Treatment based on phosphate homeostasis-promoting agents in the brain

Abstract

This invention relates to the use of cerebral phosphate homeostasis promoters in the field of biomedicine, specifically in cerebral calcification diseases, including reagents that can modulate alternative splicing in the introns of the SLC20A2 gene or reagents that target astrocytes. Furthermore, this invention provides reagents that can modulate alternative splicing in the introns of the SLC20A2 gene and their use. Moreover, this invention provides a cerebral calcification model based on alternative splicing in the introns of the human SLC20A2 gene, a method for producing the same, and its use.

Description

TECHNICAL FIELD

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the priority of Chinese Patent Application No. 202310890680.8 filed on July 19, 2023 and Chinese Patent Application No. 202311370896.8 filed on October 20, 2023, and the entire contents of these patent applications are hereby incorporated by reference in their entirety into the present specification.

[0002] The present invention relates to the field of biomedicine, and specifically, to the use of a cerebral phosphorus homeostasis promoter, which comprises a reagent capable of regulating alternative splicing in the intron of the SLC20A2 gene or a reagent targeting astrocytes, in cerebral calcification diseases. The present invention also provides a reagent capable of regulating alternative splicing in the intron of the SLC20A2 gene and use thereof. Furthermore, the present invention provides a cerebral calcification model based on alternative splicing in the intron of the human SLC20A2 gene, a method for preparing the same, and use thereof.

BACKGROUND ART

[0003] Cerebral calcification is a common disease in clinical imaging that is associated with age and aging. In cranial CT observations of elderly people aged 60 and above, more than 20% present with punctate calcification, and 2-4% or more are accompanied by large-sized patchy calcification. Some neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are complicated by cerebral calcification in more than 20% of patients, and the lesions expand with age. Therefore, cerebral calcification is a neurodegenerative disease that should be emphasized in clinical practice and requires treatment.

[0004] Hereditary cerebral calcification disorders are neurodegenerative diseases caused by gene mutations, and include many types, such as primary familial brain calcification (PFBC) and Aicardi-Goutieres syndrome (AGS). Among these, PFBC is a relatively representative type, with an incidence of approximately 6 in 1000. The pathological features of PFBC are bilateral symmetrical calcifications commonly found in the basal ganglia, thalamus, and cerebellar dentate nucleus regions, although calcifications may occasionally be found in the subcortical regions of some patients. As the disease progresses, the calcified foci may merge and enlarge. High-resolution brain histopathological imaging studies have found that calcium salt granules are mainly deposited in the brain spaces surrounding terminal arterioles, veins, and capillaries. The clinical symptoms of hereditary cerebral calcification vary widely, mainly including motor impairments, mental disorders, cognitive impairments, epileptic seizures, dizziness, and headaches. In severe cases, personality and behavioral changes occur, eventually leading to mental illness or dementia. Some patients develop physical disabilities and become unable to live independently, while others show no clear symptoms.

[0005] Hereditary cerebral calcification disorders are sporadic (often due to de novo mutations) or familial and exhibit a high degree of genetic heterogeneity. In the last decade or so, pathogenic genes for PFBC discovered in human genetics research include SLC20A2, XPR1, PDGFB, PDGFRB, MYORG, and JAM2. These genes suggest that the pathogenesis of cerebral calcification manifests itself in two main ways. One is that damage to the integrity of the blood-brain barrier (BBB) ​​structure or a decrease in pericyte coverage increases BBB permeability, and gene mutations in PDGFRB, PDGFB, and JAM2 may be present in patients associated with this pathway, all of which are expressed in cells that make up the neurovascular unit.

[0006] The second pathway involves impaired transport of inorganic phosphate (Pi) ions in the cerebrum and the resulting disruption of brain phosphate homeostasis (BPH), typically resulting in a marked increase in Pi levels in cerebrospinal fluid (CSF). Patients exhibiting this pathway may have mutations in the SLC20A2, MYORG, or XPR1 genes, the two genes encoding the Na-Pi cotransporter PiT2 protein and the phosphate efflux transporter protein XPR1 and its regulator MYORG, respectively. Furthermore, statistical analysis of clinical calcification and symptoms shows that patients with Pi transport-related gene mutations have larger calcification foci and more severe clinical symptoms compared to patients with gene mutations associated with the BBB pathway. Of all reported cases of familial calcification, SLC20A2 mutations account for 61% of all genetic elements, XPR1 mutations account for 6%, and MYORG mutations account for 13%.

[0007] Currently, clinical management of PFBC primarily focuses on treating symptoms, and there are no effective drugs that can remove or reduce abnormal calcium deposits. In particular, understanding the mechanisms of BPH disruption may offer opportunities for the development of appropriate clinical treatments.

[0008] Therefore, in this field, there is a strong need for the development of novel strategies for addressing cerebral calcification that are convenient, effective, and aimed at restoring phosphorus homeostasis. [Overview of the project] [Problems that the invention aims to solve]

[0009] In a first aspect, the present invention provides the use of cerebral phosphate homeostasis promoters in the manufacture of drugs for preventing and / or treating cerebral calcification diseases. [Means for solving the problem]

[0010] In some embodiments, the brain phosphate homeostasis enhancer is a reagent capable of modulating alternative splicing in the introns of the SLC20A2 gene. In some embodiments, the alternative splicing includes cryptic exon inclusion, and the reagent modulates (e.g., represses or enhances) the splicing of the cryptic exon from the precursor mRNA of the SLC20A2 gene. In some embodiments, the reagent thereby modulates (e.g., represses or enhances) the level of mature mRNA processed from the precursor mRNA, and / or modulates (e.g., represses or enhances) the expression of the PiT2 protein.

[0011] In some embodiments, the alternative splicing includes alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3. In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene. In some embodiments, the hidden exon has the sequence shown in SEQ ID NO: 2.

[0012] In some embodiments, the alternative splicing includes alternative splicing in intron 10. In some embodiments, the alternative splicing in intron 10 is the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene. In some embodiments, the hidden exon has the sequence shown in SEQ ID NO: 3.

[0013] In some embodiments, the reagent specifically binds to a target region in the SLC20A2 gene or its precursor mRNA, the target region being located in an intron region between two typical exon regions, and the intron region containing the hidden exon. In some embodiments, the target region is located within a region from approximately 100 nucleotides upstream of the hidden exon to approximately 100 nucleotides downstream of the hidden exon. In some embodiments, the target region is selected from a region spanning the splice site of the hidden exon, a region within the hidden exon, a region within 100 nucleotides upstream of the hidden exon, or a region within 100 nucleotides downstream of the hidden exon. In some embodiments, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD).

[0014] In some embodiments, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA. In some embodiments, the target region is located within the sequence shown in SEQ ID NO: 8.

[0015] In some embodiments, the target region is located within the c.1795-1803 to c.1795-1527 region of the SLC20A2 gene or its precursor mRNA. In some embodiments, the target region is located within the sequence indicated by SEQ ID NO: 9.

[0016] In some embodiments, the reagent is selected from antisense oligonucleotides (ASOs) or gene editing tools (e.g., CRISPR / Cas, base editors, or Cas protein-independent RNA editing tools). In some embodiments, the reagent is an antisense oligonucleotide (ASO). In some embodiments, the reagent is a DNA editor, e.g., CRISPR / Cas9. In some embodiments, the reagent is an RNA editor, e.g., CRISPR / Cas13. In some embodiments, the reagent is an adenine / cytosine base editor. In some embodiments, the reagent is a Cas protein-independent RNA editing tool.

[0017] In some embodiments, the reagent is an antisense oligonucleotide (ASO) that specifically binds to the target region in the precursor mRNA of SLC20A2. In some embodiments, the ASO consists of 10 to 30 (e.g., 15 to 30, 15 to 25, 18 to 30, 18 to 25, 20 to 25) consecutive nucleotides, preferably 18 to 25 consecutive nucleotides. In some embodiments, the ASO includes modifications to the main chain nucleotide and nucleotide linkage, ribose modification, ribose side chain base modification, or a combination thereof.

[0018] In some embodiments, the ASO specifically binds to a target region located within the c.289+801~c.289+1135 region of the SLC20A2 precursor mRNA. In some embodiments, the ASO has at least 80% complementarity (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) of the nucleotide sequence in SEQ ID NO: 8. In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 13-46, 63-65 or a sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) thereto.

[0019] In some embodiments, the ASO specifically binds to a target region located within the c.1795-1803 to c.1795-1527 region of the SLC20A2 precursor mRNA. In some embodiments, the ASO has at least 80% complementarity (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) of the nucleotide sequence in SEQ ID NO: 9. In some embodiments, the ASO includes the nucleotide sequence represented by any of SEQ ID NOs: 47-61, 66-68, or a sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) thereto.

[0020] In some embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool. In some embodiments, the gene editing tool is a CRISPR / Cas system comprising a Cas protein and a guiding RNA (gRNA), the gRNA being capable of binding to the target region in the SLC20A2 gene or its precursor mRNA. In some embodiments, the gRNA binds to the target region and guides the binding of the editor complex to exert editing activity. In some embodiments, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 precursor mRNA, or within the region c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA. In some embodiments, the gene editing tool disrupts the function of a splicing acceptor (SA) or splicing donor (SD) sequence of a hidden exon in the genome or precursor mRNA sequence by mutating the SA or SD sequence, thereby suppressing the integration of the hidden exon into the mRNA. In some embodiments, the gene editing tool is a DNA base editor comprising (i) a fusion protein containing a DNA base editing domain (e.g., deaminase) and a DNA binding domain (e.g., a Cas protein, e.g., Cas9 or Cas12), and (ii) a guiding RNA (gRNA) that can bind to the target region in the SLC20A2 gene. In some embodiments, the gene editing tool is an RNA base editing tool. In some embodiments, the RNA base editing tool includes a CRISPR / Cas system-based RNA base editor, or a CRISPR / Cas system-independent editor, such as an oligonucleotide-based base editor.

[0021] In some embodiments, the brain phosphate homeostasis promoter is an astrocyte phosphate transport promoter.

[0022] In some embodiments, the aforementioned cerebral phosphorus homeostasis promoter is selected from the following group: (Z1) PiT2 protein, a nucleic acid molecule encoding the PiT2 protein, an expression vector expressing the PiT2 protein, a PiT2 promoter, or a combination thereof; (Z2) MYORG protein, a nucleic acid molecule encoding the MYORG protein, an expression vector expressing the MYORG protein, a MYORG promoter, or a combination thereof; (Z3) XRP1 protein, a nucleic acid molecule encoding the XRP1 protein, an expression vector expressing the XRP1 protein, a XRP1 promoter, or a combination thereof; (Z4) Any combination of Z1 to Z3 described above.

[0023] In some embodiments, the aforementioned PiT2 protein comprises the amino acid sequence set forth in SEQ ID NO: 155. In some embodiments, the aforementioned MYORG protein comprises the amino acid sequence set forth in SEQ ID NO: 154. In some embodiments, the aforementioned XRP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 153.

[0024] In some embodiments, the promoters described in Z1 to Z3 include small molecule compounds, gene editing reagents, small nucleic acid-based drugs (for example, antisense nucleotide-based drugs), or combinations thereof.

[0025] In some embodiments, the astrocyte phosphate transport promoter is an expression vector comprising an exogenous gene and an enhancer element selectively expressed by astrocytes and / or a promoter selectively expressed by astrocytes, the exogenous gene being selected from nucleic acid molecules encoding PiT2 protein, MYORG protein, or XRP1 protein, or a combination thereof. In some embodiments, the exogenous gene is a nucleic acid molecule encoding PiT2 protein. In some embodiments, the expression vector is a viral vector, such as AAV. In some embodiments, the promoter selectively expressed by astrocytes is gfaABC1D. In some embodiments, the gfaABC1D promoter comprises the sequence shown in SEQ ID NO: 156.

[0026] In some embodiments, the astrocyte phosphate transport promoter is a small nucleic acid drug, the small nucleic acid drug is linked to a targeting molecule, and the targeting molecule is a target for astrocytes. In some embodiments, the small nucleic acid drug is an ASO. In some embodiments, the small nucleic acid drug is packaged entirely or partially within the targeting molecule, or is compounded with the targeting molecule. In some embodiments, the targeting molecule is selected from an antibody or antibody fragment, a polysaccharide, an aptamer, or nanoparticles.

[0027] In some embodiments, the cerebral calcification disorders include primary familial cerebral calcification (PFBC), primary basal ganglia calcification (PBGC), or other types of cerebral calcification disorders associated with cerebral phosphate homeostasis defects (e.g., upregulation of inorganic phosphate ions in cerebrospinal fluid). The other types of cerebral calcification disorders may include other types of cerebral calcification disorders due to abnormalities in glial cell phosphate transport function, such as certain cases of sporadic cerebral calcification, cerebral calcification due to tumors or inflammation, and Aicardi-Goutieres syndrome (AGS).

[0028] In a second aspect, the present invention provides a method for preventing and / or treating a cerebral calcification disorder, comprising the step of administering an effective amount of a cerebral phosphate homeostasis enhancer to a subject of interest. In some embodiments, the cerebral phosphate homeostasis enhancer is as defined in the first aspect above. In some embodiments, the cerebral calcification disorder includes primary basal ganglia calcification (PBGC), primary familial cerebral calcification (PFBC), or other types of cerebral calcification disorders associated with cerebral phosphate homeostasis defects (e.g., upregulation of inorganic phosphate ions in cerebrospinal fluid). The other types of cerebral calcification disorders may include other types of cerebral calcification disorders due to abnormalities in glial cell phosphate transport function, such as certain cases of sporadic cerebral calcification, cerebral calcification due to tumors or inflammation, and Aicardi-Goutieres syndrome (AGS).

[0029] In a third aspect, the present invention provides a drug kit for treating cerebral calcification disease, comprising the following: (a) A first container, and a first detection reagent for detecting gene mutations in the first container, which is a reagent for detecting the presence or absence of gene mutations in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and (b) A second container, and a drug composition contained in the second container comprising an astrocyte phosphate transport promoter as defined in the first aspect and a pharmaceutically acceptable carrier and / or excipient.

[0030] In some embodiments, optionally, the drug kit may further include a third container and a solution in the third container that assists in the dissolution or delivery of the drug in the second container into the body.

[0031] In a fourth aspect, the present invention provides a method for screening candidate drugs for the prevention and / or treatment of cerebral calcification disease, comprising the following steps: (a) In the test group, the test drug was added to a culture system of astrocytes or a derived cell line, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group was measured. In the control group, the test drug was not added to a similar cell culture system, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group was measured. If the cellular phosphorus transport levels (E1) and / or activity (A1) in the test group are significantly higher than in the control group, it indicates that the test compound has a promoting effect on the expression and / or activity of phosphorus transport levels and is a candidate drug for preventing and / or treating cerebral calcification disease.

[0032] In a fifth aspect, the present invention provides the use of a reagent capable of modulating alternative splicing in the introns of the SLC20A2 gene in the manufacture of a drug, wherein the drug is for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject. Furthermore, the present invention provides a method for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject, comprising the step of administering an effective amount of a reagent capable of modulating alternative splicing in the introns of the SLC20A2 gene to the required subject.

[0033] In some embodiments, reagents capable of modulating alternative splicing in the introns of the SLC20A2 gene are as defined in the first aspect.

[0034] In some embodiments, the PiT2 dysfunction relates to a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. In some embodiments, the mutation is located in an exon region, and the subject is heterozygous with respect to the mutation in the exon region. In some embodiments, the mutation is located in a target region in an intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region. In some embodiments, the target region is located within the region c.289+801~c.289+1135 of the SLC20A2 precursor mRNA, or within the region c.1795-1803~c.1795-1527 of the SLC20A2 precursor mRNA. In some embodiments, the PiT2 dysfunction is haploinsufficiency of SLC20A2. In some embodiments, regulation of functional PiT2 protein or RNA expression is beneficial for diseases associated with the PiT2 dysfunction.

[0035] In some embodiments, the disease is cerebral calcification, such as primary familial cerebral calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0036] In some embodiments, the disease is a disease associated with an abnormality in the Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis.

[0037] In a sixth aspect, the present invention relates to a reagent that can specifically bind to a target region in the SLC20A2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, and the intron region contains a hidden exon.

[0038] In some embodiments, the hidden exon is located within intron 2, preferably at c.289+901 to c.289+1035 of the SLC20A2 gene or its precursor mRNA, and preferably the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA.

[0039] In some embodiments, the hidden exon is located within an intron 10, preferably at c.1795-1703 to c.1795-1627 of the SLC20A2 gene or its precursor mRNA, and preferably the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA.

[0040] In some embodiments, the target region is as defined in the first aspect.

[0041] In some embodiments, the reagent is an ASO as defined in the first aspect.

[0042] In the seventh aspect, the present invention provides a drug composition comprising a reagent or combination thereof that can specifically bind to a target region in the SLC20A2 gene or its precursor mRNA as described in the sixth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0043] In the eighth aspect, the present invention provides the use of a reagent that can specifically bind to a target region in the SLC20A2 gene or its precursor mRNA as described in the sixth aspect, or a drug composition as described in the seventh aspect, wherein the drug is for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject. Furthermore, the present invention provides a method for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject, comprising the step of administering to a subject in need a reagent that can specifically bind to a target region in the SLC20A2 gene or its precursor mRNA as described in the sixth aspect, or a drug composition as described in the seventh aspect.

[0044] In some embodiments, the disease associated with the PiT2 dysfunction is cerebral calcification, such as primary familial cerebral calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0045] In some embodiments, the diseases associated with the PiT2 dysfunction are diseases associated with Pi imbalance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis.

[0046] In some embodiments, the drug treats the disease by regulating the expression of PiT2 protein or functional RNA in the subject's cells.

[0047] In some embodiments, the subject includes a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. In some embodiments, the mutation is located in an exon region, and the subject is heterozygous with respect to the mutation in the exon region. In some embodiments, the mutation is located in the target region of an intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region. In some embodiments, the target region is located within the region c.289+801~c.289+1135 of the SLC20A2 precursor mRNA, or within the region c.1795-1803~c.1795-1527 of the SLC20A2 precursor mRNA. In some embodiments, the PiT2 functional defect is a haploinsufficiency of SLC20A2.

[0048] In the ninth aspect, the present invention provides a method for regulating the expression of the PiT2 protein in cells, comprising the step of contacting the cells with a reagent capable of regulating alternative splicing in the introns of the SLC20A2 gene in vitro. In some embodiments, the reagent capable of regulating alternative splicing in the introns of the SLC20A2 gene is as defined in the first aspect.

[0049] In the ninth aspect, the present invention provides a method for regulating the expression of the PiT2 protein in cells, comprising the step of contacting the cells in vitro with a reagent described in the sixth aspect. In some embodiments, the cells contain a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations or structural polymorphisms, preferably the mutation is located in an exon region and the cells are heterozygous with respect to the mutation in the exon region, preferably the mutation is located in a target region in an intron and the cells are heterozygous, homozygous or compound heterozygous with respect to the mutation in the intron located in the target region.

[0050] In its tenth aspect, the present invention provides a method for identifying a reagent capable of regulating the expression of PiT2 in cells, comprising: i) identifying a reagent that targets a target region in the PiT2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, the intron region contains a hidden exon, and the target region is located within a region from approximately 100 nucleotides upstream of the hidden exon to approximately 100 nucleotides downstream of the hidden exon; ii) delivering the reagent identified in step i) to the cells; and iii) measuring the expression levels of PiT2 mRNA and protein in the cells of step ii).

[0051] In some embodiments, the method further includes the step of comparing the expression level measured in step iv) iii) with the expression level of PiT2 in cells treated with a control.

[0052] In some embodiments, the expression level is measured by RT-PCR, qPCR, or Western blotting.

[0053] In some embodiments, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD).

[0054] In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene, preferably the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA, preferably the target region is located within the sequence indicated by Sequence ID No. 8.

[0055] In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene, preferably the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA, preferably the target region is located within the sequence indicated by SEQ ID NO: 9.

[0056] In its eleventh aspect, the present invention provides a method for producing a cerebral calcification model, comprising the steps of modifying a target genome such that the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced in whole or in part with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced in whole or in part with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene, thereby providing a cerebral calcification model.

[0057] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon.

[0058] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0059] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2 or intron 10 as defined in the first aspect.

[0060] In some embodiments, the replacement method includes gene knockout, gene editing, RNA interference, epigenetic repression, or any combination thereof.

[0061] In some embodiments, the model is an animal model, such as a mammal model, such as a rodent model (e.g., a mouse or rat model), or a non-human primate model.

[0062] In some embodiments, the model is a mammalian cell model, such as human epithelial fibroblasts or glial cells.

[0063] In its twelfth aspect, the present invention provides a genetically modified non-human animal whose genome is such that the intron sequence between exons 2 and 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exons 10 and 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene.

[0064] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2 or intron 10 as defined in the first aspect.

[0065] In some embodiments, the genomic fragment of the human SLC20A2 gene intron substituted between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10.

[0066] In some embodiments, the genomic fragment of the intron of the human SLC20A2 gene that is substituted between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0067] In some embodiments, the non-human animal is a rodent, such as a mouse or a rat.

[0068] In some embodiments, the non-human animal is a non-human primate.

[0069] In some embodiments, the genetically modified non-human animal serves as an animal model for cerebral calcification.

[0070] In a thirteenth aspect, the present invention provides genetically modified cells whose genomes include those in which the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or in which the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene contains a mutation that can alter the splicing of the SLC20A2 gene.

[0071] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon.

[0072] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0073] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2 or intron 10 as defined in the first aspect.

[0074] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene is introduced exogenously into the cells.

[0075] In some embodiments, the cells are mammalian cells.

[0076] In some embodiments, the cells are human epithelial fibroblasts or glial cells.

[0077] In some embodiments, the genetically modified cells serve as a cellular model for brain calcification.

