; other methods, such as injection through the lymphatic system, or ventricular / spinal lumbar puncture, are still under development, some of which can achieve whole-brain delivery in mice, and may be able to achieve human brain delivery in the future. Using specific AAV serotypes (AAV2, AAV5, AAV9, etc.), or artificially modified versions of any of these serotypes (including AAV PHP.eB, AAV.CPP.16 and AAV.CPP.21 vectors), combined with cell type-specific promoters (such as gfaABC1D), selective expression of targeted cell types can be achieved. Of course, further development of viral technology, or the specific infection of astrocytes can be achieved through the modification of serotypes alone, which can reduce the dependence on selective promoters.

[0189] Viral vectors have the ability to enter cells, while non-viral vectors such as plasmids can be combined with other adjuvants to assemble virus-like or lipid vectors, which can also be taken up by target cells. Such technologies have been proven to be effective in some human tissues; in the near future, such technologies may be used for selective delivery of astrocytes throughout the brain. Such adjuvants include polycationic liposomes, lipid nanoparticles (Lipid nanoparticles, LNP), etc. The carrier used in the present invention is preferably suitable for use in vivo, and preferably suitable for use in humans.

[0190] The vector will preferably contain one or more regulatory sequences to direct the expression of the nucleic acid sequence in astrocytes. The vector can be connected to the regulatory sequence in an "operably connected" manner, which includes one or more sequence modules such as promoters, introns, enhancers, translation regulatory elements, RNA stability regulatory sequences, ligand-induced expression regulatory sequences, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites. The vector may also include a selective screening marker, for example, to support the effective assembly of the vector by the virus in a viral production system (e.g., 293 cells), or an element of inducible expression in astrocytes, or an element of negatively regulating the expression of the target gene in non-astrocytic cells.

[0191] "Operably linked" means that the nucleic acid sequence is linked to other sequence modules in a certain order or structure based on its functional role, so that they regulate the expression or function of the target gene in a spatiotemporal orderly manner according to the designed goal. For example, a nucleic acid sequence operably linked to a promoter will have a transcriptional regulatory function that is affected by the promoter or affects the activity of the promoter.

[0192] The promoter mediates the transcriptional expression of the open expression frame nucleic acid sequence encoding the target gene connected to it. The promoter can be constitutive or inducible. The promoter can guide selective expression in astrocytes, or include oligodendrocytes and their precursor cells at the same time. In the latter case, the design and screening of the promoter needs to be tested by expression of astrocytes and oligodendrocytes at the same time. A suitable promoter is the key to achieving astrocyte-specific expression in the present invention. For example, in the mouse model described in the present invention, the selective expression of astrocytes depends on gfaABC1D to drive the transcription of the PIT2 encoding gene; some studies have verified that the selectivity of the gfaABC1D promoter is conservative and available in human neural cells. Considering that single-cell research is promoting more optional promoters to be screened out, more promoters can be used in the future to achieve selective expression of astrocytes, such as promoters derived from genes such as ALDH1L1, AQP4, S100B and screened and modified.

[0193] From the perspective of technical feasibility, a variety of expression vectors can be used to achieve the expression of PiT2 protein in astrocytes (preferably primates, more preferably human astrocytes). The present invention preferably uses adeno-associated virus as an expression vector. The present invention provides a method for constructing a recombinant adeno-associated virus expression vector loaded with a PiT2 coding sequence. The method can quickly and stably construct an AAV expression vector carrying a PiT2 coding sequence, and is used to package and obtain an AAV virus preparation for treatment.

[0194] The present invention also provides a host cell for a genetically engineered cell of AAV virus particles. Preferably, the host cell is a mammalian cell (preferably a human, more preferably a human astrocyte), which increases the expression of PiT2 protein.