[0078] In a fourteenth aspect, the present invention includes the use of genetically modified non-human animals or cells, which (i) are used as models to present or analyze the pathogenesis of cerebral calcification disease or to study the disruption of cerebral phosphate homeostasis or targets of cerebral calcification disease; (ii) are used as models to screen candidate drugs or treatment plans for treating cerebral calcification disease or diseases associated with disruption of cerebral phosphate homeostasis; and / or (iii) are used as models to screen candidate drugs or treatment plans for diseases associated with disruption of phosphate homeostasis. Here, the genetically modified non-human animal or cell genome includes a case in which the intron sequence between exons 2 and 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or a case in which the intron sequence between exons 10 and 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene.

[0079] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon.

[0080] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the genomic fragment of the intron of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0081] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2 or intron 10 as defined in the first aspect.

[0082] In the fifteenth aspect, the present invention provides a nucleic acid construct comprising a minigene fragment sequence of the human SLC20A2 gene, the minigene fragment sequence comprising exon 2 and exon 3 and an intron sequence between them, or exon 10 and exon 11 and an intron sequence between them, wherein the intron sequence comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0083] In some embodiments, the nucleic acid construct includes a promoter operably linked to the minigene fragment sequence.

[0084] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2 or intron 10 as defined in the first aspect.

[0085] In some embodiments, the minigene fragment sequence includes the sequence shown in SEQ ID NO: 4 or 5.

[0086] In some embodiments, a polyA signal sequence is included downstream of the minigene fragment sequence.

[0087] In some embodiments, the nucleic acid construct further includes a reporter gene, such as a fluorescent protein (e.g., GFP, mCherry) or luciferase.

[0088] In the sixteenth aspect, the present invention provides a method for screening reagents capable of modulating SLC20A2 mRNA splicing, comprising the steps of: (i) providing cells comprising the nucleic acid construct described in the fifteenth aspect; (ii) providing a genetically modified non-human animal described in the twelfth aspect or a genetically modified cell described in the thirteenth aspect; or (iii) providing a brain calcification model obtained by the method described in the eleventh aspect; contacting a test reagent with any of (i)-(iii); and detecting alternative splicing of SLC20A2 mRNA.

[0089] In some embodiments, the cells are derived from, for example, mammals, such as rodents (e.g., mice or rats), non-human primates, or humans.

[0090] In some embodiments, a test reagent is selected that can suppress alternative splicing (for example, by reducing the amount of expression product containing hidden exon incorporation), thereby increasing the production of functional PiT2 protein.

[0091] In some embodiments, a test reagent is selected that can enhance alternative splicing (for example, by increasing the amount of expression product containing hidden exon incorporation), thereby reducing the production of functional PiT2 protein.

[0092] In some embodiments, the detection includes analyzing the length of the mRNA expression product of SLC20A2.

[0093] In some embodiments, the alternative splicing is an alternative splicing in intron 2 as defined in the first aspect, preferably the detection involves performing a PCR analysis using primers that span exon 2 and exon 3, preferably generating an amplicon containing a hidden exon incorporation of about 135 nt in length. In some embodiments, the alternative splicing is an alternative splicing in the intron 10 as defined in the first aspect, preferably the detection involves performing a PCR analysis using primers that span exon 10 and exon 11, preferably generating an amplicon containing a hidden exon incorporation of about 76 nt in length. In some embodiments, the method includes a step of contacting a test reagent with cells containing the nucleic acid construct in vitro to detect alternative splicing of the nucleic acid construct, preferably the detection includes analyzing the length of the mRNA expression product of the nucleic acid construct or analyzing the expression of a reporter gene contained in the nucleic acid construct. [Brief explanation of the drawing]

[0094] [Figure 1]Mutations in the SLC20A2 intron detected in the PFBC family by WGS. a. Kinship of six PFBC family members with SLC20A2 intron mutations. Solid, hollow, gray, or slashed symbols represent individuals affected by cerebral calcification, individuals without cerebral calcification, individuals without symptoms, and individuals who have died, respectively. Symbols with question marks (?) represent individuals for which brain CT imaging data is unavailable. "#" and "&" represent individuals selected for whole exome sequencing (WES) or whole genome sequencing (WGS), respectively. b. Representative brain CT images of patients in the six PFBC family. c. Distribution of mutations in five introns at the SLC20A2 locus. Light purple frames represent exons, and the numbers below them are the exon numbers. Lines within exons indicate introns. In the display of intron mutations, italicized bold indicates mutations that are not expected to result in new splice acceptors or donors, suggesting they exert their effect by affecting latent hidden exons, while italicized regular mutations are mutations that may result in splice acceptors or donors. [Figure 2]Hidden exons in SLC20A2 alter normal splicing of SLC20A2 mRNA and PiT2 protein expression through intron mutations. ab. Schematic diagrams of the construction and transcripts of the minigenes with intron mutations in SLC20A2, c. 289+937 G>A and c. 289+1007 C>G (a). (b) shows the alterations in the splicing product after intron mutations detected by PCR of the minigenes. In RT-PCR analysis performed on samples of Neuro-2A cells transfected with SLC20A2 minigenes having c. 289+937 G>A, c. 289+1007 C>G and c. 289 +1021 G>C mutations, the lower band represents the exon of accurate splicing, and the upper band represents the PCR product of the transcript with a cryptic exon inserted between exons 2 and 3 of SLC20A2. Schematic diagram of a SLC20A2 minigene construct containing human SLC20A2 intron 10 and c.1795-1698 A>G mutations (c) and PCR detection analysis of the transcript (d). The lower band represents the product of the transcript with accurate splicing, and the upper band represents the PCR product of the transcript with a cryptic exon inserted between exons 10 and 11 of SLC20A2. e. Schematic diagram of an SLC20A2 CDS construct with abnormal splicing due to mutations in two introns (both with one HA tag at the N-terminus). f. After transfusion of Neuro-2A cells with HA-labeled SLC20A2 CDS constructs, the effect on the PiT2 protein product of the endogenous SLC20A2 mutation was detected by Western blotting (WB). Immunoblotting results for the SLC20A2 gene product PiT2 in fibroblast extracts from PFBC patients with gh.c.289+1007 C>G mutation (P1) and SLC20A2 gene hetero-deletion (P2) or healthy controls (C1 and C2) showed that intron mutations lead to a decrease in PiT2 expression similar to exon mutations, (h) is the statistical result. [Figure 3]Hidden exons crpE2 and crpE10 are present in SLC20A2 transcripts generated from human brain-derived cells. a. The presence or absence of hidden exons between adjacent exons in the mRNA of the SLC20A2 gene was analyzed by RT-PCR in HEK293T and A172 cells. CHX, cyclohexylamine (a widely used compound that can suppress the degradation of abnormal transcripts). Generally, the bottom band represents a normal transcript, and the top band represents an abnormal transcript containing a hidden exon (denoted by an asterisk and named crpE2 and crpE10). b. Sanger sequencing of the top bands of the hidden exons (crpE2 and crpE10) in HEK293T and A172 cells in Figure a revealed that their sequences are similar to those of the hidden exons found in the mini-gene experiment in Figure 2, indicating that these hidden exons are endogenous and that mutations only significantly increase the probability of their integration into the mRNA. Human cell lines including c.HEK293T, HeLa, HepG2, and A172, and RT-PCR products of crpE2 and crpE10 from human fibroblasts derived from healthy control groups (C1, C2); one PFBC patient with an SLC20A2 heterozygous c.289+1007 C>G mutation (P1); and one PFBC patient with an SLC20A2 heterozygous deletion (P2). [Figure 4]ASO suppresses the integration of hidden exons of SLC20A2 in the minigene. a. Schematic diagram of hidden exons with the c.289 +1007 C>G mutation in the SLC20A2 genome sequence in the minigene, compared to PMO-modified ASOs (PMO-1, PMO-2, PMO-3) and MOE-modified ASOs (MOE-A, MOE-B, MOE-C). b, c. In Neuro-2A cells transfected with the SLC20A2 c.289+1007 C>G minigene, PMO-modified ASOs (PMO-1, PMO-2, PMO-3) (b) or MOE-modified ASOs (MOE-A, MOE-B, MOE-C) (c) were involved in the integration of crpE2 hidden exons after RNA extraction from treated cells, and RT-PCR analysis and statistical results were obtained for the integration status of crpE2 hidden exons. The upper band represents the non-productive transcript crpE2 (729 bp), and the lower band represents the normal transcript (594 bp). d. Schematic diagram of hidden exons with mutations at c.1795-1698 in the SLC20A2 genome sequence in minigenes, representing PMO-modified ASOs (PMO-7, PMO-8, PMO-9) and MOE-modified ASOs (MOE-G, MOE-H, MOE-I). e, f. RT-PCR analysis and statistical results for the incorporation status of crpE10 hidden exon after RNA extraction from treated cells with either PMO-modified ASOs (PMO-7, PMO-8, PMO-9) (e) or MOE-modified ASOs (MOE-G, MOE-H, MOE-I) (f), compared to control ASOs (PMO-NC and MOE-NC). The upper band represents transcripts with crpE10 insertion (261 bp), and the lower band represents normal transcripts (185 bp). The red line represents MOE-modified ASOs, and the blue line represents PMO-modified ASOs. The quantified percentage of splicing events involving hidden exons was calculated from the intensity of the RT-PCR bands, specifically as follows: (transcript with hidden exons / (transcript with hidden exons + normal transcript)) × 100. Relative PiT2 protein was evaluated using tubulin as an internal reference (i.e., PiT2 / tubulin).*: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, Student's t-test. [Figure 5] ASO upregulates PiT2 protein expression by suppressing the incorporation of hidden exons in SLC20A2 mRNA due to intron mutations. a. Schematic diagram showing that after culturing fibroblasts from PFBC patients (P1) with the SLC20A2 c.289 +1007 C>G intron mutation, MOE-modified ASOs (MOE-A, MOE-B, MOE-C) can target hidden exons in precursor mRNA transcribed from the SLC20A2 gene in both chromosomes with the c.289 +1007 C>G intron mutation and normal chromosomes without the mutation, further suppressing abnormal splicing of SLC20A2. b. Stable cultured SLC20A2 c. Fibroblasts from PFBC patients (P1) with the 289+1007 C>G intron mutation were treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or control MOE-NC), RNA was extracted, and RT-PCR detection and statistical analysis were performed on mRNA levels of SLC20A2 incorporating the crpE2 hidden exon. c. Western blotting results and statistical analysis of PiT2 protein levels in fibroblasts induced from P1 after involvement of MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. The red line represents MOE-modified ASOs. The quantified value (%) of splicing events incorporating hidden exons was calculated from the intensity of the RT-PCR bands, specifically as follows: (transcript incorporating hidden exon / (transcript incorporating hidden exon + normal transcript)) × 100. Relative PiT2 protein was evaluated using tubulin as an internal reference (i.e., PiT2 / tubulin). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, Student's t-test. [Figure 6]ASOs can also increase PiT2 expression by modulating the splicing event involving the hidden exon of SLC20A2 in normal human fibroblasts. a. Schematic diagram showing improved expression of SLC20A2 mRNA and PiT2 protein in precursor mRNA transcribed from the SLC20A2 gene on normal chromosomes after culturing human fibroblasts derived from healthy controls, by modulating the splicing event of the hidden exon crpE2 or crpE10 with MOE-modified ASOs (crpE2 target: MOE-A, MOE-B, MOE-C; crpE10 target: MOE-J, MOE-K, MOE-L). b. RT-PCR detection and statistical analysis of expression levels of SLC20A2 mRNA transcripts with the inserted crpE2 hidden exon after treating human fibroblasts derived from healthy controls (C1) with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or MOE-NC in the control group). c. Western blotting detection and statistical analysis of PiT2 protein levels after treating healthy control (C1) human fibroblasts with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. d. RT-PCR detection and statistical analysis of expression levels of SLC20A2 mRNA transcripts with inserted crpE2 hidden exon after treating healthy control (C2) human fibroblasts with MOE-modified (MOE-A, MOE-B, MOE-C, or MOE-NC in the control group). The upper band is a transcript with inserted crpE2 (699 bp), and the lower band is a normal transcript (564 bp). e. Western blotting detection and statistical analysis of PiT2 protein levels in healthy control (C2) human fibroblasts after treatment with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. f. RT-PCR detection and statistical analysis of expression levels of SLC20A2 mRNA transcripts with inserted crpE10 hidden exon after treatment with healthy control (C1) human fibroblasts after treatment with MOE-modified (MOE-G, MOE-H, MOE-I) compared to MOE-NC.The upper band represents the transcript with crpE10 insertion (199 bp), and the lower band represents the normal transcript (123 bp). Compared to g.MOE-NC, the levels of PiT2 protein in human fibroblasts from healthy control C1 after involvement of MOE modification ASOs (MOE-G, MOE-H, MOE-I). The quantified value (%) of the splicing event with hidden exon incorporation was calculated from the intensity of the RT-PCR bands, specifically as follows: (transcript with hidden exon incorporation / (transcript with hidden exon incorporation + normal transcript)) × 100. Relative PiT2 protein was evaluated using tubulin as an internal reference (i.e., PiT2 / tubulin). *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001, Student's t-test. [Figure 7]This is an expanded screening of ASO sequences that regulate SLC20A2 splicing. a. Minigenes are transfused into N2A cells, and the response of the splicing product of the minigene expression to ASOs is analyzed by PCR to identify new ASO sequences that target crpE2 splicing regulation (cr5E2-m1, cr5E2-5, cr5E2-6, cr5E2-8, 5crE2-9, cr5E2-10, cr5E2-12, 5crE2-13, cr5E2-13L, cr5E2-13S, cr5E2-14, cr5E2-16, cr5E2-17, cr5E2-18, cr5E2-19, cr5E2-20, cr5E2-21, cr5E2-22, cr5E This includes sequences 2-23, cr5E2-24, cr3E2-5, cr3E2-8, cr3E2-9, cr3E2-10, cr3E2-11, cr3E2-12, cr3E2-13, cr3E2-14, cr3E2-15, cr3E2-16), and novel ASO sequences targeting crpE10 splicing regulation (cr5E10-9, cr5E10-11, cr5E10-12, cr5E10-13, cr5E10-15, cr5E10-16, cr5E10-19, cr3E10-9, cr3E10-14, cr3E10-15), and these sequences and related information are shown in the appendix. b. Further screening of ASO sequences screened with minigenes in healthy human fibroblasts. The regulatory effect of ASO on the splicing of endogenously expressed SLC20A2 was analyzed and evaluated by multiple PCR and WB detection of the protein. Only experimental results for some sequences are shown above. c. After a comprehensive analysis of two screenings, several ASO sequences with good effects were obtained. [Figure 8]ASO modulates SLC20A2 mRNA splicing in patients with SLC20A2 heterozygous deletion, thereby upregulating PiT2 protein expression. a. Schematic diagram showing how SLC20A2 mRNA and PiT2 protein expression can be improved by targeting the splicing event of the hidden exon crpE2 in precursor mRNA transcribed from the SLC20A2 gene on another normal chromosome using MOE-modified ASOs (MOE-A, MOE-B, MOE-C) after culturing fibroblasts from patients with SLC20A2 heterozygous deletion (P2), treating them with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) or control MOE-NC, extracting RNA, and performing RT-PCR detection and statistical analysis of SLC20A2 mRNA levels incorporating the crpE2 hidden exon. c. Western blotting results and statistical analysis of PiT2 protein levels in P2 fibroblasts after involvement of MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. The red line represents MOE-modified ASOs. The quantified value (%) of splicing events with hidden exons was calculated from the intensity of the RT-PCR band, specifically as follows: (transcript with hidden exons / (transcript with hidden exons + normal transcript)) × 100. Relative PiT2 protein was evaluated using tubulin as an internal reference (i.e., PiT2 / tubulin). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, Student's t-test. [Figure 9]This study describes the construction of a humanized SLC20A2 intron knock-in (SLC20A2-KI) model mouse and the analysis of SLC20A2 expression. a. Schematic diagram of the construction of the SLC20A2-KI mouse model of intron 2 of the humanized SLC20A2 gene. Using CRISPR / Cas9, an intron 2 region of the SLC20A2 genome was created, and homologous recombination-based repair was guided by the introduced target donor sequence. The sequences of intron 2 and the bridging regions with exons 2 and 3 on both sides of mouse SLC20A2 were then replaced with the sequence of intron 2 of human SLC20A2 with the 289+1007 C>G mutation. Finally, the resulting transcript matched the reading frame of the entire mouse Slc20A2 gene. b. Expression levels of SLC20A2 transcriptions with humanized crpE2 incorporated were analyzed in the brain tissue of heterozygous and homozygous SLC20A2-KI mice and wild-type mice by RT-PCR. The upper band represents the abnormal transcript (crpE2) due to the humanized intron 2 of the c.289+1007 C>G mutation, and the lower band represents the SLC20A2 transcript corresponding to the normal one. WT: wild-type; HET: heterozygous; HOM: homozygous; Ctx: cerebral cortex; Hip: hippocampus; Tha: thalamus. c, d. qPCR analysis of SLC20A2 transcription levels in the cerebrum of WT, HET, and HOM SLC20A2-KI mice (c) and Western blotting results for PiT2 levels (d) both indicate that the humanized mutant mice have expression defects of Slc20a2 mRNA and PiT2 protein similar to Slc20a2 KO mice. e. Compared to wild-type mice, heterozygous and homozygous SLC20A2-KI mice exhibit a stepwise increase in CSF Pi levels, similar to Slc20a2 KO mice. [Figure 10]This study analyzes brain calcification in humanized SLC20A2 (SLC20A2-KI) model mice using micro-CT and RIS staining. a. Micro-CT scans (voxel resolution: 9 μm × 9 μm × 9 μm) of the cerebrum after perfusion fixation with PFA in homozygous SLC20A2-KI mouse models at 5, 7, and 9.5 months of age. From left to right are micro-CT images of the dorsal coronal plane of the midbrain, the basal coronal plane of the forebrain, and the sagittal plane of one hemiceral region, respectively. White dots represent calcification nodules. b. Images of brain calcification obtained by VS120 microscopy after staining brain sections of homozygous SLC20A2-KI mice at 5, 7, and 9.5 months of age with the fluorescent dye RIS-AF647 (an imaging reagent with an AlexaFluo-647 nm fluorescent group that can bind to calcium diphosphonate salts). The images represent the analysis results for three mice from each group. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, Student's t-test. Scale: 1 mm. [Figure 11] This study analyzes histochemical staining of brain calcifications in humanized SLC20A2 (SLC20A2-KI) model mice. Homozygous SLC20A2-KI mice underwent OCT frozen sections of their cerebral tissue at 7 months (a), 10 months (b), and 12 months (c) of age. These sections were then histochemically stained with five different stains: alizarin red, fonkossa, Alcian blue, H&E, and PAS. Alizarin red and fonkossa primarily indicated calcification deposition, while Alcian blue and PAS primarily indicated mucopolysaccharide components attached to calcified foci, a common characteristic of calcified deposits. Scale: 500 μm. [Figure 12]The involvement of ASO in the ventricles repairs SLC20A2 expression in the brains of SLC20A2-KI mice, thereby downregulating cerebrospinal fluid phosphate ion levels. a. Experimental design plan for intraventricular (ICV) injection of ASO into homozygous SLC20A2-KI mice. Mice were administered ICV with MOE-NC, MOE-A, or MOE-B at 3 months of age for a treatment period of 4 months. The dosage of each MOE was 50 μg, 100 μg, 200 μg, or 400 μg. Brain tissue was collected from 7-month-old mice and analyzed for cryptic exon inclusion of SLC20A2 transcripts, PiT2 protein levels, cerebrospinal fluid phosphate ion levels, and the status of brain calcification. b. At 5 months of age, 2 months after ICV injection, the distribution of MOE-A throughout the mouse cerebrum (cortex, hippocampus, thalamus, and cerebellum) was analyzed by in-situ hybridization in SLC20A2-KI mice. The RNA probe was designed to specifically bind to the MOE-A sequence. MOE-A was stained red, and DAPI was stained blue. c. 4 months after administration of MOE-NC, MOE-A, or MOE-B, the expression status of normal SLC20A2 mRNA transcripts was analyzed by fluorescence quantitative PCR compared to age-appropriate WT mice and uninvolved homozygous SLC20A2-KI mice. d. RT-PCR was used to analyze the status of transcripts incorporating the hidden exon crpE2 and normal SLC20A2 transcripts 4 months after injection of MOE-NC, MOE-A, and MOE-B via ICV. The top band represents a transcript of mRNA incorporating the hidden exon crpE2 (crpE2), and the bottom band represents a normal SLC20A2 transcript. e. Four months after injection of MOE-A via ICV into SLC20A2-KI mice, the level of PiT2 protein in the thalamus showed clear restoration of expression compared to control MOE and untreated mice, demonstrating the effectiveness of ASO. f. In addition to target gene expression, biomarkers directly related to cerebral calcification, namely phosphate ion levels in cerebrospinal fluid, also decreased significantly, further demonstrating the effectiveness of ASO. [Figure 13]The involvement of ASO in the ventricles significantly suppresses the development and progression of intracranial calcification in the brains of SLC20A2-KI mice. a. Sagittal view of the brain from micro-CT imaging of brain calcification 4 months after injection of MOE-NC, MOE-A, and MOE-B at different doses (50, 100, 200, 400 μg) by ICV into 3-month-old SLC20A2-KI mice. White dots represent nodules of brain calcification. b. Quantification of the number and area of ​​calcification nodules distributed in the thalamus, hypothalamus, midbrain, and basal prebrain 4 months after injection of MOE-NC, MOE-A, and MOE-B at different doses (50, 100, 200, 400 μg) by ICV into 3-month-old SLC20A2-KI mice, i.e., at 7 months of age. Images are representative of the analysis of 2-3 mice from each group. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, Student's t-test. Scale: 1 mm. [Figure 14] ICV injection of ASO drugs suppresses further deterioration of cerebral calcification in SLC20A2-KI mice that already exhibit cerebral calcification. a. Experimental design of ICV injection of PMO-modified ASO in homozygous SLC20A2-KI mice. Mice already exhibited obvious calcification deposition in multiple regions of the whole brain at 5 months of age, at which point they received ICV injection of PMO-NC or PMO-1 for a duration of 2 months. The dose of each MOE was 25 μg or 75 μg. Brain tissue was collected from 7-month-old mice and analyzed for the content of hidden exons and the status of cerebral calcification. CSF was extracted and analyzed before PMO administration and at the end of administration (i.e., 5 and 7 months). b. Changes in CSF Pi levels before and after administration of PMO-NC and PMO-1 in homozygous SLC20A2-KI mice. c. Sagittal views of micro-CT imaging of cerebral calcification in 7-month-old homozygous SLC20A2-KI mice after intervention with ICV injections of control and therapeutic ASO doses of 25 μg and 75 μg. White dots represent nodules of cerebral calcification. Images are representative of the analysis of 2-3 mice from each group. ****: P<0.0001, Student's t-test. Scale: 1 mm. [Figure 15]This shows the effect of whole or conditional knockout of Pit2 (Slc20a2) on brain phosphate homeostasis. A shows that the mean level of inorganic phosphate ions (Pi) in cerebrospinal fluid (CSF) of whole PiT2 knockout (Pit2 KO) mice was ~1.8 mM, significantly higher than the Pi concentration in CSF in WT mice (~0.5 mM). B shows that there was no significant change in inorganic phosphate ions (Pi) in the serum of whole PiT2 knockout mice. C shows the changes in Pi concentration in brain CSF of Pit2 conditional knockout (Pit2 cKO) mice of different cell types, where the increase in Pi in CSF is most pronounced (approximately 170% increase) in astrocyte conditional knockout mice (achieved by induction with tamoxifen using Aldh1L1-CreERT2), with a mean level of approximately 1.35 mM. Changes in CSF Pi in other conditional knockout mice, including oligodendrocyte (Plp1-CreERT2), excitatory neuron (Camk2a-iCre), inhibitory neuron (Vgat-Cre), and perivascular cell (Pdgfrb-Cre) mice, were moderate (the increase was between 40-60%), suggesting that astrocyte-mediated Pi transport is most important for maintaining brain phosphate homeostasis. D indicates that there were no significant differences in serum Pi concentrations among different conditional knockout mouse breeds. [Figure 16] Intravenous injection of AAV virus mediates the selective expression of PiT2 in astrocytes in the brain. A shows the serotype of AAV (PHP.eB), the structure of the expression vector, and a schematic diagram of intravenous injection in mice. B shows a sagittal brain section of a mouse, fixed with PFA, one month after intravenous injection. Immunofluorescence staining shows broad expression of the PiT2-V5 signal (red) in astrocytes throughout the mouse brain, with blue Hoechsts representing cell nuclei. [Figure 17]The inhibitory effect of selective compensatory expression of AAV on intracranial calcification in mice. Micro-CT scans of the mouse cerebrum after perfusion showed that Pit2 KO mice treated with a control virus (expressing only GFP) (control group) exhibited clear distribution of multiple calcification foci in both the coronal and sagittal planes of intracranial calcification at 5 and 7 months of age. Among these, significant large-area calcification deposition was observed in the dorsal midbrain, basal forebrain, thalamus, and hypothalamus. At 7 months of age, mice showed a larger calcification coverage area and larger calcification granules than at 5 months of age. However, Pit2 KO mice that successfully received therapeutic AAV injection (replacement group) showed significant suppression of cerebral calcification at 5 and 7 months of age. [Modes for carrying out the invention]

[0095] I. Definition In this invention, unless otherwise specified, scientific and technical nouns used herein have their ordinary meanings as understood by those skilled in the art. At the same time, to better understand this invention, definitions and interpretations of related terms are provided below.

[0096] As used herein, the term “SLC20A2” refers to the gene encoding solute carrier family 20 member 2 (see, for example, the human gene NCBI ID: 6575), also known as PiT2, and the protein product it encodes is a 652-amino acid phosphate transporter, called the PiT2 protein or PiT-2 protein (see, for example, NP_006740). The mRNA sequence of SLC20A2 is well known to those skilled in the art, see, for example, NCBI: NM_006749, NM_001257181, or NM_001257180. In this specification, when describing the location of the mRNA sequence of the SLC20A2 gene, for example, to describe the location of a hidden exon, refer to the sequence shown in NCBI: NM_006749. An exemplary amino acid sequence of the PiT2 protein is shown in SEQ ID NO: 155.

[0097] As used herein, the term "XPR1" refers to the gene encoding the phosphate efflux protein XPR1. The sequences of the XPR1 gene (e.g., Gene ID: 9213) and the XPR1 protein (e.g., NP_004727.2) are both known to those skilled in the art, and refer to public databases, e.g., HGNC:12827. An exemplary amino acid sequence of the XPR1 protein is shown in SEQ ID NO: 153.

[0098] As used herein, the term "MYORG" refers to the gene encoding the MYORG regulator of the phosphate efflux protein XPR1. The sequences of the MYORG gene (e.g., Gene ID: 57462) and the MYORG protein (e.g., NP_065753.2) are both known to those skilled in the art, and refer to public databases, e.g., HGNC:19918. An exemplary amino acid sequence of the MYORG protein is shown in SEQ ID NO: 154.

[0099] As used herein, the expression “modulation of splicing” refers to any change affecting the level of any RNA or mRNA variant by the native precursor mRNA of SLC20A2. For example, modulation may mean causing an increase or decrease in the mRNA level of an abnormal SLC20A2, including, for example, the incorporation of a hidden exon; causing an increase or decrease in the mRNA level of normal full-length SLC20A2; and / or causing an increase or decrease in the RNA or mRNA level of an abnormal SLC20A2, including an immature stop codon (nonsense codon). Of course, any change in the ratio between SLC20A2 splicing variants is also considered a result of splicing modulation. Each possibility represents a single embodiment of the present invention. In some embodiments, modulation means increasing the mRNA level of normal full-length SLC20A2 and / or decreasing the mRNA level of abnormal (e.g., including the incorporation of a hidden exon) SLC20A2.

[0100] As used herein, the term “complementary” or “complementarity” refers to the ability of nucleic acids, such as oligonucleotides and polynucleotides, to form base pairs with one another. Base pairing is typically formed by hydrogen bonds between nucleotide units in parallel, opposite-direction polynucleotide chains. Complementary polynucleotide chains can be base-matched in the Watson-Crick scheme (e.g., A to T, A to U, C to G) or in any other way that can result in a double-stranded structure. As is known to those skilled in the art, when using RNA rather than DNA, the base complementary to adenosine is said to be uracil rather than thymine. However, where U is indicated in the preceding and following sentences herein, unless otherwise specified, it implies the ability to substitute for T. “At least partially complementary” means two fully complementary or mismatched base pairs of six, five, four, three, two, or one or fewer, that simultaneously retain the ability to hybridize under the relevant conditions. Those skilled in the art can determine the optimal conditions for testing the complementarity of two sequences by the final application of the hybridized nucleotides. Such conditions may include, for example, strict conditions such as 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing in an in vitro test system. Other conditions may also include, for example, physiologically relevant conditions that may be encountered in vivo.

[0101] As used herein, unless otherwise specified, the left end of a single-stranded nucleic acid sequence (e.g., precursor mRNA transcript, oligonucleotide, ASO, etc.) is called the 5' end, and the left direction of a single-stranded or double-stranded nucleic acid sequence is called the 5' direction. Similarly, the right end or right direction of a nucleic acid sequence (single-stranded or double-stranded) is called the 3' end or 3' direction. Generally, the region or sequence at 5' of a reference point in a nucleic acid is called "upstream," and the region or sequence at 3' of a reference point in a nucleic acid is called "downstream." Generally, the 5' direction or end of mRNA is the location of the initiation codon / start codon, while the 3' end or direction is the location of the stop codon. In some aspects, nucleotides upstream of a reference point in a nucleic acid can be represented by negative numbers, while nucleotides downstream of a reference point can be represented by positive numbers. For example, a reference point (e.g., an exon-exon junction in mRNA) can be represented as the "zero" position, and the nucleotide directly adjacent to the reference point upstream can be represented as "minus one," for example, "-1," while the nucleotide directly adjacent to the reference point downstream can be represented as "plus one," for example, "+1."

[0102] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is known in the art. A “pharmaceutically acceptable carrier and / or excipient” does not exert a therapeutic effect at expected doses, nor is it intended to exert a therapeutic effect. Such ingredients may perform the following functions: a) assist in the processing of the drug delivery system during the manufacturing process; b) ensure, support, or enhance the stability, bioavailability, or patient acceptability of the active ingredient; c) assist in product identification; and / or d) enhance the effect of any other attributes of the active ingredient, such as overall safety, efficacy, or delivery, during storage and use. For example, the siRNA of the present invention can be packaged with a delivery carrier. “pharmaceutically acceptable carriers and / or excipients” include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining reagents, absorption-delaying reagents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal reagents, such as p-hydroxybenzoate, trichloro-t-butanol, phenol, and sorbic acid. Reagents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Reagents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyhydric alcohols (e.g., glycerin).

[0103] As used herein, the term “treatment” means a method performed to obtain a beneficial or necessary clinical outcome. For the purposes of the present invention, beneficial or necessary clinical outcomes include, but are not limited to, symptom relief, reduction of disease extent, stabilization of disease state (i.e., no further deterioration), delay or mitigation of disease development, improvement or mitigation of disease state, and symptom relief (whether partial or in whole), whether detectable or undetectable. “Treatment” also means extending survival time compared to expected survival time (if no treatment is performed).

[0104] As used herein, the term “effective dose” means an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, an effective dose for treating a disease is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who is already afflicted with the disease, for example, an amount necessary to improve at least one clinical parameter of the disease in the patient or to reduce the severity of at least one clinical parameter of the disease in the patient. All measurements of such effective doses are within the scope of the skills of those skilled in the art. For example, an effective dose for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient’s immune system, the patient’s general condition, such as age, weight and sex, the means of administering the drug, and any other treatments administered concurrently.

[0105] II. Therapies to modulate alternative splicing In some aspects, the present invention relates to the regulation of alternative splicing and its use in the treatment of diseases, the manufacture of disease models, and the like.

[0106] Genetic studies have revealed that PFBC may be caused by autosomal heterozygous mutations in SLC20A2, PDGFRB, PDGFB, or XPR1, or autosomal biallelic mutations in MYORG, JAM2, or CMPK2. In particular, dominant heterozygous mutations in the SLC20A2 gene account for 61% of already reported PFBC cases, and it has been shown that PFBC can be treated with gene therapy targeting the corresponding gene. However, a lack of understanding of the mechanisms of PFBC development and a lack of effective delivery tools have significantly hindered the development of gene editing therapy for PFBC.

[0107] The inventors of this application performed whole-genome sequencing (WGS) on 135 PFBC patients who were confirmed to be exon mutation- or CNV-negative by whole-exome sequencing (WES). They discovered five intronic mutations in the SLC20A2 gene from six autosomal dominant PFBC families. Minigene detection and patient-derived cell experiments confirmed the presence of hidden exon sequences in the intronic sequences of SLC20A2 precursor mRNA, and that these intronic mutations increase the probability of incorporation of these hidden exon sequences during mRNA processing. Because these hidden exon sequences contain immature stop codons, the mRNA sequences with abnormal splicing cannot be translated to produce the functional protein product PiT2. Thus, the inventors of this application discovered immature termination of PiT2 translation due to the incorporation of previously unreported hidden exons in abnormal splicing of SLC20A2 mRNA, and this mechanism provides a novel strategy for regulating SLC20A2 gene mRNA splicing and expression.

[0108] Based on the above findings, the inventors of this application were able to enhance the expression of normal SLC20A2 mRNA and PiT2 protein in cells containing SLC20A2 intron mutations and fibroblasts derived from patients with SLC20A2 haploinsufficiency by regulating splicing mediated by ASO. Furthermore, in a humanized SLC20A2 mouse model mimicking PFBC patients with SLC20A2 deficiency, intraventricular (ICV) administration of ASO reduced abnormal SLC20A2 transcripts and suppressed the development and progression of cerebral calcification.

[0109] As a result, the inventors of this application have provided a therapy that suppresses cerebral calcification by reducing the integration of hidden exons in introns by regulating the alternative splicing process of SLC20A2, and have provided a promising therapeutic means for diseases associated with PiT2 dysfunction (e.g., PFBC due to SLC20A2 haploinsufficiency).

[0110] Furthermore, based on the alternative splicing discovered in the SLC20A2 intron, the inventors of the present application further provide cell and animal models of cerebral calcification and methods for producing them, which can be used to present and analyze the onset stages of cerebral calcification disease, as models for studying targets of cerebral phospholipid disruption or cerebral calcification disease, and for drug screening.

[0111] II-A. Use of modulated alternative splicing in disease treatment In some respects, the present invention relates to reagents, methods, and uses for restoring or enhancing the expression and function of the SLC20A2 gene protein product PiT2 by regulating the splicing of the precursor mRNA of the SLC20A2 gene. One aspect of the present invention provides the use of a reagent capable of modulating alternative splicing in the introns of the SLC20A2 gene in the manufacture of a drug for preventing and / or treating a disease associated with PiT2 dysfunction in a subject, or for preventing and / or treating a disease associated with PiT2 dysfunction in a subject. The present invention also provides a method for preventing and / or treating a disease associated with PiT2 dysfunction in a subject, comprising the step of administering to the required subject an effective amount of a reagent capable of modulating alternative splicing in the introns of the SLC20A2 gene. The PiT2 dysfunction includes a deficiency in expression level or activity.

[0112] In some embodiments, the alternative splicing includes cryptographic exon inclusion, and the reagent modulates (e.g., represses or enhances) the splicing of the cryptographic exon from the precursor mRNA of the SLC20A2 gene. In some embodiments, the reagent modulates (e.g., represses or enhances) the level of mature mRNA processed from the precursor mRNA, and / or modulates (e.g., represses or enhances) the expression of the PiT2 protein.

[0113] In some embodiments, the reagent inhibits the incorporation of the hidden exons in mature SLC20A2 mRNA, increasing the level of accurately spliced ​​mature SLC20A2 mRNA, decreasing the level of abnormally spliced ​​mature SLC20A2 mRNA, and / or increasing the production of functional PiT2 protein. In this specification, the expression "increases the level of accurately spliced ​​mature SLC20A2 mRNA" means that the level of accurately spliced ​​mature SLC20A2 mRNA after treatment with the reagent provided by the present invention is increased compared to the level before treatment or after a control treatment.

[0114] In some embodiments, the reagent promotes the incorporation of the hidden exon in mature SLC20A2 mRNA, thereby suppressing the level of accurately spliced ​​mature SLC20A2 mRNA, where the hidden exon sequence contains a premature termination codon (PTC), and the incorporation of the hidden exon leads to a decrease or suppression of functional PiT2 protein. In this specification, the expression "reducing the level of abnormally spliced ​​mature SLC20A2 mRNA" means a decrease in the level of abnormally spliced ​​mature SLC20A2 mRNA after treatment with the reagent provided by the Invention, compared to the level before treatment or after a control treatment. Abnormally spliced ​​mature SLC20A2 mRNA includes the incorporation of a hidden exon between exons 2 and 3 or between exons 10 and 11 as defined herein.

[0115] Alternative splicing in intron 2 In some embodiments, the alternative splicing includes alternative splicing in intron 2.

[0116] In some embodiments, alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3.

[0117] In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0118] In some embodiments, the c.289+1007 C>G mutation in the SLC20A2 gene promotes alternative splicing in intron 2.

[0119] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0120] In some embodiments, the reagent inhibits alternative splicing in intron 2. As used herein, "inhibits alternative splicing in intron 2" means that a 135 nt hidden exon sequence (SEQ ID NO: 2) found in intron 2 of the SLC20A2 gene reduces the incidence of its addition to mature SLC20A2 mRNA.

[0121] Alternative splicing in intron 10 In some embodiments, the alternative splicing includes alternative splicing in the intron 10.

[0122] In some embodiments, alternative splicing in intron 2 is the incorporation of a hidden exon between exons 10 and 11.

[0123] In some embodiments, the hidden exons are located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0124] In some embodiments, the c.1795-1698 A>G mutation in the SLC20A2 gene promotes alternative splicing in intron 10.

[0125] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3.

[0126] In some embodiments, the reagent inhibits alternative splicing in intron 10. As used herein, "inhibits alternative splicing in intron 10" means that a 76nt hidden exon sequence (SEQ ID NO: 3) found in intron 10 of the SLC20A2 gene reduces the incidence of its addition to mature SLC20A2 mRNA.

[0127] In some embodiments, the alternative splicing includes alternative splicing in intron 2 or alternative splicing in intron 10. In some embodiments, the reagent is a reagent that inhibits alternative splicing in intron 2 or a reagent that inhibits alternative splicing in intron 10.

[0128] target area In some embodiments, the reagent specifically binds to a target region in the SLC20A2 gene or its precursor mRNA, the target region being located in an intron region between two typical exon regions, and the intron region containing the hidden exon.

[0129] In some embodiments, the target region consists of approximately 100 or fewer nucleotides upstream of the hidden exon (for example, 90 or fewer, approximately 80 or fewer, approximately 70 or fewer, approximately 60 or fewer, approximately 50 or fewer, approximately 40 or fewer, approximately 30 or fewer, approximately 20 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) It is located within the region from that point to the region of approximately 100 or fewer nucleotides downstream of the aforementioned hidden exon (for example, approximately 90 or fewer, approximately 80 or fewer, approximately 70 or fewer, approximately 60 or fewer, approximately 50 or fewer, approximately 40 or fewer, approximately 30 or fewer, approximately 20 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer). In some embodiments, the target region is selected from a region that straddles the splice site of the hidden exon, a region within the hidden exon, a region within 100 nucleotides upstream of the hidden exon, or a region within 100 nucleotides downstream of the hidden exon.

[0130] In some embodiments, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD).

[0131] In some embodiments, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA.

[0132] In some embodiments, the target region is located within the sequence shown in Sequence ID 8.

[0133] In some embodiments, the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA.

[0134] In some embodiments, the target region is located within the sequence indicated by sequence number 9.

[0135] In some embodiments, the target region includes a region that spans the 5' splice site (splicing acceptor site, SA) of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 25 nucleotides upstream of the 5' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) to approximately 25 nucleotides downstream of the 5' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) of the 5' splicing site of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 20 nucleotides upstream of the 5' splicing site of the hidden exon to approximately 20 nucleotides downstream of the 5' splicing site of the hidden exon.

[0136] In some embodiments, the target region includes a region that spans the 3' splice site (splicing donor site, SD) of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 25 nucleotides upstream of the 3' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) to approximately 25 nucleotides downstream of the 3' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) of the 3' splicing site of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 20 nucleotides upstream of the 3' splicing site of the hidden exon to approximately 20 nucleotides downstream of the 3' splicing site of the hidden exon.

[0137] In some embodiments, the target region is located within the hidden exon. In some embodiments, the target region or its periphery includes splicing adjustment elements, such as splicing enhancer elements or splicing silencer elements.

[0138] In some embodiments, the target region is located within an intron region upstream or downstream of the hidden exon. In some embodiments, the target region or its periphery includes splicing modulating elements, such as a splicing branch site, a splicing enhancer element, or a splicing silencer element.

[0139] reagent In some embodiments, the reagent is an antisense oligonucleotide (ASO). In some embodiments, the reagent is a gene editing tool, such as a DNA or RNA editing system, such as CRISPR / Cas, a base editor, or a Cas protein-independent RNA editing tool. In some embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In some embodiments, the reagent is an RNA editor, such as CRISPR / Cas13. In some embodiments, the reagent is an adenine / cytosine base editor. In some embodiments, the reagent is a CRISPR / Cas system-independent RNA editing tool.

[0140] ASO In some embodiments, the reagent is an antisense oligonucleotide polyreoside (ASO) that specifically binds to the target region in the precursor mRNA of SLC20A2, and the ASO specifically binds to the target region according to the principle of base complementarity and further exerts the function of regulating the splicing of the precursor mRNA through steric hindrance.

[0141] In some embodiments, the ASO consists of 10 to 30 consecutive nucleotides (for example, 15 to 30, 15 to 25, 18 to 30, 18 to 25, or 20 to 25), preferably consisting of 18 to 25 consecutive nucleotides.

[0142] In this specification, the term ASO includes any form of oligo molecule containing nucleic acid bases that can hybridize complementaryally with the nucleic acid in the target mRNA. ASO may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of both or three of the above. The term "naturally occurring nucleotide" includes deoxyliponucleotides and liponucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar groups and / or modified backbone. Chemical modifications of ASO are readily conceivable to those skilled in the art.

[0143] In some embodiments, the ASO includes modifications of the main chain nucleotide and nucleotide linkage, ribose modification, ribose side chain base modification, or a combination thereof. In some embodiments, the main chain modification is selected from thiophosphate ester linkage or phosphorodiamidate linkage. In some embodiments, the sugar moiety modification includes, but is not limited to, 2'-O-methoxyethyl (2'-MOE), morpholino, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), constrained ethyl (cEt), loc nucleic acid (LNA), and peptide nucleic acid.

[0144] In some embodiments, each monomer of ASO is modified in a similar manner, for example, each linkage in the main chain of ASO contains a thiophosphate ester linkage, or each ribose moiety contains a 2'-O-methyl modification. Such modifications present in any of the monomer components of ASO are called “homogeneous modifications.” In some embodiments, a combination of different modifications may be required, for example, ASO may contain a combination of a phosphorodiamidate linkage and a sugar moiety containing a morpholine ring (morpholino). A combination of different modifications to ASO is called “mixed modifications” or “mixed chemistries.”

[0145] In some embodiments, the ASO includes a 2'-MOE modification. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification. In some embodiments, the modification of the ASO includes a combination of 2'-MOE and loc nucleic acid (LNA). In some embodiments, the ASO has a thiophosphate ester linkage backbone.