[0195] Gene therapy vectors

[0196] The gene therapy vector in the present invention is a viral expression vector. According to the present invention, the viral expression vector is an adeno-associated virus (AAV) vector. At the time of writing this patent, the technology of achieving a combination of serotype and promoter for whole-brain astrocyte-specific expression has been realized in mice, such as using a combination of PHP.eB or PHP.AX plus the promoter gfaABC1D; however, the effectiveness of this combination in primates or humans has not been effectively proven, and engineering transformation screening is still needed. However, it can be predicted that it may be modified from the AAV vectors in the group consisting of the serotypes AAV1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 listed below, or their derived chimeric AAVs such as AAV2-AAV3, AAVhu.14, AAV3a / 3b, AAVHSC15, AAV-HSC17, AAVhu.37, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6 (Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2 (Y-> F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45, and some serotypes derived from non-human primates, such as AAVrh.10, AAVrh32.33, AAVrh.8. The screening of more effective delivery serotypes depends on the directed evolution and screening of capsid protein coding sequences, especially based on in situ screening and testing in tissues and cell types of interest.

[0197] At present, gene therapy vectors are mainly delivered to astrocytes throughout the brain through intravenous injection. With technological advances, other injection routes may also be able to achieve efficient infection and expression of astrocytes throughout the brain, including injection through the head and neck lymphatic system, sinuses, intraventricular injection or lumbar puncture.

[0198] Formulations and compositions

[0199] The present invention provides a preparation or composition, which contains (a) a therapeutically effective amount of a brain phosphorus homeostasis promoter and (b) a pharmaceutically acceptable carrier or excipient.

[0200] As used herein, the brain phosphorus homeostasis promoter includes an astrocyte phosphorus transport promoter. In some embodiments, the brain phosphorus homeostasis promoter and the astrocyte phosphorus transport promoter can be used interchangeably.

[0201] In some embodiments, the brain phosphorus homeostasis promoter comprises:

[0202] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0203] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0204] (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof;

[0205] (Z4) Any combination of the above Z1 to Z3.

[0206] In some embodiments, the brain phosphorus homeostasis promoter can promote and / or repair the phosphorus transport function of astrocytes, thereby maintaining and / or restoring brain phosphorus homeostasis, preventing and / or treating brain calcification caused by brain phosphorus homeostasis defects.

[0207] In another preferred embodiment, the pharmaceutical preparation is used to treat hereditary brain calcification. The hereditary brain calcification is primary familial brain calcification (PFBC). Although not fully verified, the preparation of the present invention may be suitable for the treatment of other types of brain calcification caused by abnormal glial cell phosphorus transport function, such as some sporadic brain calcification cases, brain calcification caused by tumors or inflammation, and Aicardi-Goutières syndrome (AGS). This speculation needs to be supported by further research data.

[0208] For the convenience of clinical application, the pharmaceutical composition of the present invention can be contained in an injection container or a disposable injection device (such as an injection needle). The injection device can contain a single dose of the pharmaceutical composition. The injection device can be contained in a medicine box for easy storage and use. During transportation, the tiny container containing the drug suspension needs to be placed in a refrigerated condition (such as dry ice or a refrigerated transport box). It should be stored in a -80°C refrigerator for a long time, and can be stored in a -20 degree or 4 degree refrigerator for a short time.

[0209] The medicine box or test kit of the present invention may also include instructions for use, so as to help those skilled in the art understand the purpose of the preparation and the correct storage and use methods.

[0210] The "active ingredient" in the pharmaceutical composition of the present invention refers to the vector of the present invention, such as a viral vector (including an adeno-associated viral vector). The "active ingredient", preparation and / or composition of the present invention can be used to treat brain calcification. "Safe and effective amount" means: the amount of the active ingredient is sufficient to significantly improve the main clinical observation indicators (such as cerebrospinal fluid phosphate levels, other biomarkers of calcification), the progression of brain calcification or the severity of the main symptoms, without causing serious side effects. "Pharmaceutically acceptable drug carrier (carrier) or excipient (excipient)" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredient of the present invention and with each other, which can facilitate better delivery and function of the active ingredient without significantly reducing the efficacy of the active ingredient or producing side effects.

[0211] The composition can be a liquid or solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art, or will be reliable and useful as verified by clinical trials.

[0212] In the present invention, the vector can be injected intravenously, or other injection methods that have been proven to be effective, to achieve delivery and expression of astrocytes throughout the brain. In any mode of administration, preferably, the vector is provided as an injectable liquid. Preferably, the injection liquid can be injected as a concentrated solution after dilution, or provided as a ready-to-use injection solution loaded in a syringe.