[0146] In some embodiments, the ASO includes a 2'-OMe modification. In some embodiments, each nucleotide of the ASO has a 2'-OMe modification. In some embodiments, the modification of the ASO includes a combination of 2'-OMe and loc nucleic acid (LNA). In some embodiments, the ASO has a thiophosphate ester linkage backbone.

[0147] In some embodiments, the ASO comprises a combination of a phosphorodiamidate linkage and a sugar moiety containing a morpholine ring (morpholino), such as a phosphorodiamidate morpholino oligomer (PMO).

[0148] In some embodiments, the ASO specifically binds to a target region located within the c.289+801~c.289+1135 region of the SLC20A2 precursor mRNA.

[0149] In some embodiments, the ASO has at least 80% complementarity with the nucleotide sequence in SEQ ID NO: 8. Herein, complementarity (the degree of complementarity between one polynucleotide and another) can be quantified by the ratio (e.g., percentage) of bases that form hydrogen bonds as expected by universally accepted base-pairing rules in a chain. The sequence of the ASO does not necessarily have to be 100% complementary to the sequence of the target nucleic acid with which it hybridizes. In some embodiments, the ASO may have sequence complementarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with respect to the target region in the target nucleic acid sequence it targets. For example, if 18 of the 20 nucleic acid bases of the oligo compound complement the target region, the ASO specifically hybridized thereby exhibits 90% complementarity.

[0150] In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 13-46, 63-65, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto. In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 19-22, 25, 26, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto.

[0151] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 13-46 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto, and comprises a 2'-MOE modification and / or a thiophosphate ester linkage. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification and a thiophosphate ester linkage backbone.

[0152] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 63-65 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith, and the ASO is a PMO.

[0153] In some embodiments, the ASO specifically binds to a target region located within the c.1795-1803~c.1795-1527 region of the SLC20A2 precursor mRNA.

[0154] In some embodiments, the ASO has at least 80% complementarity (for example, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) with the nucleotide sequence in SEQ ID NO: 9.

[0155] In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 47-61, 66-68, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith. In some embodiments, the ASO includes a nucleotide sequence represented by SEQ ID NO: 50, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith.

[0156] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 47-61 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto, and comprises a 2'-MOE modification and / or a thiophosphate ester linkage. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification and a thiophosphate ester linkage backbone.

[0157] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 66-68 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith, and the ASO is a PMO.

[0158] Gene editing tools In some embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool, which typically includes (i) a module having base editing activity and (ii) a module capable of binding to the target region in the SLC20A2 gene. The gene editing tool disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of a hidden exon in the genome or precursor mRNA sequence by mutating the SA or SD sequence and suppressing the integration of the hidden exon into mRNA.

[0159] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0160] CRISPR / Cas system In some embodiments, the reagent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), the gRNA being capable of binding to the target region in the SLC20A2 gene or its precursor mRNA.

[0161] In some embodiments, the gRNA binds to a target region and guides the binding of the Cas enzyme to exert editing activity.

[0162] In some embodiments, the CRISPR system disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon by mutating it, thereby suppressing the integration of the hidden exon into mRNA.

[0163] In some embodiments, the target region is located within the c.289+801~c.289+1135 region of the SLC20A2 precursor mRNA.

[0164] In some embodiments, the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA.

[0165] DNA base editor In some embodiments, the reagent is a DNA base editor. The DNA base editor typically comprises (i) a fusion protein containing a DNA base editing domain (e.g., deaminase) and a DNA binding domain (e.g., a Cas protein, e.g., Cas9 or Cas12), and (ii) a guiding RNA (gRNA) that can bind to the target region in the SLC20A2 gene.

[0166] In some embodiments, the gRNA binds to a target region and guides the binding of the fusion protein, thereby exerting base-editing activity.

[0167] In some embodiments, the DNA base editor disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon by causing a base mutation, thereby suppressing the incorporation of the hidden exon into mRNA.

[0168] In some embodiments, the Cas protein is a catalytically inactive Cas protein or Cas nickase protein.

[0169] In some embodiments, the base editor includes an ABE (Adenine base editor) or a CBE (Cytosine base editor), or a novel editor obtained by molecular evolution / mutation screening.

[0170] RNA editing tools In some embodiments, the reagent is an RNA editing tool. RNA editing tools include CRISPR / Cas system-based RNA base editors, CRISPR / Cas system-independent editors, such as oligonucleotide-based base editors.

[0171] In some embodiments, the CRISPR / Cas system-based RNA base editor comprises (i) a fusion protein containing an RNA base editing domain (e.g., RNA deaminase) and an RNA binding domain (e.g., Cas13), and (ii) a guiding RNA (gRNA) that can bind to the target region in the precursor mRNA of SLC20A2. The gRNA binds to the target region and guides the binding of the fusion protein to exert base editing activity.

[0172] In some embodiments, the oligonucleotide-based base editor includes (i) a sequence module capable of recruiting an endogenous RNA base editor protein (e.g., RNA deaminase, e.g., ADAR) and (ii) a guide sequence module capable of binding to the target region in the precursor mRNA of SLC20A2. The guide sequence module binds to the target region and guides the binding of the RNA base editor protein to exert base editing activity.

[0173] In some embodiments, the RNA editing tool disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon in the precursor mRNA by nucleotide mutation, thereby suppressing the integration of the SA or SD into the pre-mRNA and the hidden exon mRNA.

[0174] application In some embodiments, the PiT2 functional defect relates to a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms.

[0175] In some embodiments, the mutation is located in an exon region, and the subject is heterozygous with respect to the mutation in the exon region.

[0176] In some embodiments, the mutation is an intron mutation, for example, a mutation in intron 2 and / or intron 10. In some embodiments, the mutation is located in the target region of the intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region.

[0177] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0178] In some embodiments, the PiT2 functional defect is a haploinsufficiency of SLC20A2.

[0179] In some embodiments, the drug treats the disease by increasing the expression of PiT2 protein or functional RNA in the subject's cells. Therefore, in some embodiments, the disease according to the present invention may be any disease due to or related to any PiT2 dysfunction, as long as the treatment of the disease benefits from the regulation (e.g., increase) of the expression of functional PiT2 protein or RNA.

[0180] In some embodiments, the disease associated with the PiT2 dysfunction is cerebral calcification. In some embodiments, the cerebral calcification is primary familial cerebral calcification (PFBC).

[0181] In some embodiments, the diseases associated with the PiT2 dysfunction are diseases associated with Pi imbalance. In some embodiments, the diseases associated with Pi imbalance include chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis.

[0182] In some embodiments, the reagents described in any of the above embodiments may be prepared in dosage forms suitable for their intended route of administration, for example, by local administration (e.g., direct injection or implantation), systemic administration, or by extra-gastrointestinal routes including subcutaneous, intravenous, intraperitoneal or intracranial (e.g., intraventricular, intrameningeal or intrashelteral), intramuscular, transdermal, tracheal (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0183] In some embodiments, the reagent can be administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. After compounding the required amount of the active ingredient in a suitable solvent, a formulation suitable for inhalation administration can be produced by sterile filtration. Typically, the formulation for inhalation administration is a sterile solution at physiological pH and has low viscosity. Tension can be balanced by adding salts to the formulation. In some cases, the solubility of the active ingredient may be increased by adding a surfactant or co-solvent to improve the aerosol properties. In some cases, viscosity may be controlled by adding excipients to ensure the size and distribution of atomized droplets.

[0184] In some embodiments, the reagent may be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intra-articular, intraocular, intraperitoneal, intraventricular (e.g., by lumbar puncture), or local administration. Drug compositions suitable for use by injection include sterile aqueous solutions or dispersions and sterile powders for preparing sterile, injectable solutions or dispersions in situ. For intravenous administration, suitable carriers include physiological saline, antimicrobial water, or phosphate-buffered saline (PBS). The drug composition needs to maintain stability under production and storage conditions and prevent contamination by microorganisms such as bacteria and fungi. For example, a sterile injectable solution can be prepared by combining the required dose of the active ingredient in a suitable solvent, and optionally, other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining reagents, absorption-delaying reagents, preservatives, or any combination thereof), followed by filtration for sterilization. Furthermore, to facilitate storage and use, the sterile injection solution may be prepared as a sterile lyophilized powder (for example, by vacuum drying or lyophilization).

[0185] In some embodiments, the reagent is selected from ASOs, and the drug composition is preferably prepared in the form of a solution, emulsion, micro-emulsion, foam, or liposome-containing formulation (e.g., cationic or non-cationic liposomes). In some embodiments, the drug composition is preferably prepared for administration by intraventricular injection (e.g., by lumbar puncture), intraperitoneal injection, intramuscular injection, intrasacral injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0186] In another aspect, the present invention provides a method for regulating the expression of the PiT2 protein in cells, comprising the step of contacting the cells with a reagent capable of regulating alternative splicing in the introns of the SLC20A2 gene in vitro.

[0187] In some embodiments, the cells contain a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. In some embodiments, the cells are heterozygous with respect to the mutation. In some embodiments, the cells are homozygous with respect to the mutation, where the mutation is located within the target region.

[0188] II-B. Reagents targeting the SLC20A2 target region and their use In some respects, the present invention relates to reagents, methods, and uses for restoring or enhancing the function of the SLC20A2 gene product by targeting a target region in SLC20A2.

[0189] In one aspect of the present invention, a reagent is provided that can specifically bind to a target region in the SLC20A2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, and the intron region contains a hidden exon.

[0190] In some embodiments, the hidden exon is located within intron 2.

[0191] In some embodiments, the hidden exons are located at c.289+901 to c.289+1035 of the SLC20A2 gene or its precursor mRNA.

[0192] In some embodiments, the c.289+1007 C>G mutation in the SLC20A2 gene promotes the incorporation of the hidden exon in intron 2.

[0193] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0194] In some embodiments, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA.

[0195] In some embodiments, the target region is located within the sequence shown in Sequence ID 8.

[0196] In some embodiments, the hidden exon is located within the intron 10.

[0197] In some embodiments, the hidden exons are located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene or its precursor mRNA.

[0198] In some embodiments, the c.1795-1698 A>G mutation in the SLC20A2 gene promotes the incorporation of hidden exons in intron 10.

[0199] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3.

[0200] In some embodiments, the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA.

[0201] In some embodiments, the target region is located within the sequence indicated by sequence number 9.

[0202] In some embodiments, the target region is located within the region from approximately 100 nucleotides upstream of the hidden exon to approximately 100 nucleotides downstream of the hidden exon.

[0203] In some embodiments, the target region is selected from a region that straddles the splice site of the hidden exon, a region within the hidden exon, a region within 100 nucleotides upstream of the hidden exon, or a region within 100 nucleotides downstream of the hidden exon.

[0204] In some embodiments, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD).

[0205] In some embodiments, the target region includes a region that spans the 5' splice site (splicing acceptor site, SA) of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 25 nucleotides upstream of the 5' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) to approximately 25 nucleotides downstream of the 5' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) of the 5' splicing site of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 20 nucleotides upstream of the 5' splicing site of the hidden exon to approximately 20 nucleotides downstream of the 5' splicing site of the hidden exon.

[0206] In some embodiments, the target region includes a region that spans the 3' splice site (splicing donor site, SD) of the hidden exon. Preferably, the target region is located within the region from approximately 25 nucleotides upstream of the 3' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) to approximately 25 nucleotides downstream of the 3' splicing site of the hidden exon (for example, approximately 22 or fewer, approximately 20 or fewer, approximately 18 or fewer, approximately 15 or fewer, approximately 12 or fewer, approximately 10 or fewer, approximately 9 or fewer, approximately 8 or fewer, approximately 7 or fewer, approximately 6 or fewer, approximately 5 or fewer, approximately 4 or fewer, approximately 3 or fewer, approximately 2 or fewer, approximately 1 or fewer) to approximately nucleotides downstream of the 3' splicing site of the hidden exon. In some embodiments, the target region is located within a region extending from approximately 20 nucleotides upstream of the 3' splicing site of the hidden exon to approximately 20 nucleotides downstream of the 3' splicing site of the hidden exon.

[0207] In some embodiments, the target region is located within the hidden exon. Preferably, the target region or its periphery includes splicing adjustment elements, such as splicing enhancer elements or splicing silencer elements.

[0208] In some embodiments, the target region is located within an intron region upstream or downstream of the hidden exon. Preferably, the target region or its periphery includes splicing modulating elements, such as a splicing branch site, a splicing enhancer element, or a splicing silencer element.

[0209] In some embodiments, the length of the target region is 100 nucleotides or less, for example, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, or 25 or less nucleotides.

[0210] In some embodiments, the reagent is selected from antisense oligonucleotides (ASOs) or gene editing tools (e.g., CRISPR / Cas, base editors, or Cas protein-independent RNA editing tools).

[0211] In some embodiments, the reagent is an antisense oligonucleotide (ASO). In some embodiments, the reagent is a gene editing tool, such as a DNA or RNA editing system, such as CRISPR / Cas, a base editor, or a Cas protein-independent RNA editing tool. In some embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In some embodiments, the reagent is an RNA editor, such as CRISPR / Cas13. In some embodiments, the reagent is an adenine / cytosine base editor. In some embodiments, the reagent is a Cas protein-independent RNA editing tool.

[0212] ASO In some embodiments, the reagent is an antisense oligonucleotide (ASO) that specifically binds to the target region in the precursor mRNA of SLC20A2, thereby regulating the splicing and expression of the SLC20A2 gene.

[0213] In some embodiments, the ASO consists of 10 to 30 consecutive nucleotides (for example, 15 to 30, 15 to 25, 18 to 30, 18 to 25, or 20 to 25), preferably consisting of 18 to 25 consecutive nucleotides.

[0214] In some embodiments, the ASO includes modifications of the main chain nucleotide and nucleotide linkage (e.g., phosphodiester bond), ribose modification, ribose side chain base modification, or a combination thereof. In some embodiments, the main chain modification is selected from thiophosphate ester linkage or phosphorodiamidate linkage. In some embodiments, the sugar moiety modification is selected from, but is not limited to, 2'-O-methoxyethyl (2'-MOE), morpholino, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), constrained ethyl (cEt), loc nucleic acid (LNA), and peptide nucleic acid.

[0215] In some embodiments, each monomer of ASO is modified in a similar manner, for example, each linkage in the main chain of ASO contains a thiophosphate ester linkage, or each ribose moiety contains a 2'-O-methyl modification. Such modifications present in any of the monomer components of ASO are called “homogeneous modifications.” In some embodiments, a combination of different modifications may be required, for example, ASO may contain a combination of a phosphorodiamidate linkage and a sugar moiety containing a morpholine ring (morpholino). A combination of different modifications to ASO is called “mixed modifications” or “mixed chemistries.”

[0216] In some embodiments, the ASO includes a 2'-MOE modification. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification. In some embodiments, the modification of the ASO includes a combination of 2'-MOE and loc nucleic acid (LNA). In some embodiments, the ASO has a thiophosphate ester linkage backbone.

[0217] In some embodiments, the ASO includes a 2'-OMe modification. In some embodiments, each nucleotide of the ASO has a 2'-OMe modification. In some embodiments, the modification of the ASO includes a combination of 2'-OMe and loc nucleic acid (LNA). In some embodiments, the ASO has a thiophosphate ester linkage backbone.

[0218] In some embodiments, the ASO comprises a combination of a phosphorodiamidate linkage and a sugar moiety containing a morpholine ring (morpholino), such as a phosphorodiamidate morpholino oligomer (PMO).

[0219] In some embodiments, the ASO specifically binds to a target region located within the c.289+801~c.289+1135 region of the SLC20A2 precursor mRNA.

[0220] In some embodiments, the ASO has sequence complementarity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the nucleotide sequence in SEQ ID NO: 8. In some embodiments, the ASO is 100% complementary with the nucleotide sequence in SEQ ID NO: 8.

[0221] In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 13-46, 63-65, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto. In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 19-22, 25, 26, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto.

[0222] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 13-46 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto, and comprises a 2'-MOE modification and / or a thiophosphate ester linkage. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification and a thiophosphate ester linkage backbone.

[0223] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 63-65 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith, and the ASO is a PMO.

[0224] In some embodiments, the ASO specifically binds to a target region located within the c.1795-1803~c.1795-1527 region of the SLC20A2 precursor mRNA.

[0225] In some embodiments, the ASO has sequence complementarity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the nucleotide sequence in SEQ ID NO: 9. In some embodiments, the ASO is 100% complementary with the nucleotide sequence in SEQ ID NO: 9.

[0226] In some embodiments, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 47-61, 66-68, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith. In some embodiments, the ASO includes a nucleotide sequence represented by SEQ ID NO: 50, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith.

[0227] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 47-61 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity thereto, and comprises a 2'-MOE modification and / or a thiophosphate ester linkage. In some embodiments, each nucleotide of the ASO has a 2'-MOE modification and a thiophosphate ester linkage backbone.

[0228] In some embodiments, the ASO comprises a nucleotide sequence represented by any of SEQ ID NOs: 66-68 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith, and the ASO is a PMO.

[0229] Gene editing tools In some embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool, which typically includes (i) a module having base editing activity and (ii) a module capable of binding to the target region in the SLC20A2 gene. The gene editing tool disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of a hidden exon in the genome or precursor mRNA sequence by mutating the SA or SD sequence and suppressing the integration of the hidden exon into mRNA.

[0230] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0231] CRISPR / Cas system In some embodiments, the reagent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), the gRNA being capable of binding to the target region in the SLC20A2 gene or its precursor mRNA.

[0232] In some embodiments, the gRNA binds to a target region and guides the binding of the Cas enzyme to exert editing activity.

[0233] In some embodiments, the CRISPR system disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon by mutating it, thereby suppressing the integration of the hidden exon into mRNA.

[0234] In some embodiments, the target region is located within the c.289+801~c.289+1135 region of the SLC20A2 precursor mRNA.

[0235] In some embodiments, the target region is located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA.

[0236] DNA base editor In some embodiments, the reagent is a DNA base editor. The DNA base editor typically comprises (i) a fusion protein containing a DNA base editing domain (e.g., deaminase) and a DNA binding domain (e.g., a Cas protein, e.g., Cas9 or Cas12), and (ii) a guiding RNA (gRNA) that can bind to the target region in the SLC20A2 gene.

[0237] In some embodiments, the gRNA binds to a target region and guides the binding of the fusion protein, thereby exerting base-editing activity.

[0238] In some embodiments, the DNA base editor disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon by causing a base mutation, thereby suppressing the incorporation of the hidden exon into mRNA.

[0239] In some embodiments, the Cas protein is a catalytically inactive Cas protein or Cas nickase protein.

[0240] In some embodiments, the base editor includes an ABE (Adenine base editor) or a CBE (Cytosine base editor), or a novel editor obtained by molecular evolution / mutation screening.

[0241] RNA editing tools In some embodiments, the reagent is an RNA editing tool. RNA editing tools include, for example, RNA base editors based on the CRISPR / Cas system, and base editors based on oligonucleotides.

[0242] In some embodiments, the CRISPR / Cas system-based RNA base editor comprises (i) a fusion protein containing an RNA base editing domain (e.g., RNA deaminase) and an RNA binding domain (e.g., Cas13), and (ii) a guiding RNA (gRNA) that can bind to the target region in the precursor mRNA of SLC20A2. The gRNA binds to the target region and guides the binding of the fusion protein to exert base editing activity.

[0243] In some embodiments, the oligonucleotide-based base editor includes (i) a sequence module capable of recruiting an endogenous RNA base editor protein (e.g., RNA deaminase, e.g., ADAR) and (ii) a guide sequence module capable of binding to the target region in the precursor mRNA of SLC20A2. The guide sequence module binds to the target region and guides the binding of the RNA base editor protein to exert base editing activity.

[0244] In some embodiments, the RNA editing tool disrupts the function of the splicing acceptor (SA) or splicing donor (SD) sequence of the hidden exon by causing a base mutation, thereby suppressing the incorporation of the hidden exon into mRNA.

[0245] In another aspect, the present invention provides a drug composition comprising the above-described reagent or combination thereof and a pharmaceutically acceptable carrier and / or excipient.

[0246] In some embodiments, the drug composition may be prepared in a dosage form suitable for its intended route of administration, for example, by local administration (e.g., direct injection or implantation), systemic administration, or by extra-gastrointestinal routes including subcutaneous, intravenous, intraperitoneal or intracranial (e.g., intraventricular, intrameningeal or intrashelteral), intramuscular, transdermal, tracheal (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0247] In some embodiments, the drug composition can be administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. After compounding the required amount of the active ingredient in a suitable solvent, a formulation suitable for inhalation administration can be produced by sterile filtration. Typically, the formulation for inhalation administration is a sterile solution at physiological pH and has low viscosity. Tension can be balanced by adding salts to the formulation. In some cases, the solubility of the active ingredient may be increased by adding a surfactant or co-solvent to improve the aerosol properties. In some cases, viscosity may be controlled by adding excipients to ensure the size and distribution of atomized droplets.

[0248] In some embodiments, the drug composition may be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intra-articular, intraocular, intraperitoneal, intraventricular (e.g., by lumbar puncture), or local administration. Drug compositions suitable for use by injection include sterile aqueous solutions or dispersions, as well as sterile powders for preparing sterile injectable solutions or dispersions in situ. For intravenous administration, suitable carriers include physiological saline, antimicrobial water, or phosphate-buffered saline (PBS). The drug composition must maintain stability under production and storage conditions and prevent contamination by microorganisms such as bacteria and fungi. For example, a sterile injectable solution can be prepared by combining the required dose of the active ingredient in a suitable solvent, and optionally, other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining reagents, absorption-delaying reagents, preservatives, or any combination thereof), followed by filtration for sterilization. Furthermore, to facilitate storage and use, the sterile injection solution may be prepared as a sterile lyophilized powder (for example, by vacuum drying or lyophilization).