[0213] Some examples of pharmaceutically acceptable drug carriers include: cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0214] The composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0215] The nucleic acid or fusion nucleic acid encoding PiT2 provided by the present invention can express PiT2 protein in vitro or in vivo, and the preparation containing the optimized PiT2 coding sequence can be used to prepare a drug for treating hereditary brain calcification.

[0216] Treatment

[0217] The present invention provides a method for preventing brain calcification from occurring in advance, or treating brain calcification, especially hereditary brain calcification. From the perspective of adaptability, this method is applicable to all groups with imbalance of phosphorus homeostasis in the brain due to defects in glial cell phosphorus transport, including patients with primary familial brain calcification (PFBC) associated with PiT2, MYORG or XPR1 gene mutations, and may also include non-PFBC types of brain calcification driven by imbalance of phosphorus homeostasis in the brain. The method includes introducing a promoter of the present invention, such as a vector containing an optimized sequence encoding PiT2, MYORG or XPR1, into astrocytes for expression. The method may include delivering a nucleic acid expression vector administered to astrocytes of the brain by intravenous injection (or other technical means), or a promoter that regulates the expression of PiT2, MYORG or XPR1 in astrocytes, such as an antisense nucleotide (ASO) drug, or a gene editing or base editing agent that can regulate the splicing of target gene mRNA.

[0218] The composition of the present invention can be administered alone or in combination with other therapeutic drugs (eg, formulated in the same pharmaceutical composition).

[0219] As used herein, treating a disease means administering a therapeutically effective amount of a promoter as described herein to improve the pathological process, or alleviate one or more symptoms of the disease, including restoring the phosphorus transport capacity of astrocytes, repairing phosphorus homeostasis imbalance in the brain, and reducing the occurrence of brain calcification.

[0220] The method of the present invention comprises introducing a nucleic acid sequence encoding PiT2, MYORG or XPR1 protein into astrocytes of the brain. Preferably, the method comprises contacting the cell with a vector (preferably a virus, more preferably an adeno-associated virus) comprising a nucleic acid sequence encoding PiT2 protein. Preferably, the cell is an astrocyte.

[0221] The present invention is based on delivering optimized nucleic acid sequences encoding PiT2, MYORG or XPR1, or promoters that can regulate PiT2, MYORG or XPR1 gene expression to astrocytes, and selectively expressing them in astrocytes to reconstruct the phosphorus transport capacity of astrocytes, repair the imbalance of phosphorus homeostasis in the brain, and ultimately curb the further progression of brain calcification in the brain and alleviate related clinical symptoms.

[0222] The main advantages of the present invention include:

[0223] The present invention first discovered that the phosphorus transport process mediated by astrocytes, including the intake of inorganic phosphate (Pi) from the extracellular interstitial fluid by the PiT2 transporter expressed on the membrane of the glial leaflets, and the discharge of the Pi taken in by glial cells to the outside of the brain through the outlet of the blood-brain barrier by the XPR1 transporter on the membrane of the endfoot structure, is an important link in the regulation of phosphorus homeostasis in the brain. Conditional knockout experiments in mouse animal models showed that after the phosphorus transport ability mediated by astrocytes was blocked compared with other types of cells, the phosphate in the cerebrospinal fluid showed the most significant increase, that is, homeostasis imbalance. At the same time, expression studies based on in situ hybridization showed that astrocytes highly expressed the phosphorus intake transporter PiT2 and the excretion transporter XPR1; while neurons highly expressed another intake transporter PiT1.

[0224] Based on the findings in item (1), by delivering viruses into the astrocytes of Pit2-KO mice to specifically express the PiT2 protein in astrocytes, the level of phosphate transport in the brain of Pit2-KO mice can be significantly reduced, and the occurrence and progression of brain calcification can be inhibited.

[0225] In terms of method, the present invention is based on the core cellular pathological mechanism that brain calcification occurs due to imbalance of phosphorus homeostasis. By selectively expressing PiT2 on astrocytes, the phosphorus transport capacity of astrocytes is rebuilt, the imbalanced phosphorus homeostasis in the brain is repaired, and the most underlying driving factor of calcification is fundamentally eliminated.