[0249] In some embodiments, the reagent is selected from ASOs, and the drug composition is preferably prepared in the form of a solution, emulsion, micro-emulsion, foam, or liposome-containing formulation (e.g., cationic or non-cationic liposomes). In some embodiments, the drug composition is preferably prepared for administration by intraventricular injection (e.g., by lumbar puncture), intraperitoneal injection, intramuscular injection, intrasacral injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0250] In another aspect, the present invention provides the use of such reagents or drug compositions in the manufacture of drugs for preventing and / or treating diseases associated with PiT2 dysfunction (e.g., deficiency of quantity or activity) in subjects. The present invention also provides a method for preventing and / or treating diseases associated with PiT2 dysfunction in subjects, comprising the step of administering an effective amount of such reagents or drug compositions to the required subjects.

[0251] In some embodiments, the disease associated with the PiT2 dysfunction is cerebral calcification, for example, primary familial encephalopathy (PFBC). In some embodiments, the disease associated with the PiT2 dysfunction is a disease associated with Pi imbalance, for example, chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis. In some embodiments, the drug treats the disease by regulating (e.g., increasing) the expression of PiT2 protein or functional RNA in the subject's cells.

[0252] In some embodiments, the subject includes a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms.

[0253] In some embodiments, the mutation is located in an exon region, and the subject is heterozygous with respect to the mutation in the exon region.

[0254] In some embodiments, the mutation is an intron mutation, for example, a mutation in intron 2 and / or intron 10. In some embodiments, the mutation is located in the target region of the intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region.

[0255] In some embodiments, the PiT2 functional defect is a haploinsufficiency of SLC20A2.

[0256] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0257] In another aspect, the present invention also provides a method for regulating the expression of the PiT2 protein in cells, comprising the step of contacting the cells with the reagent or drug composition described above in vitro.

[0258] In some embodiments, the cells contain a mutation in the SLC20A2 gene. In some embodiments, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. In some embodiments, the mutation is located in an exon region, and the cells are heterozygous with respect to the mutation in the exon region. In some embodiments, the mutation is an intron mutation, for example, a mutation in intron 2 and / or intron 10. In some embodiments, the mutation is located in the target region of the intron, and the cells are heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region.

[0259] II-C. Screening Methods In some respects, the present invention relates to a method for screening reagents that regulate PiT2 expression.

[0260] In one aspect of the present invention, there is provided a method for identifying a reagent capable of modulating the expression of PiT2 in a cell, comprising the steps of: i) identifying a reagent that targets a target region in the PiT2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, the intron region contains a cryptic exon, and the target region is within a region from a position of about 100 nucleotides or less upstream of the cryptic exon to a position of about 100 nucleotides or less downstream of the cryptic exon; ii) delivering the reagent identified in step i) to the cell; and iii) measuring the expression level of PiT2 in the cell obtained in step ii).

[0261] In some embodiments, the method is performed in vitro.

[0262] In some embodiments, the method further comprises the step of iv) comparing the expression level measured in step iii) with the expression level of PiT2 in a cell treated with a control. An increase relative to the control group indicates that the reagent is an activator. A decrease relative to the control group indicates that the reagent is an inhibitor.

[0263] In some embodiments, the expression level of PiT2 refers to the expression level of functional PiT2, including, for example, the mRNA level of a correctly spliced product or the level of the protein encoded thereby.

[0264] In some embodiments, the expression level is measured by RT-PCR, qPCR or Western blotting.

[0265] In some embodiments, the target region is selected from a region upstream of the 5' splice site (splicing acceptor site, SA) of the cryptic exon, a region spanning the 5' splice site, a region within the cryptic exon, a region spanning the 3' splice site, or a region downstream of the 3' splice site (splicing donor site, SD).

[0266] In some embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene. In some embodiments, the cryptic exon has the sequence set forth in SEQ ID NO: 2. In some embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA. In some embodiments, the target region is located within the sequence set forth in SEQ ID NO: 8.

[0267] In some embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene. In some embodiments, the cryptic exon has the sequence set forth in SEQ ID NO: 3. In some embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA. In some embodiments, the target region is located within the sequence set forth in SEQ ID NO: 9.

[0268] In some embodiments, the reagent targets the target region in the precursor mRNA of SLC20A2. In some embodiments, the reagent is an antisense oligonucleotide (ASO). In some embodiments, the reagent is an RNA editor, for example, CRISPR / Cas13. In some embodiments, the reagent is a Cas protein-independent RNA editing tool.

[0269] In some embodiments, the reagent targets the target region in the SLC20A2 gene. In some embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In some embodiments, the reagent is a base editor, such as an adenine / cytosine base editor.

[0270] In some respects, the present invention relates to a method for screening reagents that modulate mRNA splicing of SLC20A2.

[0271] In one aspect of the present invention, the present invention provides a nucleic acid construct comprising a minigene fragment sequence of the human SLC20A2 gene, wherein the minigene fragment sequence comprises exon 2 and exon 3 and an intron sequence between them, or exon 10 and exon 11 and an intron sequence between them, wherein the intron sequence includes a mutation that can alter the splicing of the SLC20A2 gene.

[0272] In some embodiments, the nucleic acid construct includes a promoter operably linked to the minigene fragment sequence.

[0273] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3. In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0274] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0275] In some embodiments, the mutation includes a c.289+1007 C>G mutation in the SLC20A2 gene.

[0276] In some embodiments, the minigene fragment sequence including exons 2 and 3 and the intron sequence between them includes the sequence shown in SEQ ID NO: 4.

[0277] In some embodiments, the alternative splicing includes alternative splicing in the intron 10.

[0278] In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0279] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3.

[0280] In some embodiments, the mutation includes the c.1795-1698 A>G mutation in the SLC20A2 gene.

[0281] In some embodiments, the minigene fragment sequence including exons 10 and 11 and the intron sequence between them includes the sequence shown in SEQ ID NO: 5.

[0282] In some embodiments, a polyA signal sequence is included downstream of the minigene fragment sequence.

[0283] In some embodiments, the nucleic acid construct further includes a reporter gene, such as a fluorescent protein (e.g., GFP, mCherry) or luciferase.

[0284] In another aspect, the present invention provides expression vectors comprising nucleic acid constructs as described above. In this specification, the term “vector” means a nucleic acid transport tool into which polynucleotides can be inserted. A vector is called an expression vector if it causes the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transtransfer so that the genetic material elements it carries are expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages, e.g., λ phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). The vector may contain multiple expression-regulating elements, including, but not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. Furthermore, the vector may also contain a replication start site.

[0285] In another aspect, the present invention provides a method for screening reagents capable of modulating mRNA splicing of SLC20A2, comprising the steps of: (i) providing cells containing the nucleic acid construct described above; (ii) providing a genetically modified non-human animal or cell of the present invention; or (iii) providing a cerebral calcification model obtained by a method for producing a cerebral calcification model of the present invention; contacting a test reagent with any of (i)-(iii); and detecting alternative splicing of SLC20A2 mRNA. In some embodiments, the method is for purposes other than diagnosis and treatment. In some embodiments, the method is performed in vitro.

[0286] In some embodiments, the cells are derived from, for example, a mammal, such as a rodent (e.g., a mouse or rat), a non-human primate, or a human.

[0287] In some embodiments, a test reagent capable of inhibiting alternative splicing (e.g., reducing the amount of expression products including cryptic exon incorporation) is selected, thereby increasing the production of functional PiT2 protein.

[0288] In some embodiments, a test reagent capable of enhancing alternative splicing (e.g., increasing the amount of expression products including cryptic exon incorporation) is selected, thereby reducing the production of functional PiT2 protein.

[0289] In some embodiments, the detection comprises analyzing the length of the SLC20A2 mRNA expression product.

[0290] In some embodiments, the alternative splicing is alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is incorporation of a cryptic exon between exon 2 and 3. In some embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene. In some embodiments, the cryptic exon has the sequence set forth in SEQ ID NO: 2.

[0291] In some embodiments, the detection comprises performing PCR analysis using primers spanning exon 2 and exon 3. In some embodiments, an amplicon incorporating a cryptic exon of about 135 nt in length is generated. In some embodiments, the primers comprise the forward primer set forth in SEQ ID NO: 123 and the reverse primer set forth in SEQ ID NO: 124.

[0292] In some embodiments, the alternative splicing includes alternative splicing in intron 10. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene. In some embodiments, the hidden exon has the sequence shown in SEQ ID NO: 3.

[0293] In some embodiments, the detection involves performing a PCR analysis using primers that span exon 10 and exon 11. In some embodiments, an amplicon is generated incorporating a hidden exon approximately 76 nt in length. In some embodiments, the primers include a forward primer shown in SEQ ID NO: 127 and a reverse primer shown in SEQ ID NO: 128.

[0294] In some embodiments, the method includes the step of detecting alternative splicing of the nucleic acid construct by contacting a test reagent with cells containing the nucleic acid construct in vitro. In some embodiments, the method includes the step of providing cells containing the nucleic acid construct by introducing (e.g., transtransferring) the nucleic acid construct or a vector containing the nucleic acid construct into cells. In some embodiments, the detection includes performing the above-described analysis on the length of the mRNA expression product of the nucleic acid construct. In some embodiments, the detection includes analyzing the expression of a reporter gene contained in the nucleic acid construct. In some embodiments, the expression of the reporter gene indicates alternative splicing. In some embodiments, the expression of the reporter gene indicates exact splicing.

[0295] II-D. Cerebral calcification model and its manufacture In some respects, the present invention relates to a brain calcification model constructed based on alternative splicing in the intron of the SLC20A2 gene.

[0296] In one aspect of the present invention, a method for producing a brain calcification model is provided, comprising the step of modifying a target genome such that the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene, thereby providing a brain calcification model. In some embodiments, the target genome is homozygous or heterozygous with respect to the replacement.

[0297] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon. In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a region from about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon.

[0298] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3. In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0299] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0300] In some embodiments, the mutation includes a c.289+1007 C>G mutation in the human SLC20A2 gene.

[0301] In some embodiments, the genomic fragment of the human SLC20A2 gene intron substituted between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10.

[0302] In some embodiments, the alternative splicing includes alternative splicing in the intron 10.

[0303] In some embodiments, alternative splicing in intron 2 involves the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the human SLC20A2 gene.

[0304] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3.

[0305] In some embodiments, the mutation includes the c.1795-1698 A>G mutation in the human SLC20A2 gene.

[0306] In some embodiments, the intronic genome fragment of the human SLC20A2 gene substituted between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0307] In some embodiments, the donor sequence for replacement has the following structure: exon 2 (3'-part)-intron-crpE2 (or crpE10)-intron-exon 3 (5'-part).

[0308] In some embodiments, the donor sequence for replacement has the following structure: exon 10 (3'-part)-intron-crpE2 (or crpE10)-intron-exon 11 (5'-part).

[0309] In some embodiments, the expression of the modified endogenous Slc20a2 gene is under the control of a regulatory element located at the endogenous Slc20a2 locus in the target genome.

[0310] Methods for replacing the intron sequences of the endogenous Slc20a2 gene with the corresponding intron sequences of the human SLC20A2 gene can be carried out using various suitable genome editing techniques known in the art. In some embodiments, such replacement methods include gene knockout, gene editing, RNA interference, epigenetic repression, or any combination thereof.

[0311] In some embodiments, the gene knockout includes homologous recombination-based stem cell targeting techniques, gene editing techniques, induction of genomic mutations by chemical molecules, site-specific inactivation of transposons, or any combination thereof.

[0312] In some embodiments, the gene editing includes a CRISPR / Cas system (e.g., Cas9, Cas12a (Cpf1)), a TALEN, a zinc finger nuclease, base editing, or any combination thereof.

[0313] In some embodiments, the replacement method involves introducing a targeting vector into cells that carries an insertion nucleic acid (e.g., a genomic fragment sequence of the corresponding intron of the human SLC20A2 gene to be introduced) with 5' and 3' homology arms ligated to its sides. In some embodiments, the insertion nucleic acid further contains a selectivity marker gene (e.g., a self-deletion cassette of a selectivity marker gene), the gene having site-directed recombination sites (e.g., loxP, Frt, etc.) ligated to its sides. In some embodiments, the insertion nucleic acid further contains a reporter gene.

[0314] In some embodiments, the model is a non-human animal model, and the target genome is the genome of a non-human animal.

[0315] In some embodiments, the model is a mammalian model. In some embodiments, the animal model is a rodent model, such as a mouse or rat model. In some embodiments, the animal model is a non-human primate model.

[0316] In some embodiments, the method includes the steps of modifying the genome of embryonic stem (ES) cells of a non-human animal so that the modified genome contains the substitution in the endogenous Slc20a2 gene, and obtaining a humanized brain calcification animal model by constructing a non-human animal model using the non-human animal ES cells containing the modified genome. In some embodiments, the targeting vector can be introduced into the non-human animal ES cells, for example, by electroporation.

[0317] In some embodiments, the model is a cell model, and the target genome is the endogenous genome of the target cell. In some embodiments, the genomic fragment (with or without mutations) of the corresponding intron of the replacement human SLC20A2 gene is exogenously introduced into the target cell.

[0318] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human epithelial fibroblasts or glial cells.

[0319] In some embodiments, the method includes the step of obtaining a cell model containing the modified genome by modifying the genome of a target cell so that the modified genome contains the substitution in the endogenous Slc20a2 gene.

[0320] In another aspect, the present invention provides a genetically modified non-human animal whose genome is such that the intron sequence between exons 2 and 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exons 10 and 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene contains a mutation that can alter the splicing of the SLC20A2 gene. In some embodiments, the non-human animal is homozygous or heterozygous with respect to the replacement.

[0321] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon. In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a region from about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon.

[0322] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3. In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the human SLC20A2 gene.

[0323] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0324] In some embodiments, the mutation includes a c.289+1007 C>G mutation in the SLC20A2 gene.

[0325] In some embodiments, the genomic fragment of the human SLC20A2 gene intron substituted between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10.

[0326] In some embodiments, the alternative splicing includes alternative splicing in the intron 10.

[0327] In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0328] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3.

[0329] In some embodiments, the mutation includes the c.1795-1698 A>G mutation in the SLC20A2 gene.

[0330] In some embodiments, the intronic genome fragment of the human SLC20A2 gene substituted between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0331] In some embodiments, the non-human animal is a rodent, such as a mouse or a rat.

[0332] In some embodiments, the non-human animal is a non-human primate.

[0333] In some embodiments, the genetically modified non-human animal serves as an animal model for cerebral calcification.

[0334] In another aspect, the present invention provides genetically modified cells whose genomes include those in which the intron sequence between exons 2 and 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or in which the intron sequence between exons 10 and 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene contains a mutation that can alter the splicing of the SLC20A2 gene. In some embodiments, the cells are homozygous or heterozygous with respect to the replacement.

[0335] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a hidden exon. In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene includes at least a region from about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon.

[0336] In some embodiments, the genomic fragment of the corresponding intron of the human SLC20A2 gene is exogenously introduced into the cells. "Exogenously introduced" refers to nucleic acids originating from cells other than the target cells. Exogenous nucleic acids may be artificially generated or recombinantly produced, or isolated from heterogeneous cells and introduced into the target cells.

[0337] In some embodiments, mutations that can alter the splicing of the SLC20A2 gene induce or enhance alternative splicing in intron 2. In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 2 and 3. In some embodiments, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0338] In some embodiments, the hidden exon has the sequence shown in Sequence ID No. 2.

[0339] In some embodiments, the mutation includes a c.289+1007 C>G mutation in the SLC20A2 gene.

[0340] In some embodiments, the genomic fragment of the human SLC20A2 gene intron substituted between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10.

[0341] In some embodiments, the alternative splicing includes alternative splicing in the intron 10.

[0342] In some embodiments, the alternative splicing in intron 2 is the incorporation of a hidden exon between exons 10 and 11. In some embodiments, the hidden exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0343] In some embodiments, the hidden exon has the sequence shown in Sequence ID 3. In some embodiments, the mutation includes the c.1795-1698 A>G mutation in the SLC20A2 gene.

[0344] In some embodiments, the intronic genome fragment of the human SLC20A2 gene substituted between exon 10 and exon 11 includes the sequence shown in SEQ ID NO: 11.

[0345] In some embodiments, the cells are mammalian cells.

[0346] In some embodiments, the cells are human epithelial fibroblasts or glial cells.

[0347] In some embodiments, the genetically modified cells serve as a cellular model for brain calcification.

[0348] II-E. Use of cerebral calcification models In some respects, the present invention also relates to the use of the above-mentioned cerebral calcification model in areas such as disease research and drug screening.

[0349] One aspect of the present invention provides the use of the cerebral calcification model of the present invention, or genetically modified non-human animals or genetically modified cells, for presenting and analyzing the onset stages of cerebral calcification disease, or as a model for studying targets of cerebral phosphate disruption or cerebral calcification disease.

[0350] In another aspect, the present invention provides the use of the cerebral calcification model of the present invention, or genetically modified non-human animals or genetically modified cells, as a model for screening candidate drugs or treatment plans for treating cerebral calcification disorders or diseases associated with disruption of cerebral phosphate homeostasis.

[0351] In some embodiments, the disease is a cerebral calcification disease.

[0352] In some embodiments, the drug is a drug that prevents or treats cerebral calcification.

[0353] In some embodiments, the drug is a drug that restores phosphate homeostasis in the brain.

[0354] In another aspect, the present invention provides the use of the brain calcification model of the present invention, or genetically modified non-human animals or genetically modified cells, as a model for screening candidate drugs or treatment plans for diseases associated with disruption of phosphorus homeostasis.

[0355] In some embodiments, diseases associated with disruption of phosphorus homeostasis include, but are not limited to, chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis.

[0356] In some embodiments, the drug is one that modulates the expression level of the product PiT2 encoded by SLC20A2 by regulating its mRNA splicing.

[0357] In some embodiments, the drug modulates the function of the product PiT2 encoded by SLC20A2 by regulating its mRNA splicing.

[0358] In some embodiments, the drug increases the production of functional PiT2 protein by inhibiting alternative splicing of SLC20A2 mRNA.

[0359] In some embodiments, the drug inhibits the production of functional PiT2 protein by promoting alternative splicing of SLC20A2 mRNA.

[0360] In another aspect, the present invention provides a method for screening candidate drugs or treatment plans for treating cerebral calcification disorders or disorders associated with disruption of cerebral phosphate homeostasis, comprising the steps of: applying the candidate drug or treatment plan to a cerebral calcification model of the present invention, or to a genetically modified non-human animal or genetically modified cells; and evaluating whether the candidate drug or treatment plan can (i) increase the expression level of a full-length mRNA transcript of SLC20A2 and / or the protein product PiT2 encoded thereby; (ii) increase the phosphate transport capacity of target cells (e.g., glial cells, e.g., astrocytes); and / or (iii) decrease inorganic phosphate ion levels in cerebrospinal fluid.

[0361] In some embodiments, the disease is a cerebral calcification disease.

[0362] In some embodiments, the drug is a drug that prevents or treats cerebral calcification.

[0363] In some embodiments, the drug is a drug that restores phosphate homeostasis in the brain.

[0364] In some embodiments, the candidate drug or treatment plan can increase the expression level of the full-length mRNA transcript of SLC20A2 and / or the protein product PiT2 encoded therein, upmodulating it, for example by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc. In some embodiments, the candidate drug or treatment plan can increase the expression level of the exact splicing mRNA transcript of SLC20A2 and / or the functional PiT2 protein product encoded therein, upmodulating it, for example by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0365] In some embodiments, the candidate drug or treatment plan can increase the phosphorus transport capacity of target cells (e.g., glial cells in the brain, e.g., astrocytes), for example, by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0366] In some embodiments, the candidate drug or treatment plan can reduce inorganic phosphate ion levels in the cerebrospinal fluid, for example, by lowering abnormally elevated inorganic phosphate ion levels in the cerebrospinal fluid by at least 75%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 25%, to, for example, a level close to a normal reference level (e.g., a healthy control).

[0367] In another aspect, the present invention provides a method for screening candidate drugs or treatment plans for treating diseases associated with disruption of phosphate homeostasis, comprising the steps of: applying the candidate drug or treatment plan to a cerebral calcification model of the present invention, or to a genetically modified non-human animal or genetically modified cells; and evaluating whether the candidate drug or treatment plan can (i) increase the expression level of a full-length mRNA transcript of SLC20A2 and / or the protein product PiT2 encoded thereby; (ii) increase the phosphate transport capacity of target cells (e.g., endocerebral glial cells, e.g., astrocytes); and / or (iii) decrease inorganic phosphate ion levels in cerebrospinal fluid.

[0368] In some embodiments, diseases associated with disruption of phosphorus homeostasis include, but are not limited to, chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis.

[0369] In some embodiments, the candidate drug or treatment plan can increase the expression level of the full-length mRNA transcript of SLC20A2 and / or the protein product PiT2 encoded therein, upmodulating it, for example by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc. In some embodiments, the candidate drug or treatment plan can increase the expression level of the exact splicing mRNA transcript of SLC20A2 and / or the functional PiT2 protein product encoded therein, upmodulating it, for example by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0370] In some embodiments, the candidate drug or treatment plan can increase the phosphorus transport capacity of target cells (e.g., glial cells in the brain, e.g., astrocytes), for example, by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0371] In some embodiments, the candidate drug or treatment plan can reduce inorganic phosphate ion levels in the cerebrospinal fluid, for example, by lowering abnormally elevated inorganic phosphate ion levels in the cerebrospinal fluid by at least 75%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 25%, to, for example, a level close to a normal reference level (e.g., a healthy control).

[0372] In some embodiments, the drug is one that modulates the expression level of the product PiT2 encoded by SLC20A2 by regulating its mRNA splicing.

[0373] In some embodiments, the drug modulates the function of the product PiT2 encoded by SLC20A2 by regulating its mRNA splicing.

[0374] In some embodiments, the drug increases the production of functional PiT2 protein by inhibiting alternative splicing of SLC20A2 mRNA.

[0375] In some embodiments, the drug inhibits the production of functional PiT2 protein by promoting alternative splicing of SLC20A2 mRNA.