[0226] It should be noted that this method is not only for treating the progression of brain calcification, but also for repairing phosphorus homeostasis in the brain. In the patient's brain, first of all, due to genetic mutations or acquired factors, the phosphorus homeostasis in the brain is unbalanced, and then this imbalance induces the occurrence of brain calcification. Therefore, the most essential treatment of this method is to repair the imbalanced phosphorus homeostasis in the brain. In the future, when clinical experts agree that phosphorus homeostasis imbalance is a disease, just like elevated blood sugar is diabetes, or elevated blood lipids are a pathology that requires immediate treatment, the treatment strategy of this method may benefit more audiences.

[0227] The delivery strategy based on this method can be further optimized in the future with technological progress. However, the core method strategy described in this method, namely, targeting astrocytes to repair the imbalance of phosphorus homeostasis in the brain, is still protected by the claims of the present invention.

[0228] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0229] Example 1 Expression characteristics of genes related to hereditary brain calcification in mouse brain

[0230] Given the abnormal brain Pi homeostasis found in PFBC patients with mutations in the Pi transporter genes PIT2 or XPR1, we analyzed the expression patterns of the SLC20 sodium-phosphate cotransporter genes Pit1 (Slc20a1) and Pit2 (Slc20a2) and the Pi exporter gene Xpr1 in the mouse brain. Using publicly available mouse brain single-cell RNA-seq datasets, we generated cell type expression heatmaps of Pit1, Pit2, Xpr1, and the PFBC-associated glycosidase Myorg. The single-cell expression heatmaps suggested that both astrocytes and neurons showed enriched expression of Pit1, Pit2, and Xpr1, suggesting active phosphate transport in these two cell types.

[0231] To validate the expression of Pi transporters in astrocytes and neurons, we used RNAscope in situ hybridization (ISH) to investigate the expression of Pit1 and Pit2 in the adult mouse brain, focusing on the thalamus, cerebellum, striatum, and hypothalamus, regions known to harbor calcification deposits in PFBC patients. Astrocytes were labeled by immunostaining for the S100b marker protein. In the cerebellum, Bergmann cells, a specific astrocyte subtype, showed no ISH signal for Pit1 but exhibited a strong signal for Pit2. In the thalamus, the ISH signal for Pit2 was also highly expressed in all astrocytes, while only a minority (<20%) of astrocytes expressed a less intense Pit1 signal. This dominant expression of Pit2 over Pit1 in astrocytes was consistent in all four brain regions and was confirmed by double in situ hybridization with another astrocyte marker gene, Aldh1l1. In addition, Olig1-positive oligodendrocytes and oligodendrocyte precursor cells (OPCs) also express Pit2 but not Pit1.

[0232] In contrast, glutamatergic (Vglut2+) and GABAergic (Gad1+) neurons expressed strong Pit1 and relatively weak Pit2 expression. Considering that oligodendrocytes express mild levels of Xpr1, these results suggest a binary expression pattern, with high expression of Pit1 and Xpr1 in neurons and high expression of Pit2 and Xpr1 in astrocytes. Combined with the fact that 61% of reported PFBC patients have PIT2 but not PIT1 mutations, our finding of preferential expression of Pit2 in astrocytes suggests the importance of astrocyte-mediated imbalance of brain Pi homeostasis in the development of brain calcification.

[0233] Example 2 Preparation of mouse animal model

[0234] Slc20a2 tm1aWtsi Mice were mated with Actin-Flp to remove the En2a-LacZ-PGK-Neo cassette to generate Pit2flox mice, or bred with Ella-Cre to remove the PGK-Neo-flox-Exon3 cassette to generate Pit2 KO mice. Pit1 (Slc20a1) flox mice were purchased from the KOAP engineered mouse collection (T013241, GemPharmatech, China). Subsequently, they were bred with wild-type C57BL / 6 mice (SLAC Animals, China) for at least three generations. The conditional Cre mouse strains were obtained from the laboratory of Baljit S. Khakhtar and Aldh1l1-CreER. T2 , Plp1-CreER provided by Jia-Wei Zhou laboratory T2 , Pdgfrb-Cre prepared by Volkhard Lindner's laboratory, Camk2a-iCre prepared by G. Schutz's laboratory, Vgat-Ires-Cre (#016962) generated by Jax mouse laboratory. Mouse genotyping was performed using Taq polymerase (Vazyme, China). Tamoxifen (Tamoxifen, 20 mg / ml, corn oil as solvent) was intraperitoneally administered once a day for 5 consecutive days at a dose of 1 mg / 10 g body weight.

[0235] The animal care and experimental procedures were approved by the Animal Care and Use Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

[0236] Example 3

[0237] 3.1 Preparation of serum and cerebrospinal fluid and determination of Pi concentration

[0238] Blood from the orbital vein of adult mice was collected and allowed to stand at room temperature for at least 1 hour, then centrifuged (2000xg, 15 minutes) to prepare serum. Cerebrospinal fluid of anesthetized adult mice was collected from the cerebellomedullary cisterna magna, and then centrifuged for 5 minutes (2000xg, 4°C) to remove cell debris. The concentration of inorganic phosphate in serum and cerebrospinal fluid was detected using the PiColorLock free inorganic phosphate (Pi) detection kit (303-0030, InnovaBiosciences), and then the value was read at 635nm using a SpectraMax (Moleculardevice) spectrophotometer, and the Pi concentration was calculated using the function obtained by the standard curve.

[0239] 3.2 Plasmid construction, viruses and delivery

[0240] The ORF of human MYORG, PIT2 or XPR1 expression plasmids was derived from the expression plasmid of Origene. For in vivo expression, the PiT2 and XPR1 expression cassettes were inserted after the gfaABC1D promoter of the AAV2 expression construct, and the expression plasmid was combined with the plasmid of the PHP.eB serotype capsid protein for AAV virus packaging. In the intraparenchymal injection, the initial titer of about 1x10e was injected into the brain using Nano-injector (Nanoject III, Drummond Scientific Company) at a speed of 60nL / min. 13 The diluted AAV2 / 5 virus was injected into the thalamus of adult mice. To achieve whole-brain re-expression of astrocytes, each 8-week-old (50-56-day-old) mouse was injected with 100ul of 2x10e 11 Virus solution of AAV5.PH.eB capsid.

[0241] 3.3 Immunostaining, Antibodies, and Microscopy

[0242] Mouse brain sections (20 / 25 μm) fixed with 4% PFA were first perforated with 1X PBS containing 0.5% Triton X-100, then blocked with PBS containing 0.1% Triton X-100 and 5% BSA, and then incubated with the corresponding primary antibody (4°C) overnight. The next day, brain sections were washed 3 times in PBS for 10 min each time, then incubated with secondary antibodies for 2 h at room temperature, then stained with Hoechst, washed 3×10 min in PBS, and finally fixed on slides. The antibodies used were: mouse-anti-V5 (R96025, Invitrogen), Rat-anti-HA (11867423001, Roche), Mouse-anti-Calnexin (ab10286, Abcam), Chicken-anti-GFP (ab13970, Abcam), Rabbit-anti-AQP4 (AB3594, Millipore), Rabbit-anti-Col4 (ab6586, Abcam), Rabbit-anti-S100β (ab41548, ab52642, Abcam). Then, a FV3000 laser confocal microscope (Olympus) was used to capture the fluorescence expression image data on the brain slice specimens. The parameters were: three-channel fluorescence 488 / 568 / 647nm, 40× or 60× objective lens, zoom factor 3-5, z-spacing 0.4μm.

[0243] 3.4 Micro-CT imaging

[0244] Micro-CT scanning of mouse brain samples fixed with 4% paraformaldehyde was performed in a high-resolution microCT scanner (Skyscan 1172, Bruker) with the following scanning parameters: source voltage 40 kV, current 250 μA, exposure time, 210 milliseconds; rotation step, 0.6°; camera pixel size, 9 × 9 × 9 μm. The images were reconstructed using the NRecon program supported by the GPUReconServer engine and output in BMP format (pixel size, 5 μm) with the following parameters: beam hardening correction rate 33%, ring artifact reduction, 8; Gaussian smoothing kernel, 2. Then, the output BMP series images were imported into Imaris software and converted into TIF series (pixel size, 20 μm). Subsequently, the TIF image stack was imported into ImageJ software for position adjustment to output the coronal and sagittal planes of the mouse MicroCT image sections. Finally, according to the standard slice values ​​of the mouse brain atlas, the sagittal planes within the lateral range of 0.1-1.6 mm midline were superimposed (75 slices, 1.5 mm thickness) with the midline of the brain as the reference to project the sagittal images of brain calcification in this area. In addition, the coronal images of brain calcification were projected from the coronal plane covering the hindbrain (75 slices, 1.5 mm thickness) or the basal forebrain and hypothalamus (150 slices, 3 mm thickness).