[0376] II-F. Beneficial Effects and Summary In this invention, we first discovered and identified the incorporation of previously unreported hidden exons in the abnormal splicing of SLC20A2 mRNA, and then invented reagents and methods to modulate this abnormal splicing. In cells from patients with cerebral calcification and in the SLC20A2-KI mouse model, modulating this abnormal splicing can upregulate the expression of the PiT2 protein, a product of the SLC20A2 gene, and reverse the increase in CSF Pi levels, thereby suppressing the progression of cerebral calcification.

[0377] In the embodiments provided by the present invention, a method for upregulating the expression of the PiT2 protein, a product of the SLC20A2 gene, by regulating the abnormal splicing of SLC20A2, can be achieved primarily by the action of antisense oligonucleotides (ASOs). However, based on the logic and implementation routes of the method described in the present invention, it will be apparent to those skilled in the art that the present invention can also be achieved by permanently altering the splicing scheme of SLC20A2 mRNA at the genomic level by DNA base editing, or by altering the splicing scheme of SLC20A2 mRNA at the RNA level by RNA base editing.

[0378] Furthermore, since PiT2 is highly distributed in the cerebrum, kidneys, cardiovascular system, and skeleton, and is involved in Pi balance, regulating splicing of PiT2 expression is useful in restoring other syndromes associated with Pi imbalance, including chronic kidney disease, cardiovascular atherosclerosis, and skeletal disorders.

[0379] In particular, the method provided by the present invention to regulate abnormal splicing of SLC20A2 mRNA can improve the expression level of PiT2 protein even in healthy cells, and by correcting SLC20A2 haploinsufficiency regardless of the specific mutation pattern, this method can save PiT2 expression in patients with SLC20A2 loss-of-function mutations caused by non-intronic mutations, which account for a larger proportion of cases.

[0380] Based on this, the present invention provides a novel strategy for treating SLC20A2 haploinsufficiency based on mRNA splicing of the SLC20A2 gene, which has significant clinical value in treating cerebral calcification disorders or diseases associated with disruption of phosphate homeostasis.

[0381] III. Therapies targeting astrocytes In certain aspects, the present invention relates to gene compensation therapies and uses that target astrocytes to reconstruct phosphate homeostasis in the brain, including suppressing the development of cerebral calcification in individuals with PiT2 mutations, and delaying or preventing the progression of cerebral calcification in patients with PFBC or cerebral calcification associated with disruption of phosphate homeostasis.

[0382] Through extensive and in-depth research and large-scale screening, the inventors have developed, for the first time, a gene compensation therapy targeting astrocytes that can effectively contribute to the progression of cerebral calcification. Further experiments have revealed, for the first time, the mechanism by which cerebral calcification is caused by gene and / or molecular mutations and / or abnormalities of PiT2, MYORG, XPR1, etc. It was found that these mutations and / or abnormalities cause phosphate transport dysfunction in astrocytes, disrupting cerebral phosphate homeostasis and leading to cerebral calcification. In terms of methodology, the present invention provides a strategy to suppress or inhibit the occurrence and progression of calcification by restoring cerebral phosphate homeostasis by increasing the expression levels of PiT2, MYORG, or XPR1 genes in cerebral astrocytes.

[0383] In the embodiments provided by the present invention, a recombinant AAV virus was constructed to cause astrocytes to selectively express the PiT2 protein. Experimental results showed that intravenous injection of the recombinant AAV virus, which can cross the blood-brain barrier, enabled the expression of the compensatory gene protein product PiT2, which is selectively carried by the expression module expressing the PiT2 protein, in astrocytes in the cerebrum of mice. As a result, PiT2-KO mice, in which astrocytes in the brain have received expression compensation, achieve restoration of cerebral phosphate homeostasis and more fundamentally and effectively inhibit the development and progression of cerebral calcification. The strategies presented in the present invention also support alternative embodiments, namely, the delivery of gene expression promoters to astrocytes, such as antisense nucleotide (ASO) drugs, or modification of RNA splicing based on gene / base editing, to regulate the expression levels of PiT2, MYORG, or XPR1 in astrocytes, thereby similarly achieving the objective of restoring cerebral phosphate homeostasis and preventing cerebral calcification. These data indicate that the gene compensation therapy of the present invention is useful in restoring phosphate homeostasis in the brain, suppressing the development of cerebral calcification or preventing its further progression, and improving associated neurological symptoms, and therefore has outstanding value for clinical implementation.

[0384] III-(A). Astrocyte Phosphate Transport Enhancers One aspect of the present invention provides an astrocyte phosphate transport promoter. In this specification, the astrocyte phosphate transport promoter is a reagent capable of promoting phosphate transport in astrocytes. The reagent can, for example, achieve specific expression of astrocytes, for example, by using astrocyte-specific expression elements (e.g., promoters). Alternatively, it can specifically target astrocytes, for example, by specifically binding to specific receptors on the membrane of astrocytes.

[0385] In some embodiments, the cerebral phosphate homeostasis promoter is selected from the following group: (Z1) PiT2 protein, nucleic acid molecule encoding PiT2 protein, expression vector expressing PiT2 protein, PiT2 promoter, or combination thereof; (Z2) MYORG protein, nucleic acid molecule encoding MYORG protein, expression vector expressing MYORG protein, MYORG enhancer, or combination thereof; (Z3) XRP1 protein, nucleic acid molecule encoding the XRP1 protein, expression vector expressing the XRP1 protein, XRP1 enhancer, or combination thereof; (Z4) Any combination of Z1 to Z3 above.

[0386] In some embodiments, the promoter described in any of Z1 to Z3 can activate or upregulate the expression of the gene, and / or activate or enhance the activity of the protein product of the gene.

[0387] In some embodiments, the promoter may exert its activating effect by any mechanism, for example, by activating gene expression at the RNA or protein level (e.g., by enhancing gene transcription and / or enhancing the translation of the mRNA product of the gene).

[0388] In some embodiments, the promoter enhances the biological function of the gene by activating its expression level. In such embodiments, the expression level can be measured at the nucleic acid level or the protein level. Methods for measuring expression at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR, or real-time (real) RT-PCR. Methods for measuring expression at the protein level include, but are not limited to, Western blotting or polyacrylamide gel electrophoresis with protein staining techniques, such as a combination of Coomassie brilliant blue or silver staining, mass spectrometry, and ELISA.

[0389] In some embodiments, the accelerator described in any of Z1 to Z3 comprises a small molecule compound, a gene editing reagent (e.g., a base editing reagent), a small nucleic acid drug (e.g., an antisense nucleotide drug), or a combination thereof. In some embodiments, the accelerator is a small molecule compound. In some embodiments, the accelerator is a gene editing reagent (e.g., a base editing reagent). In some embodiments, the accelerator is a small nucleic acid drug (e.g., an antisense nucleotide drug).

[0390] In some embodiments, the astrocyte phosphate transport promoter is selected from promoters that regulate the expression levels of PiT2, MYORG, or XPR1 in astrocytes.

[0391] In some embodiments, the astrocyte phosphate transport promoter described in any of the above embodiments can achieve specific expression of astrocytes, for example, by an astrocyte-specific expression element (e.g., a promoter). In such embodiments, the astrocyte phosphate transport promoter may also be an expression vector comprising an exogenous gene and an enhancer element and / or a promoter selectively expressed by astrocytes operably linked thereto, wherein the exogenous gene is selected from nucleic acid molecules encoding the PiT2 protein, MYORG protein, or XRP1 protein, or a combination thereof.

[0392] In some embodiments, the exogenous gene is a nucleic acid molecule encoding the PiT2 protein.

[0393] In some embodiments, the promoter selectively expressed by the astrocytes is gfaABC1D. In some embodiments, the promoter selectively expressed by the astrocytes is a promoter derived from a gene such as ALDH1L1, AQP4, or S100B.

[0394] In some embodiments, the astrocyte phosphate transport promoter is an expression vector (e.g., a viral vector, e.g., AAV) comprising a nucleic acid molecule encoding the PiT2 protein and a promoter (e.g., gfaABC1D) that is operably linked to it and selectively expressed by astrocytes.

[0395] In some embodiments, the expression vector is a viral vector, such as a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a box virus vector (e.g., a vaccinia virus vector), or a herpes simplex virus vector.

[0396] In some embodiments, the expression vector is AAV. Adeno-associated virus (AAV), also known as adeno-related virus, belongs to the genus Dependvirus of the family Parvoviridae and is the simplest single-stranded DNA defect virus discovered to date. The wild-type AAV genome consists of approximately 4.7 kb of linear single-stranded DNA (ssDNA) with terminal inversion repeats (ITRs) on both sides, or with the coding sequences for the Rep and Cap proteins between the two ITR elements. Through the regulation of selectable promoters and splicing variants at multiple locations, the Rep gene can express four different proteins that can be involved in the replication, transcription, and integration of the viral genome. Meanwhile, with the help of AAP, a key assembly activator protein 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 in a 1:1:10 ratio. Adeno-associated virus (AAV) is considered an ideal delivery vector in gene therapy strategies due to its characteristics: it is smaller than other viral vectors, has little to no immunogenicity, a very low probability of genome integration, and can translocate into dividing and unedivided cells.

[0397] The ITR elements of a virus contain cis-elements necessary for genome replication and packaging. Therefore, AAVs can be modified into viral delivery vectors by removing all viral coding gene sequences between the ITR elements and replacing them with the exogenous DNA sequences of the desired gene. These exogenous DNA sequences encode a standard expression cassette, allowing for the expression of a specific target gene in target cells. These engineered recombinant adeno-associated virus vectors (rAAVs) are considered one of the most promising gene delivery vectors due to their good safety profile, broad host cell range (dividing and unedivided cells), low immunogenicity, and long duration of exogenous gene expression in the body. They are widely used in gene therapy and vaccine research worldwide. After more than a decade of research, the biological properties of recombinant adeno-associated viruses are well understood, and a wealth of data has been accumulated, particularly regarding their effectiveness in various cell, tissue, and in vivo experiments. In medical research, rAAVs are used in gene therapy studies for many diseases (including in vivo and in vitro experiments).

[0398] AAV vectors can be manufactured by standard methods in this art. The production, purification, and use of many serotypes of adeno-associated viruses have all been tested in numerous laboratory, animal, and human experiments. Methods for purifying vectors are described in various technical books and biotechnical methodology texts, for example, in U.S. Patent Nos. 6566118, 6989264, and 6995006, the contents of which are incorporated herein by reference in their entirety. The manufacture of recombinant AAV vectors is described, for example, in PCT application No. PCT / US2005 / 027091, the contents of which are incorporated herein by reference in their entirety. Embodiments of using recombinant AAV vectors to deliver target genes in vitro and intra vivo have already been described in the literature and patents (see, for example, International Patent Application Publications No. WO91 / 18088 and WO93 / 09239, U.S. Patents No. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference). These patents describe engineered AAV plasmids of various origins (varieties, serotypes, mutation-optimized strains, etc.) with the original rep and / or cap genes removed from the AAV genome, and are used to support exogenous target genes for applications in which recombinant AAV viruses are produced to deliver target genes in vitro (into cultured cells) or intra vivo (directly into living organisms). Based on safety considerations, AAV viruses for therapeutic purposes must be of the replication defect type, and the production of these recombinant AAVs is generally carried out by cotransferring two plasmids into a stable cell line infected with a human helper virus (e.g., adenovirus). Plasmid A is a plasmid with terminal inverted repeat (ITR) elements at both ends, carrying a specific promoter and target expression gene coding sequence, and plasmid B is a plasmid carrying the AAV genome replication gene (rep) and the capsid protein coding gene (cap). In some production systems, based on further safety requirements, the replication gene (rep) and the capsid protein coding gene (cap) may be constructed on separate plasmids.

[0399] In some embodiments, to obtain more favorable delivery and expression effects to target tissue and cell types, recombinant AAV virus expression vectors may be combined with viral capsid protein coding plasmids of different serotypes, where these serotypes or capsid protein coding 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), where these capsid proteins may be induced coding sequences modified by mutation screening or directed evolution in molecular biology. Therefore, this publication may use recombinant AAV viruses containing any of the above serotypes, or AAV serotypes with better efficacy that may be developed in the future. Basic methods for developing, screening, and producing serotypes of such viral capsid proteins are known in this field and are described in published academic literature and U.S. Patent No. 6,596,535.

[0400] In some embodiments, the AAV (particularly the capsid protein) is selected from serotype AAVs capable of crossing the blood-brain barrier, such as PHP.eB or PHP.AX.

[0401] In some embodiments, the astrocyte phosphate transport promoter is an AAV (e.g., PHP.eB or PHP.AX) comprising a nucleic acid molecule encoding the PiT2 protein and a promoter (e.g., gfaABC1D) that is operably linked to it and selectively expressed by astrocytes.

[0402] In some embodiments, the astrocyte phosphate transport promoter described in any of the above embodiments can specifically target astrocytes, for example, by specifically binding to a specific receptor on the membrane of astrocytes. In such embodiments, the astrocyte phosphate transport promoter is linked to a targeting molecule, the targeting molecule having targeting properties for astrocytes. In some embodiments, the astrocyte phosphate transport promoter is packaged entirely or partially within the targeting molecule, or is compounded with the targeting molecule. In some embodiments, the targeting molecule is selected from an antibody or an antibody fragment, a polysaccharide, an aptamer, or nanoparticles.

[0403] In some embodiments, the astrocyte phosphate transport promoter is a small nucleic acid drug, the small nucleic acid drug is linked to a targeting molecule, and the targeting molecule has targeting properties for astrocytes.

[0404] In some embodiments, the small nucleic acid drug is an ASO. In some embodiments, the ASO upregulates the expression levels of PiT2, MYORG, or XPR1.

[0405] In some embodiments, the astrocyte phosphate transport promoter may be a gene editing or base editing preparation capable of regulating mRNA splicing of a target gene, and by being linked to a targeting molecule having the above-described targeting properties for astrocytes, it regulates the expression levels of PiT2, MYORG, or XPR1 in astrocytes. In some embodiments, the gene editing or base editing preparation typically comprises (i) a module having base editing activity and (ii) a module capable of binding to a target gene. Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0406] III-(B). Expression vectors and host cells In one aspect of the present invention, a concept for constructing and designing a plasmid for expressing a phosphate transporter (e.g., PiT2 protein) in astrocytes is provided, the plasmid comprising a promoter that selectively drives transcription of a target gene in astrocytes, and an open expression frame sequence encoding the phosphate transporter (e.g., PiT2 protein). From the provided sequence information, technicians can construct nucleic acid sequences or expression plasmids suitable for transduction into target cells using publicly available molecular cloning techniques.

[0407] In some embodiments, 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, and is used to introduce into astrocytes of the cerebrum of a human or primate animal to express a target gene, and may also be used as a therapeutic vector. The vector may be viral or non-viral (e.g., plasmid or mRNA sequence of LNP packaging). Viral vectors include those derived from the following types of viruses: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpesvirus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, boxvirus, and SV40. Preferably, the viral vector is replication defective, but may be replication deficient, replicable, or conditionally replicable. Viruses can usually remain outside the chromosome and not be integrated into the host cell genome.

[0408] Suitable viral vectors for delivering the nucleic acid sequence encoding the PiT2 protein to astrocytes are AAV vectors, such as intravenously injectable and blood-brain barrier-crossing AAV serotypes, e.g., PHP.eB or PHP.AX, which have proven effective in mice. Intravenous injection and subsequent crossing of the blood-brain barrier enable efficient whole-brain viral delivery. Some methods, such as lymphatic injection or ventricular / lumbar puncture, are still under development and some have achieved whole-brain delivery in mice, potentially enabling delivery to the human brain in the future. By using specific AAV serotypes (e.g., AAV2, AAV5, AAV9), or modified versions of any of these serotypes (including AAV PHP.eB, AAV.CPP.16, and AAV.CPP.21 vectors), and combining them with cell-type specific promoters (e.g., gfaABC1D), selective expression of target cell types can be achieved. Of course, with further advancements in viral technology, if specific infection of astrocytes can be achieved solely through serotype modification, the reliance on selective promoters can be reduced.

[0409] While viral vectors have the ability to enter cells, non-viral vectors, such as plasmids, can also be taken up by target cells when combined with other adjuvants to assemble viral analogs or lipid vectors, and such techniques have proven effective in some human tissues. In the not-too-distant future, such techniques may be used for the selective delivery of astrocytes throughout the brain. Such adjuvants include polycationic liposomes, lipid nanoparticles (LNPs), and the like. The vectors used in this invention are preferably suitable for in vivo use and preferably suitable for human use.

[0410] The vector preferably comprises one or more regulatory sequences to guide the expression of a nucleic acid sequence in astrocytes. The vector may be linked to the regulatory sequences in a “manipulable linkage” manner, and the regulatory sequences comprise one or more sequence modules such as promoters, introns, enhancers, translational regulatory elements, RNA stability regulatory sequences, ligand-induced expression regulatory sequences, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites. The vector may further comprise, for example, selective screening markers, elements of inducible expression in astrocytes, or elements that negatively regulate the expression of a target gene in non-astroglial cells, to support the vector's effective assembly by a virus in a viral production system (e.g., 293 cells).

[0411] "Manipulative linking" means that a nucleic acid sequence is linked to other sequence modules in a specific order or structure based on its functional action, in order to regulate the expression or function of a target gene in a spatiotemporally ordered manner as intended by the design. For example, a nucleic acid sequence manipulatively linked to a promoter may be influenced by the promoter or have a transcriptional regulatory function that affects the promoter's activity.

[0412] A promoter mediates the transcriptional expression of the nucleic acid sequence of an open expression frame of a downstream coding target gene linked to it. Promoters can be constitutive or inductive. Promoters can guide selective expression in astrocytes, or simultaneously in oligodendrocytes and their precursor cells. In the latter case, screening of promoter designs must be validated simultaneously by expression in astrocytes and oligodendrocytes. A suitable promoter is key to achieving specific astrocyte expression in the present invention; for example, in the mouse model described in the present invention, selective astrocyte expression depends on the drive of gfaABC1D to the expression of the PIT2 coding gene, and some studies have verified that the selectivity of the gfaABC1D promoter is conserved and useful in human neurons. Considering that advances in single-cell studies will lead to the screening of more selectable promoters, in the future, more promoters, such as those screened and modified from genes like ALDH1L1, AQP4, and S100B, will be available for achieving selective astrocyte expression.

[0413] From a technical feasibility standpoint, many expression vectors can be applied to achieve the expression of PiT2 protein in astrocytes (preferably primate, more preferably human astrocytes). In this invention, adeno-associated virus is preferably used as the expression vector. This invention provides a method for constructing a recombinant adeno-associated virus expression vector carrying the PiT2 coding sequence. This method allows for the rapid and stable construction of an AAV expression vector carrying the PiT2 coding sequence, and then packaging to obtain a therapeutic AAV virus preparation.

[0414] Furthermore, the present invention provides a host cell containing the vector described above.

[0415] Furthermore, the present invention provides an AAV vector packaging system that includes the following: Exogenous gene expression plasmid: comprising a nucleic acid sequence encoding a phosphate transport promoter (e.g., PiT2 protein) and an astrocyte-specific promoter (e.g., gfaABC1D) operably linked thereto, preferably further comprising terminal inversion repeat sequences (ITRs) located at both ends; Packaging plasmid: Contains Rep and Cap genes.

[0416] In some embodiments, the exogenous gene expression plasmid is AAV2. In some embodiments, the packaging plasmid expresses a PHP.eB serotype capsid protein.

[0417] In some embodiments, the AAV vector packaging system further includes an auxiliary plasmid, the auxiliary plasmid containing genes that support the expression of Rep and Cap.

[0418] Furthermore, the present invention provides a host cell comprising the AAV vector packaging system described above. In some embodiments, the host cell is a genetically engineered cell for generating AAV virus particles. Preferably, the host cell is a mammalian cell (preferably human, more preferably human astrocyte) that enhances the expression level of the PiT2 protein.

[0419] III-(C). Gene therapy vectors In one aspect of the present invention, a gene therapy vector is provided, in particular a gene therapy vector for preventing and / or treating cerebral calcification diseases. The gene therapy vector is selected from expression vectors that enable the selective expression of astrocyte phosphate transport promoters (e.g., PiT2 protein) as described above.

[0420] In some embodiments, the gene therapy vector is a viral expression vector. In some embodiments, the viral expression vector is an adeno-associated virus (AAV) vector. Techniques for serotype and promoter combinations that achieve specific expression of astrocytes throughout the whole brain are already feasible in mice, for example, using the combination of PHP.eB or PHP.AX with promoter gfaABC1D. However, the efficacy of these combinations in primates or humans has not yet been clearly demonstrated, and further engineering modification screening is needed. However, only AAV vectors in the following serotypes AAV1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or their induced chimeric AAV configurations, 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, and AAV clones can be predicted. Potential modifications include serotypes 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, and AAVrh.8. More effective delivery serotype screening involves targeted evolution and screening of capsid protein coding sequences, particularly in-situ screening and validation performed in the tissues and cell types of interest.

[0421] Currently, gene therapy vectors are primarily administered via intravenous injection to deliver astrocytes throughout the brain. With technological advancements, other injection routes, including those through the head and neck lymphatic system, nasal sinuses, ventricular injection, or lumbar puncture injection, may also be capable of efficiently infecting and expressing astrocytes throughout the brain.

[0422] III-(D). Formulations and Compositions In one aspect of the present invention, a formulation or composition is provided comprising (a) an effective amount of a brain phosphate homeostasis promoter as an active ingredient, and (b) a pharmaceutically acceptable carrier or excipient. In this specification, the brain phosphate homeostasis promoter includes an astrocyte phosphate transport promoter.

[0423] In some embodiments, the formulation or composition is a drug composition.