[0245] 3.5 Fluorescence imaging of calcified deposits

[0246] The mouse brain after micro-CT scanning was frozen with OCT reagent and then cut into 25 μm thick brain slices using a freezing microtome. The brain slices at specific positions were selected, incubated with 0.2 μM RIS-AF647 dye (BV500101, Biovinc, USA) for 5 h, stained with Hoechst, and finally mounted on glass slides. In fluorescence mode, VS120 slice imaging system (Olympus) was used to collect calcification images of brain slices using a 20x objective. In order to quantify brain calcification, the RIS staining channel of VS120 images was batch converted into monochrome grayscale images in Tif format. Next, the number and area of ​​calcified nodules were analyzed in the four determined regions of interest (ROI) using the analyze particles of ImageJ software. Each slice was registered to the corresponding coordinate position from the midline to the lateral direction of the brain according to the sagittal brain region topological characteristics of the adult mouse brain atlas. Finally, GraphPad Prism was used to perform statistical analysis on the number and area of ​​calcified nodules in each group.

[0247] 3.6 Statistics

[0248] All data are mean ± standard deviation. Some data are indicated in the figure legends as mean ± sem. The samples between two groups were statistically analyzed by t-test between two groups, and the samples of multiple groups were analyzed by one-way analysis of variance. * indicates p < 0.01, ** indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001. For all tests, p < 0.05 (**) was considered statistically significant.

[0249] 3.7 Results

[0250] The results of the effects of PiT2 knockout in astrocytes on brain phosphorus homeostasis are shown in Figure 1 As shown. Among them, Figure A shows that the level of inorganic phosphate (Pi) in the cerebrospinal fluid (CSF) of PiT2 knockout mice increased significantly, to about 1.8mM, while the CSF Pi concentration in the brain of WT mice was about 0.5mM. Figure B shows that there was no significant change in inorganic phosphate (Pi) in the serum of PiT2 knockout mice. Figure C shows the concentration of CSF Pi in the brain of PiT2 conditional knockout mice of different cell types, among which astrocyte conditional knockout (Aldh1L1-CreER T2 ) mice showed the most significant increase in CSF Pi (170%), reaching 1.35 mM. Other conditional knockout mice, including oligodendrocytes (Plp1-CreER T2 CSF Pi in mice knocked out from the conditional knockout mice was only modestly increased (40-60%) in excitatory neurons (Camk2a-iCre), inhibitory neurons (Vgat-Cre), and pericytes (Pdgfrb-Cre). Figure D shows that there was no significant difference in serum Pi concentrations between the different conditional knockout mouse strains.

[0251] The results of intravenous injection of AAV virus-mediated selective expression of PiT2 in brain astrocytes are as follows Figure 2 Immunofluorescence showed that the PiT2-V5 signal (red) was widely expressed in astrocytes throughout the mouse brain, and blue Hoechst marked the cell nuclei.

[0252] The results of the inhibitory effect of selective compensatory expression of AAV in astrocytes on calcification in the mouse brain are as follows: Figure 3As shown in the figure. The coronal and sagittal distribution of microCT images of brain calcification in Pit2 KO mice receiving control treatment (Ctrl) at 5 and 7 months of age showed that the dorsal midbrain, basal forebrain, thalamus, and hypothalamus of the mice showed significant large-scale calcification deposition, and the calcification coverage area and calcification particles of 7-month-old mice were larger than those of 5-month-old mice. However, brain calcification of Pit2 KO mice receiving therapeutic AAV injection (Replenished) was significantly inhibited at 5 and 7 months of age.

[0253] discuss

[0254] The present invention uses in situ hybridization to analyze the expression characteristics of homologous genes of specific hereditary brain calcification-related genes, especially genes such as PIT1, PIT2, XPR1, MYORG, etc. related to phosphorus transport metabolism, in the mouse brain, and finds that astrocytes show the characteristics of selectively high expression of specific gene combinations, that is, astrocytes highly express Na-Pi co-transporters Pit2 (Slc20a2) and Xpr1, but Pit1 (Slc20a1) is not expressed in astrocytes, or only a very small number of cells express it. In addition, in mice with conditional knockout of the Pit2 gene in astrocytes, a significant increase in the level of inorganic phosphate Pi in cerebrospinal fluid occurs. Therefore, glial cells, especially astrocytes, are confirmed to be the most critical cell type for phosphorus transport metabolism closely related to brain calcification and phosphorus homeostasis regulation in the brain.

[0255] Based on the discovery of the above-mentioned cellular mechanism, the present invention adopts a method that can now achieve a large proportion of infection of specific types of cells in the whole brain, that is, the method of intravenous injection of AAV virus. After these viruses penetrate the blood-brain barrier, they can selectively express the protein product of the compensatory gene human PIT2 carried by them in the astrocytes in the brain of mice. It was further found that the mice that successfully compensated for the expression of PIT2 in the astrocytes in the brain had significantly inhibited brain calcification, indicating that the repair of phosphorus homeostasis in the brain is an effective strategy to inhibit the occurrence of brain calcification, which has a very outstanding clinical transformation value.

[0256] The present invention is based on the world's first discovery of an important physiological mechanism of astrocyte-mediated regulation of brain phosphorus homeostasis, and the discovery in a mouse model that astrocyte expression compensation can effectively reconstruct brain phosphorus homeostasis in Pit2 KO mice and effectively inhibit the occurrence and further progression of brain calcification. It can then be used as an innovative therapy for the intervention of brain calcification, bringing good news to the majority of PFBC patients.

[0257] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a brain phosphorus homeostasis promoter, characterized in that: Used for preparing medicines for preventing and / or treating brain calcification diseases.

2. The use according to claim 1, characterized in that The brain phosphorus homeostasis promoter is selected from the following group: (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof; (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof; (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof; (Z4) Any combination of the above Z1 to Z3.

3. The use according to claim 1, characterized in that The promoter includes small nucleic acid drugs.

4. The use according to claim 1, characterized in that The brain calcification diseases include primary familial brain calcification (PFBC).

5. A preparation and composition, characterized in that: The preparations and compositions contain a promoter of phosphorus homeostasis in the brain.

6. The preparation and composition according to claim 5, characterized in that: The brain phosphorus homeostasis promoter is selected from the following group: (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof; (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof; (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof; (Z4) Any combination of the above Z1 to Z3.

7. A medicine kit for treating brain calcification diseases, characterized in that: The medicine kit comprises: (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect whether a genetic mutation exists in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and (b) a second container, and a pharmaceutical composition in the second container, wherein the pharmaceutical composition comprises a pharmaceutical ingredient capable of regulating and repairing phosphorus homeostasis imbalance in the brain and a pharmaceutically acceptable carrier.

8. The medicine box according to claim 7, characterized in that: The medicine kit further comprises: (c) a third container, and a solution in the third container for assisting the dissolution or in vivo delivery of the drug in the second container.

9. A method for screening candidate compounds for preventing and / or treating brain calcification diseases, characterized in that: The method comprises the steps of: (a) In a test group, a test compound is added to the culture system of astrocytes or a cell line derived therefrom, and the phosphorus transport level (E1) and / or activity (A1) in the cells of the test group is observed; in a control group, no test compound is added to the culture system of the same cells, and the phosphorus transport level (E0) and / or activity (A0) in the cells of the control group is observed; Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test compound has a promoting effect on the expression and / or activity of the phosphorus transport level and is a candidate compound that can potentially be used to prevent and / or treat brain calcification diseases.

10. The method according to claim 9, characterized in that The method comprises step (b): administering the candidate compound determined in step (a) to a mammal model to determine its effect on the mammal.

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