[0424] In some embodiments, the formulation or composition can maintain and / or restore cerebral phosphate homeostasis by promoting and / or repairing the phosphate transport function of astrocytes, thereby preventing and / or treating cerebral calcification caused by defects in cerebral phosphate homeostasis. In some embodiments, the formulation or composition is used for the treatment of hereditary cerebral calcification, which is primary familial brain calcification (PFBC). The formulations of the present invention may be applicable to the treatment of other types of cerebral calcification due to abnormalities in glial cell phosphate transport function, such as some cases of sporadic cerebral calcification, cerebral calcification due to tumors or inflammation, and Aicardi-Goutieres syndrome (AGS).

[0425] To facilitate clinical application, the formulation or composition may be contained in an injection container or a disposable injection device (e.g., an injection needle). The injection device may contain a single dose of the formulation or composition. To facilitate storage and use, the injection device may be contained in a drug kit. During transport, the microcontainer containing the drug suspension must be kept under refrigerated conditions (e.g., dry ice or a refrigerated transport box). For long-term storage, it should be kept in a refrigerator at -80°C, and for short-term storage, it can be kept in a refrigerator at -20°C or 4°C.

[0426] In some embodiments, the “active ingredient” in the formulation or composition is a gene therapy vector as described above, for example, a viral vector (including an adeno-associated virus vector). The “active ingredient,” formulation, and / or composition can be used for the treatment of cerebral calcification. “Effective dose” means that the amount of the active ingredient is clearly sufficient to improve a major clinical observation indicator (e.g., cerebrospinal fluid phosphate ion levels, other biomarkers of calcification), the progression of cerebral calcification, or the severity of major symptoms, preferably without causing serious side effects.

[0427] In some embodiments, the formulation or composition may be a liquid or a solid, for example, a powder, gel, or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients are known to those skilled in the art or have been proven reliable and useful by clinical trials.

[0428] In some embodiments, the formulation or composition can be delivered and expressed to whole-brain astrocytes by intravenous injection or other proven effective injection methods. In any mode of administration, the formulation or composition is preferably provided as an injectable liquid. Preferably, the injectable liquid is provided either as a concentrated solution that is diluted before injection, or as an instant injectable solution filled into a syringe.

[0429] In some embodiments, the formulation or composition may include physiologically acceptable sterile water-containing or aqueous-free solutions, dispersions, suspensions or emulsions, and sterile powders for redissolution into sterile injectable solutions or dispersions. Suitable water-containing or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyhydric alcohols, and suitable mixtures thereof.

[0430] III-(E).Therapeutic use The present invention provides a method for preventing the occurrence of cerebral calcification in advance or for treating cerebral calcification, particularly hereditary cerebral calcification, using an astrocyte phosphate transport promoter. From an applicability standpoint, the method is suitable for all groups of cerebral phosphate homeostasis disruption due to glial cell phosphate transport defects, which include patients with primary familial cerebral calcification (PFBC) associated with mutations in the PiT2, MYORG, or XPR1 genes, and may also include non-PFBC type cerebral calcification due to disruption of cerebral phosphate homeostasis. The method comprises the step of introducing and expressing a vector containing an optimized sequence encoding an astrocyte phosphate transport promoter described in the present invention, such as PiT2, MYORG, or XPR1, into astrocytes. The method may include a nucleic acid expression vector delivered to astrocytes of the cerebrum by intravenous injection (or other technical means), or an activator that modulates the expression levels of PiT2, MYORG, or XPR1 in astrocytes, such as an antisense nucleotide (ASO) drug, or a gene editing or base editing preparation that can modulate mRNA splicing of a target gene. The astrocyte phosphate transport promoter described in the present invention may be administered alone or in combination with other therapeutic agents (for example, in one drug composition or in another drug composition).

[0431] In some embodiments, the use of astrocyte phosphate transport promoters can improve the pathological progression of cerebral calcification or alleviate one or more symptoms of the disease, including restoring the phosphate transport capacity of astrocytes, repairing the disruption of cerebral phosphate homeostasis, and reducing the occurrence of cerebral calcification.

[0432] In some embodiments, the method includes the step of introducing a nucleic acid sequence encoding the PiT2, MYORG, or XPR1 protein into astrocytes of the cerebrum. Preferably, the method includes the step of contacting the cells with a vector (preferably a virus, more preferably an adeno-associated virus) containing a nucleic acid sequence encoding the PiT2 protein. Preferably, the cells are astrocytes.

[0433] In some embodiments, the method delivers an optimized nucleic acid sequence encoding PiT2, MYORG, or XPR1, or a promoter capable of regulating the expression of the PiT2, MYORG, or XPR1 gene, to astrocytes and selectively expresses it in the astrocytes to restore the phosphorus transport capacity of astrocytes, repair the disruption of cerebral phosphorus homeostasis, and ultimately prevent further progression of cerebral calcification in the cerebrum, thereby alleviating associated clinical symptoms.

[0434] In some respects, the present invention provides a method for preventing and / or treating cerebral calcification disease, comprising the step of using an effective amount of a cerebral phosphate homeostasis enhancer in a subject of interest. It also provides the use of a cerebral phosphate homeostasis enhancer for the manufacture of a drug for preventing and / or treating cerebral calcification disease.

[0435] In some embodiments, the brain phosphate homeostasis promoter is an astrocyte phosphate transport promoter as described above. In some embodiments, the prevention includes preventing calcification before it occurs in a subject with a genetic mutation. In some embodiments, the mutation includes mutations in PiT2, MYORG, XRP1, or combinations thereof. In some embodiments, the calcification includes cerebral calcification.

[0436] In some embodiments, the treatment includes preventing and / or delaying the further progression of calcification in the affected area.

[0437] In some embodiments, the treatment includes reversing and / or repairing a disruption of cerebral phosphate homeostasis and / or cerebral calcification.

[0438] In some embodiments, the astrocyte phosphate transport promoter is an expression vector expressing an exogenous gene, the exogenous gene being selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof. In some embodiments, the expression vector includes a viral vector, such as AAV. In some embodiments, the exogenous gene is operably linked to an enhancer element for selective expression of astrocytes and / or a promoter for selective expression of astrocytes. In some embodiments, the promoter is gfaABC1D. In some embodiments, the promoter is a screening-modified promoter derived from a gene such as ALDH1L1, AQP4, or S100B.

[0439] In some embodiments, the astrocyte phosphate transport promoter comprises a small molecule compound, a gene editing reagent, a small nucleic acid drug, or a combination thereof. In some embodiments, the promoter is linked to a targeting molecule, which is a target for astrocytes. In some embodiments, the promoter is packaged entirely or partially within the targeting molecule, or is compounded with the targeting molecule. In some embodiments, the targeting molecule is selected from an antibody or an antibody fragment thereof, a polysaccharide, an aptamer, or nanoparticles.

[0440] In some embodiments, the astrocyte phosphate transport promoter is a small nucleic acid drug. In some embodiments, the small nucleic acid drug includes an antisense oligonucleotide (ASO).

[0441] In some embodiments, the astrocyte phosphate transport promoter is a gene editing reagent. In some embodiments, the gene editing reagent includes a base editing reagent.

[0442] In some embodiments, the prevention or treatment involves introducing the cellular phosphate transport promoter described herein into the astrocytes of a subject.

[0443] In some embodiments, the cerebral calcification disease includes cerebral calcification due to abnormalities in cerebral phosphate homeostasis. As used herein, “abnormalities in phosphate homeostasis,” “disruption of phosphate homeostasis,” “dysregulation of phosphate homeostasis,” and “deficiency of phosphate homeostasis” are interchangeable. As used herein, “dysregulation of phosphate transport” and “deficiency of phosphate transport” are interchangeable.

[0444] In some embodiments, the cerebral calcification disorder includes primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0445] In some embodiments, the drug is administered to subjects selected from a group consisting of PFBC patients with mutations in the PIT2, MYORG, or XPR1 genes, or combinations thereof, or patients with other types of cerebral calcification disorders accompanied by defects in cerebral phosphate homeostasis (e.g., elevated inorganic phosphate ions in cerebrospinal fluid). The other types of cerebral calcification disorders may include other types of cerebral calcification due to abnormalities in glial cell phosphate transport function, such as certain cases of sporadic cerebral calcification, cerebral calcification due to tumors or inflammation, and Aicardi-Goutieres syndrome (AGS).

[0446] In some embodiments, the method further includes detecting gene mutations in pathogenic genes and / or molecules, or combinations thereof, that regulate the expression and function of PiT2, MYORG, XRP1, other PiT2 and / or XPR1 phosphate transporters in the subject's body, and if the detection results indicate the presence of such mutations in the subject's body, administering the therapy to the subject.

[0447] In some embodiments, the method further includes the steps of collecting cells from a subject, differentiating the cells into astrocytes, detecting the phosphate transport function of the astrocytes, and if the detection results show that the phosphate transport capacity C1 of the subject's astrocytes is significantly lower than the phosphate transport capacity C0 of astrocytes from a normal individual, then administering the therapy to the subject. In some embodiments, "significantly lower than" means that the ratio (C1 / C0) of the phosphate transport capacity C1 of the subject's astrocytes to the phosphate transport capacity C0 of astrocytes from a normal individual is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 4.

[0448] In some embodiments, the defect in brain phosphate homeostasis includes a defect in phosphate transport by astrocytes in the brain.

[0449] In some embodiments, the cerebral calcification disease includes cerebral calcification diseases caused by disruption of cerebral phosphate homeostasis.

[0450] In some embodiments, subjects (or patients) of cerebral calcification disease in which cerebral phosphate homeostasis is disrupted have astrocyte phosphate transport capacity C1 that is significantly lower than that of astrocytes in normal individuals. In some embodiments, "significantly lower than" means that the ratio of astrocyte phosphate transport capacity C1 to that of astrocytes in normal individuals (C1 / C0) is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 4.

[0451] In some embodiments, 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 XRP1 phosphate transporters, or a combination thereof. In some embodiments, the genetic mutation includes exon mutations, intron mutations, 5'-UTR mutations, 3'-UTR mutations, or a combination thereof.

[0452] In some embodiments, the subject includes an animal model. In some embodiments, the subject includes a mammal. In some embodiments, the subject includes a human. In some embodiments, the subject includes a non-human mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject includes a primate. In some embodiments, the subject includes a rodent. In some embodiments, the subject includes a human, mouse, rat, monkey, or a combination thereof.

[0453] In some embodiments, the administration to the subject includes intravenous injection, subcutaneous injection, head and neck lymphatic injection, nasal sinus administration, cerebral ventricle injection, cerebral parenchymal injection, lumbar puncture injection, or a combination thereof.

[0454] In some respects, the present invention also provides a drug kit for preventing and / or treating cerebral calcification diseases. The drugs include: (a) A first container, and a first detection reagent for detecting gene mutations in the first container, which is a reagent for detecting the presence or absence of gene mutations in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and (b) A second container, and a drug composition contained in the second container, comprising a drug component capable of regulating and restoring a disruption of cerebral phosphate homeostasis and a pharmaceutically acceptable carrier or excipient.

[0455] In some embodiments, the drug component includes a cerebral phosphate homeostasis promoter.

[0456] In some embodiments, the drug component comprises an astrocyte phosphate transport promoter as described herein.

[0457] In some embodiments, the drug kit further comprises (c) a third container and a solution in the third container that assists in the dissolution or delivery of the drug composition in the second container into the body.

[0458] In some embodiments, the drug kit optionally further comprises (d) a fourth container and a fourth detection reagent for detecting the phosphate transport function of astrocytes in the fourth container.

[0459] In some embodiments, the drug composition in the second container is administered to subjects selected from a group consisting of PFBC patients with mutations in the PIT2, MYORG, XPR1 genes, or combinations thereof, or patients with other types of cerebral calcification disorders accompanied by deficiencies in cerebral phosphate homeostasis (e.g., elevated inorganic phosphate ions in cerebrospinal fluid). In some embodiments, the deficiency in cerebral phosphate homeostasis includes deficiencies in phosphate transport by astrocytes in the brain. In some embodiments, the cerebral calcification disorder includes cerebral calcification disorders due to disruption of cerebral phosphate homeostasis. In some embodiments, subjects (or patients) with cerebral calcification disorders with disrupted cerebral phosphate homeostasis have astrocyte phosphate transport capacity C1 significantly lower than the astroclastic phosphate transport capacity C0 of astrocytes in a normal individual. In some embodiments, "significantly lower than" means that the ratio of astroclastic phosphate transport capacity C1 to astroclastic phosphate transport capacity C0 of astrocytes in a normal individual (C1 / C0) is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 4. In some embodiments, 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 XRP1 phosphate transporters, or a combination thereof. In some embodiments, the genetic mutation includes exon mutations, intron mutations, 5'-UTR mutations, 3'-UTR mutations, copy number variations (CNVs), or a combination thereof.

[0460] In some embodiments, a first detection reagent is used to detect gene mutations in pathogenic genes and / or molecules, or combinations thereof, that regulate the expression and function of PiT2, MYORG, XRP1, other PiT2 and / or XPR1 phosphate transporters in the body of a subject of interest, and if the detection results indicate the presence of such mutations in the subject, the subject is deemed suitable for treatment with the drug composition in the drug kit.

[0461] In some embodiments, target cells are collected, differentiated into astrocytes, and the phosphate transport function of the astrocytes is detected using the fourth detection reagent. If the detection result shows that the phosphate transport capacity C1 of the target astrocytes is significantly lower than the phosphate transport capacity C0 of astrocytes from a normal individual, then the target is deemed suitable for treatment with the drug composition in the drug kit. In some embodiments, "significantly lower than" means that the ratio (C1 / C0) of the phosphate transport capacity C1 of the astrocytes derived from the target to the phosphate transport capacity C0 of astrocytes from a normal individual is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 4. In some embodiments, the cells include primary cells and passaged cells. In some embodiments, the cells include somatic cells, germ cells, gametes, and stem cells. In some embodiments, the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cells include embryonic stem cells, adipose-derived stem cells, neural stem cells, mesenchymal stem cells, oligodendrocytes, hematopoietic stem cells, and immune cells (e.g., T cells, B cells, NK cells). In some embodiments, the cells include healthy, normal human cells and cancerous human cells. In some embodiments, the cells include suspended cells and adherent cells. In some embodiments, the cells do not include embryonic stem cells. In some embodiments, the cells do not include human embryonic stem cells.

[0462] In some embodiments, the subject includes an animal model. In some embodiments, the subject includes a mammal. In some embodiments, the subject includes a human. In some embodiments, the subject includes a non-human mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject includes a primate. In some embodiments, the subject includes a rodent. In some embodiments, the subject includes a human, mouse, rat, monkey, or a combination thereof.

[0463] In some embodiments, the first container and the second, third, and fourth containers are the same or different containers.

[0464] In some embodiments, the drug composition in the second container comprises a single and / or complex formulation comprising a guide RNA or its expression vector, and / or a gene editing protein or its expression vector. In some embodiments, the gene editing protein comprises a base editing protein. In some embodiments, the single and / or complex formulations in the second container may be stored separately in different drug containers depending on the requirements for drug transport and storage.

[0465] In some embodiments, the drug composition in the second container is a single formulation containing a drug useful for treating other cerebral calcification disorders.

[0466] In some embodiments, the dosage form of the drug composition in the second container is selected from the group consisting of lyophilized formulations, liquid formulations, or combinations thereof.

[0467] In some embodiments, the dosage form of the drug composition in the second container includes an injectable dosage form, a transdermal adsorbent dosage form, a nasal inhalation dosage form, or a combination thereof.

[0468] In some embodiments, the drug kit further includes instructions.

[0469] III-(F). Drug Screening The present invention provides a method for screening candidate drugs for preventing and / or treating cerebral calcification diseases, comprising the following steps: (a) In the test group, the test drug was added to a culture system of astrocytes, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group was observed. In the control group, the test compound was not added to the same cell culture system, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group was observed. If the cellular phosphorus transport levels (E1) and / or activity (A1) in the test group are significantly higher than in the control group, it indicates that the test compound is a candidate drug for preventing and / or treating cerebral calcification disorders that promotes the expression and / or activity of phosphorus transport levels.

[0470] In some embodiments, the test drug is a compound.

[0471] In some embodiments, "significantly higher than" means E1 / E0 ≥ 1.5, preferably ≥ 2.

[0472] In some embodiments, "significantly higher than" means A1 / A0 ≥ 1.5, preferably ≥ 2.

[0473] In some embodiments, the cells include astrocytes.

[0474] In some embodiments, the cells are cells that are cultured outside the body.

[0475] In some embodiments, the method includes step (b) of applying the candidate drug identified in step (a) to a mammalian model and measuring its effect on the mammal. In some embodiments, the mammal is a mammal suffering from a cerebral calcification disease.

[0476] In some embodiments, the method described above is non-diagnostic and non-therapeutic.

[0477] III-(G). Exemplary Forms The present invention relates to the following exemplary embodiments.

[0478] Embodiment 1. Use of a cerebral phosphate homeostasis promoter, characterized in that it is for the purpose of manufacturing a drug for preventing and / or treating cerebral calcification disease.

[0479] Embodiment 2. The use described in Embodiment 1, characterized in that the cerebral phosphate homeostasis promoter is selected from the following group: (Z1) PiT2 protein, nucleic acid molecule encoding PiT2 protein, expression vector expressing PiT2 protein, PiT2 promoter, or combination thereof; (Z2) MYORG protein, nucleic acid molecule encoding MYORG protein, expression vector expressing MYORG protein, MYORG enhancer, or combination thereof; (Z3) XRP1 protein, nucleic acid molecule encoding the XRP1 protein, expression vector expressing the XRP1 protein, XRP1 enhancer, or combination thereof; (Z4) Any combination of Z1 to Z3 above.

[0480] Embodiment 3. The use of the sex-promoting agent according to Embodiment 1, characterized in that it includes a small nucleic acid drug.

[0481] Embodiment 4. The use according to Embodiment 1, characterized in that the cerebral calcification disease includes primary familial brain calcification (PFBC).

[0482] Embodiment 5. A formulation and drug composition characterized by containing a cerebral phosphate homeostasis promoter.

[0483] Embodiment 6. The formulation and composition according to Embodiment 5, characterized in that the cerebral phosphate homeostasis promoting agent is selected from the following group: (Z1) PiT2 protein, nucleic acid molecule encoding PiT2 protein, expression vector expressing PiT2 protein, PiT2 promoter, or combination thereof; (Z2) MYORG protein, nucleic acid molecule encoding MYORG protein, expression vector expressing MYORG protein, MYORG enhancer, or combination thereof; (Z3) XRP1 protein, nucleic acid molecule encoding the XRP1 protein, expression vector expressing the XRP1 protein, XRP1 enhancer, or combination thereof; (Z4) Any combination of Z1 to Z3 above.

[0484] Embodiment 7. A drug kit for treating cerebral calcification disease, characterized by comprising the following: (a) A first container, and a first detection reagent for detecting gene mutations in the first container, which is a reagent for detecting the presence or absence of gene mutations in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and (b) A second container, and a drug composition contained in the second container, comprising a drug component capable of regulating and repairing a disruption of phosphate homeostasis in the brain and a pharmaceutically acceptable carrier.

[0485] Embodiment 8. The drug kit according to Embodiment 7, further comprising (c) a third container and a solution in the third container that assists in the dissolution or delivery of the drug in the second container into the body.

[0486] Embodiment 9. A method for screening candidate compounds for preventing and / or treating cerebral calcification disease, comprising the following steps: (a) In the test group, the test compound was added to a culture system of astrocytes, and the phosphorus transport level (E1) and / or activity (A1) in the cells of the test group was observed. In the control group, the test compound was not added to the same cell culture system, and the phosphorus transport level (E0) and / or activity (A0) in the cells of the control group was observed. If the cellular phosphorus transport levels (E1) and / or activity (A1) in the test group are significantly higher than in the control group, it indicates that the test compound is a candidate compound for preventing and / or treating cerebral calcification disease that promotes the expression and / or activity of phosphorus transport levels.

[0487] Embodiment 10. The method according to Embodiment 9, characterized in that it includes step (b) of applying the candidate compound identified in step (a) to a mammalian model and measuring its effect on the mammal.

[0488] III-(H). Beneficial Effects and Summary This invention, for the first time, describes how astrocyte-mediated phosphate transport is a crucial part of regulating brain phosphate homeostasis. This process involves the uptake of inorganic phosphate ions (Pi) from extracellular interstitial fluid by the PiT2 transporter expressed on the membrane of the glial cell leaflet, and the efflux of Pi taken up by glial cells to the outside of the brain through the blood-brain barrier via the XPR1 transporter on the membrane of the endfoot structure. Conditional knockout experiments in a mouse animal model showed that when the astrocyte-mediated phosphate transport ability was blocked for other cell types, the most significant increase in cerebrospinal fluid phosphate ions occurred, indicating a breakdown of homeostasis. Simultaneously, in-situ hybridization-based expression studies showed that astrocytes highly express the uptake-type phosphate transporter PiT2 and the efflux-type phosphate transporter XPR1, while neurons highly express another uptake-type phosphate transporter, PiT1.

[0489] (1) From this discovery, it was found that delivering the virus into astrocytes of Pit2-KO mice and specifically expressing the PiT2 protein in astrocytes significantly reduced phosphate transport levels in the brains of Pit2-KO mice and inhibited the development and progression of calcification in the brain.

[0490] In this method, the present invention is based on the core cellular pathological mechanism that cerebral calcification is caused by a breakdown of phosphate homeostasis. It selectively expresses PiT2 in astrocytes, restores the phosphate transport capacity of astrocytes, repairs the disrupted cerebral phosphate homeostasis, and fundamentally eliminates the most basic driving element for the development of calcification.

[0491] It should be explained that this method not only treats the progression of cerebral calcification but also restores cerebral phosphate homeostasis. In the patient's brain, cerebral phosphate homeostasis is first disrupted due to the influence of genetic mutations or acquired factors, and this disruption leads to the development of cerebral calcification. Therefore, the most essential treatment of this method is the restoration of the disrupted cerebral phosphate homeostasis. In the future, just as elevated blood glucose is recognized as diabetes, or elevated blood lipids as a pathology requiring immediate treatment, if clinical professionals recognize the disruption of phosphate homeostasis as a disease, this treatment strategy will be useful to a wider range of recipients.

[0492] Furthermore, the core methodology revealed by this method involves targeting astrocytes to repair the disruption of phosphate homeostasis in the brain. Those skilled in the art will see that this method may be based on different delivery strategies and can be further optimized in the future as technology advances.

[0493] Examples The present invention will be described below with reference to the following examples (which are not intended to limit the present invention).

[0494] Those skilled in the art will see that the examples illustrate the present invention in an illustrative manner, but do not limit the scope of protection claimed in this application. Those skilled in the art will see that all previously disclosed teachings can be modified and altered in detail, and that any such alterations fall within the scope of protection of this invention. The entire present invention is given by the appended claims and any equivalent thereof. The experimental methods in the examples are all conventional methods unless otherwise specified. In the examples, unless specific conditions are described, the experiments were conducted under conventional or manufacturer-recommended conditions. Unless otherwise specified, the reagents or apparatus used are typically common products available commercially.

[0495] Materials and methods 1. Recruitment of subjects, whole-genome sequencing, and genetic analysis. Between 2012 and 2023, we recruited 778 patients suspected of having PFBC from the Department of Neurology, First Affiliated Hospital, Fujian Medical University. The diagnostic criteria for PFBC are as follows: a) bilateral calcifications detected by CT scan and symmetrical in the basal ganglia and / or dentate nucleus; b) total calcification score (TCS) exceeding a previously reported threshold for a specific age; c) no biochemical abnormalities and normal serum calcium, phosphorus, and parathyroid hormone (PTH) levels; d) no cause of infection, poisoning, or trauma. Neurological examination and clinical evaluation were performed by at least two senior neurologists. For all suspected patients, we first eliminated cases containing mutations in all coding regions and exon sequences of known PFBC-related genes such as SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, JAM2, and CMPK2 by whole exome sequencing (WES). Then, from the remaining sample set, we identified cases and families with PFBC-related intronic mutations. This study was authorized by the Institutional Review Board of the First Affiliated Hospital of Fujian Medical University, and written informed consent was obtained from all participants prior to enrollment.

[0496] To perform whole-genome sequencing, genomic DNA was obtained from the blood of patients with cerebral calcification. Paired DNA libraries were then prepared using human DNA samples according to the manufacturer's instructions for the relevant sequencing library construction kit (Illumina Truseq library construction). The constructed DNA libraries were subjected to second-generation sequencing with 150 bp lengths at both ends using an Illumina NovaSeq 6000. Mean sequencing depths ranged from 31.35 to 57.77, with at least 20 segments covering 90.1% to 99.2% of the entire genome. The sequencing results (without barcodes) were then aligned with the human reference genome hg19 using SpeedSeq. Single-nucleotide mutations and insertions / deletions (indels) were then analyzed using Genome Analysis Toolkit v4.1. Potential genomic structural and copy number variations in the patients' genomes were extracted using SpeedSeq. Alternatively, ANNOVAR was used to annotate single-nucleotide mutations, insertions / deletions, structural variations, and copy number variations.

[0497] For parametric linkage analysis, DNA samples were isolated from the whole blood of 45 family members of family 1, and whole-genome linkage analysis was performed using HumanOmniZhongHua-8 BeadChip (Illumina). Genotype calling and quality control were processed using the GenomeStudio genotyping module (Illumina). SNPs with Mendelian errors or calling rates less than 100% were removed. Parametric linkage analysis was performed using Merlin (v1.1.2), with a rare disease allele frequency of 0.0001, creating a rare-dominant model.

[0498] 2. Construction of mini-gene plasmids To rapidly analyze splicing events involving potential hidden exons using RT-PCR, DNA fragments of wild-type or mutant genomic sequences containing exon 2-intron 2-exon 3, exon 6-intron 6-exon 7, and exon 10-intron 10-exon 11 in the human SLC20A2 gene were constructed on mini-geneskeleton plasmids with a CMV promoter. To test the possible modifications of the SLC20A2 mRNA translation product after the incorporation of the mutation-associated hidden exon, SLC20A2 cDNA containing the corresponding hidden exon incorporation was cloned into a pCMV6-Entry plasmid (Origene) using the ClonExpress MultiS One Step Cloning Kit (Vazyme). Patient-derived mutations were then induced by overlap PCR or the Mut Express II rapid mutagenesis kit (Vazyme). Primers for amplifying these fragments are shown in Table 3.

[0499] 3. Antisense oligonucleotides (ASOs) Antisense oligonucleotide technology is a drug development strategy using chemically modified short-chain oligonucleotides (12-24 nt) that bind to their RNA targets through Watson-Crick base pairing. The sequences of the ASOs were designed according to the ASO design guidelines. ASOs with MOE and PS modifications (2'-O-methoxymethyl sugar with a thiophosphate ester backbone) were synthesized by Integrated DNA Technologies and purified by HPLC. ASOs with PMO modifications (phosphodiamide morpholine backbone) were synthesized by Gen-Tools, LLC. All ASOs were dissolved in nuclease-free water in in vitro cell culture experiments and in artificial cerebrospinal fluid (aCSF) in in vivo mouse experiments. Detailed sequences are shown in Table 1.

[0500] 4. Construction of the SLC20A2-KI mouse model To construct a humanized SLC20A2 mouse model, the genomic fragment from exon 2 to exon 3 of the SLC20A2 gene was replaced with the genomic sequence of the human SLC20A2 gene containing the c.289+1007 C>G mutation, matching the code frame, using CRISPR / Cas9 technology. In short, the target sequence with bilateral 1.4 kb homology arms was microinjected into the uterus of C57BL / 6J mice along with sgRNAs and Cas9 RNA. To screen for founder (F0) mice with the accurate gene knock-in sequence, genomic DNA extracted from the mouse tail was used for genotyping. Crossing with C57BL / 6J wild-type mice generated F1 KI mice, and germline transmission of F0 KI mice was demonstrated. To further validate target mice with more accurate homologous recombination, long-chain sequencing and Southern blotting analysis were performed on further F1 mice. We selected heterozygous and homozygous SLC20A2-KI mice and performed biochemical and phenotypic analyses. The sgRNA sequences and primers used for genotyping are shown in Table 3.

[0501] 5. Cell culture and translocation HEK293T, Neuro-2A, A172, HepG2, HeLa cells (Cellular Collection of the Chinese Academy of Sciences) and primary human fibroblasts were cultured in 6-well or 12-well plates (Corning) in DMEM supplemented with 10% (vol / vol) fetal bovine serum (FBS). The human fibroblasts were derived from PFBC patients with SLC20A2 heterozygous c.289+1007 C>G mutations and SLC20A2 heterozygous deletions, as well as from healthy control individuals donated by volunteers.

[0502] For plasmid translocation, approximately 3 μg of minigene or cDNA plasmid was translocated into HEK293T and Neuro-2A cells using Lipofectamine 3000 (Thermo Fisher). Western blotting was performed by collecting cells 24 hours after translocation. Splicing analysis by RT-PCR was performed by translocating cells with MOE or PMO-modified ASO after 24 hours of culture.

[0503] MOE-modified ASO was transfused into cells at a final concentration of 100 nM using Lipofectamine 3000 (Thermo Fisher) and continued for 48 hours. PMO-modified ASO was transfused into cells at a concentration of 4 μM PMO using 6 μL / mL Endo-Porter (Gene-Tools, LLC) for 48 hours. To suppress RNA degradation via NMD, human fibroblasts cultured with cyclohexanone (CHX, 50 μg / mL) were incubated for 6 hours prior to collection.

[0504] 6. RNA extraction and splicing analysis Following the manufacturer's instructions, total RNA was extracted from HEK293T, Neuro-2A, HepG2, HeLa, A172 cells, or human fibroblasts using TRIzol (Invitrogen). Then, 1 μg of RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). For splicing analysis, the transcribed cDNA was subjected to PCR using primer pairs spanning the corresponding exons of the SLC20A2 gene. The primer sequences are shown in Table 3.

[0505] 7. Protein extraction and Western blotting analysis Cells or mouse cerebral cells were collected in RIPA degradation buffer containing a protease inhibitor, and the degradation solution was centrifuged at 14,000 g for 15 minutes. To detect PiT2, protein extracts were incubated at 37°C for 10 minutes, then separated on a 9% SDS-PAGE gel and blotted onto PVDF film. After blocking the film with 5% skimmed milk, detection was performed using PiT2-7B5 (1:1000, monoclonal antibody obtained by immunizing mice with the loop region of the PiT2 protein), HA-tag (1:5000, #3724, Cell Signaling), GAPDH (1:3000, KC-5G5, KANGCHEN), α-tubulin (1:5000, 66031-1-Ig, Proteintech), and corresponding HRP-labeled secondary antibodies (1:2000, Cell Signaling). The bands were observed using ECL(TIANGEN) and measured using ImageJ software.

[0506] 8. Intracerebral (ICV) injection of ASO into mice All animal surgeries were performed under standard sterile conditions. Mice were anesthetized with isoflurane (5% for induction and 1.5% for maintenance), placed in a small animal stereotactic frame (Model 68528, RWD Life Science), and their body temperature was maintained with a heating pad. 2% lidocaine was injected into the incision site of the SLC20A2-KI mice, and the hair was shaved with depilatory cream. After disinfecting the scalp at the surgical site three times with betaine and 75% alcohol, a midline incision was made, the scalp was removed to expose the skull, and the periosteum and other soft tissues connected to the skull were cleaned with cotton swabs and a scalpel. After drying the skull, the skin around the skull was attached to the exposed periphery of the skull with tissue adhesive (3M Vetbond Tissue Adhesive). The plane of the skull was adjusted to the horizontal for stereotactic purposes, and bregma was checked. The frontal bone and surrounding area were coated with blue light-curing self-etching adhesive (3M ESPE Single Bond Universal, 3M Deutschland GmbH) to aid in fixation. A custom-made headplate was then flattened to the frontal bone with gutta-percha. A small annular craniotomy was carefully performed while the dura mater remained intact (AP + 0.25 mm; ML - 0.75 mm). The dura mater was protected by covering with antibiotic ointment and a fast-curing silica resin elastomer. The mice were transferred to the custom-made fixation plate with 1.5% isoflurane, and cerebrospinal fluid was collected from the tympanic cavity. The mice were then allowed to recover for at least one week before injection of ASO.

[0507] For ICV injection, mice were anesthetized with isoflurane (5% used for induction and 1% for maintenance), and their heads were fixed to a stereotactic frame with a heating pad to maintain body temperature. ASO powder was dissolved in filtered, custom-modified artificial cerebrospinal fluid (125 mM NaCl, 20 mM NaHCO3, 3 mM KCl, 1.2 mM MgSO4, 1 mM NaH2PO4, H2O, 1.5 mM CaCl2) at a concentration of 25 μg / μL. SLC20A2-ASO or control-ASO in total doses of 50 μg, 100 μg, 200 μg, or 400 μg were injected into the right ventricle (central coordinates: AP +0.25 mm; ML -0.75 mm; DV -1.85 mm) at a rate of 1 nL / s using a nano-injector (Nanoject III, Drummond Scientific Company). The glass pipette was withdrawn at least 10 minutes after injection. The craniotomy site was sealed with a flowable resin that hardens under blue light to prevent infection. After the mouse recovered from anesthesia on a heated pad, it was returned to its cage.

[0508] 9. CSF collection and Pi measurement Following a laboratory-established protocol, mice were immobilized using a stereotactic frame (Model 68528, RWD Life Science), and a collection needle was held in a three-way adjustable holder with a separate ultrafine scale. A stretched glass needle was attached to the needle and punctured into the ventricular region of anesthetized adult mice through the posterior medullary cisternum of the cerebrum. A light load was then applied to guide the flow of CSF into the glass needle. After collecting approximately 5–10 μL of CSF, the sample was centrifuged for 5 minutes (2000 g, 4°C) to remove cell debris. The concentration of inorganic phosphate in the CSF was detected using a PiColorLock gold phosphate detection kit (303-0030, Innova Biosciences) and read at 635 nm using a SpectraMax (molecular device).

[0509] 10. Detection of ASO distribution in mouse cerebrum by in-situ hybridization (ISH) All reagents used in the ISH analysis were prepared with diethyl carbonate (DEPC). Seven-month-old SLC20A2-KI mice of MOE-A and MOE-NC were anesthetized with chloral hydrate, perfused with 4% PFA, and subsequently dehydrated with 15% and 30% sucrose. miRNAscope was used according to the manufacturer's instructions. TM Brain sections with a thickness of 18 μm were prepared using Assay Red (Advanced Cell Diagnostics, Cat. No. 324500) and a custom-made MOE-A probe (Advanced Cell Diagnostics, Cat. No. 1123181-S1), and ISH was performed. Whole-brain MOE-A expression images were taken using a VS120 microscope (Olympus, Japan) with a 40× objective lens.

[0510] 11. Analysis of cerebral calcification by micro-CT and histochemical staining To assess cerebral calcification, SLC20A2-KI and WT mice were anesthetized with chloral hydrate and perfused with PBS containing 4% PFA. Micro-CT scans were performed using an X-ray micro-CT scanner (Bruker, Skyscan 1172) to obtain three-dimensional images of the mouse cerebrum. The paraffin-embedded mouse cerebrum was evaluated in high-resolution mode with an X-ray tube voltage of 90 kV, a current of 88 μA, and a voxel size of 72 μm.

[0511] To perform histochemical staining of brain calcifications, mouse brains were dehydrated with a series of gradient ethanol treatments before paraffin embedding, and then sections 4 μm thick were prepared. Brain calcifications were evaluated using widely accepted and standard methods in this field. These staining methods included alizarin red, fonkossa, Alcian blue, periodic acid-Schiff (PAS), and H&E. Each analysis was repeated three times.

[0512] 13.Statistical analysis The data is presented as the mean ± standard error (SEM) of the mean. Statistical significance is assessed by a Student's t-test. A difference of <0.05 is considered statistically significant and is expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0513] Example 1: Identification of SLC20A2 intron mutations in PFBC patients and analysis of resulting SLC20A2 splicing and translation abnormalities. After years of hard work, the inventor accumulated hundreds of cases of cerebral calcification to construct a family tree, and through exon sequencing genetic analysis, discovered pathogenic mutations in the exons of known PFBC pathogenic genes (SLC20A2, PDGFB, PDGFRB, XPR1, MYORG, and JAM2) in 33 (62.2%) of the 53 PFBC families. Furthermore, CNV analysis confirmed deletions of the lateral exon segment of the SLC20A2 gene in three other families. Of the remaining 17 families, linkage analysis was performed on a large family (Family 1) (Figure 1a) in which no exon mutations were detected. Association mapping analysis of genomic DNA samples from 45 members of the family using an Affymetrix 250K Nsp SNP chip revealed a 32Mb segment on chromosome 8 (hg19 chr8: 29042453-61341711) linked to the calcification phenotype in the family, with an LOD score of 4.5. Further WGS analysis revealed a deep-intronic variant c.289+1007 C>G in SLC20A2, and Sanger sequencing confirmed that this mutation is associated with cerebral calcification in the family. Computer-based evaluation of this mutation using SpliceAI revealed an acceptor gain score of 0.11 and a donor gain score of 0.11, indicating that an unreported hidden exon was introduced from intron 2 of SLC20A2 through splicing (Table 2).

[0514] Screening patients of the PFBC family lacking other clear genetic diagnoses using similar WGS analysis and Sanger sequencing methods revealed three additional deep intron region mutations in the SLC20A2 gene across four families (Figure 1a): c.289+937 G>A in intron 2 (families 2 and 3), c.730+768 C>T in intron 6 (families 4), and c.1795-1698 A>G in intron 10 (families 5). None of these four intron mutations (Figure 1c) are registered in major human population genetics databases, and SliceAI analysis predicted that all of them would affect the splicing process (Table 2). Furthermore, these mutations were found to be associated with cerebral calcification in these five families (Figure 1b). The ages of the subjects varied from 30 to 60 years. Four of these patients (Families 1, 3, 4, and 5) reported headaches, and one of them (Family 2) experienced dizziness. CT imaging revealed moderate to severe cerebral calcification in the basal ganglia and dentate nucleus (Families 1-5), thalamus (Families 1-3), and subcortical white matter (Families) (TCS, 16-39) (Figure 1b). Serum calcium, phosphorus, and PTH levels were normal in all patients (Table 2). These results support the possibility that intronic mutations in SLC20A2 may be the genetic cause leading to PFBC.

[0515] In mammals, the 5' splice donor site and 3' splice acceptor site of introns, which have already been identified, are currently conserved. Intron mutations affect mRNA processing by disrupting splice sites and / or splicing regulatory elements, or by altering the secondary structure of the mRNA molecule. Further sequence predictions show that the c.289+937 G>A mutation in intron 2 and the c.730+768 C>T mutation in intron 6 both modify the original splicing mechanism by directly generating new splicing acceptors or donors, while the c.289+1007 C>G mutation in intron 2 and the c.1795-1698 A>G mutation in intron 10 do not generate characteristic splicing acceptors or donors, suggesting that they may be due to an alternative mechanism, such as adjusting the probability of splicing inclusion of hidden exons.

[0516] To validate the genetic predictions, we first performed minigene analysis to explore how the introns found in the two families alter the splicing of hidden introns in SLC20A2. Specifically, we cloned the SLC20A2 genome sequence (including the linker sequences of exons 2-3 upstream and downstream of intron 2, and the linker sequences of exon 10 and exon 11 of intron 10), inserted it into a skeletal plasmid driven by the CMV promoter, and constructed wild-type plasmids of the minigenes. Site-directed mutations were then induced to generate point mutations corresponding to the c.289+937 G>A and c.1795-1698 A>G intron mutations in the two PFBC families (Figure 2a, c).

[0517] Detection of the minigene demonstrated that in families 1 and 5, the c.289+1007 C>G and c.1795-1698 A>G mutations did not lead to the development of new splice sites. However, RT-PCR and Sanger sequencing showed increased splicing incorporation of 135 nt and 76 nt hidden exons in intron 2 and intron 10, respectively (Figure 2b, d). Conversely, cells of wild-type SLC20A2 minigenes that do not express these two mutations did not show significant levels of abnormal transcripts upon detection of the aforementioned mutated minigenes, suggesting that these two mutations may alter the probability of hidden exon splicing incorporation by affecting adjacent splicing regulatory elements.

[0518] Furthermore, the effect on the protein product was further analyzed by inserting the aforementioned 135 nt and 76 nt hidden exons into the corresponding sequence positions of the CDS of SLC20A2 with the N-terminus labeled with HA. Sequence predictions indicated that the incorporation of these two hidden exons would introduce a premature termination codon (Figure 2g) into the new code frame, thus preventing the synthesis of a functional PiT2 protein through translation. When cultured Neuro-2A cells were transfused with either a minigene construct of SLC20A2 labeled with HA containing these two hidden exons, or a minigene construct of SLC20A2 containing new exons from two different novel splicing acceptor / donor structures, Western blotting results showed a shortened PiT2 protein product consistent with the predictions (Figure 2h). Based on experience, in vivo, the presence of PTC mutations in these abnormal transcripts may trigger an NMD degradation mechanism, ultimately preventing the production of significantly expressed protein products. Therefore, compared to fibroblasts from healthy controls, we found a clear decrease in full-length PiT2 levels in patient-derived fibroblasts with the c.289+1007 C>G mutation (Figure 2i). These findings together support a novel and important molecular genetic pathology mechanism: intronic mutations in the SLC20A2 gene lead to increased splicing incorporation of endogenous hidden exons, resulting in SLC20A2 haploinsufficiency and subsequent cerebral calcification of PFBC family mutation carriers.

[0519] Example 2: The human brain contains endogenous hidden exons involved in the formation of selective screening isomers of SLC20A2. In vitro evidence in the above examples suggests that the c.289+1007 C>G and c.1795-1698 A>G mutations possess the ability to introduce hidden exons during the process of mRNA splicing in intron 2 and intron 10, and computer analysis proved that the development of splice sites was not expected. The important question is whether these hidden exons are present in human cells in the first place, and whether in normal splicing these abnormal splicing isomers, which are produced at a low rate, are simply degraded.

[0520] We investigated the presence of hidden exons that regulate potential splicing in each intron of SLC20A2 in HEK293T cells (renal-derived cell lineage) and A172 cells (brain-derived cell lineage). RT-PCR analysis detected RNA transcripts in cells in which the degradation of abnormal splicing transcripts was suppressed by CHX treatment. This revealed the existence of a cryptic exon inclusion event between exon 2 and exon 3 in HEK293T and A172 cells (c.289+901~c.289+1035, named "crpE2"), and a clear cryptic exon inclusion event between exon 10 and exon 11 was observed in A172 cells (c.1795-1703~c.1795-1628, named "crpE10") (Figure 3a, b). Notably, the expression levels of the hidden exons crpE2 or crpE10 contained in these aberrant splicing transcripts were sensitive to cyclohexanone (CHX), and since CHX evaluates potential small molecule inhibitors involved in the NMD pathway, this suggests that under normal circumstances, transcripts incorporating crpE2 or crpE10 may be degraded by the NMD pathway in the cerebrum. Sanger sequencing demonstrated that crpE2 and crpE10 are indeed the 135 nt and 76 nt splicing products of minigenes with c.289+1007 C>G and c.1795-1698 A>G mutations (Figure 3b). These results indicate that the introduction of these two hidden exons does indeed exist spontaneously in human cells (and this situation is clearly increased in the presence of the c.289+1007 C>G and c.1795-1698 A>G intronic mutations).

[0521] RT-PCR analysis of a wider range of human cells (HeLa cells, HepG2 cells, HEK293T cells, A172 cells, and epithelial-derived human fibroblasts) further verified that splicing incorporation of crpE2 and crpE10 in SLC20A2 mRNA transcripts can occur in different cell types from many tissues (Figure 3c), suggesting that this splicing mechanism can be used in disease-related contexts by regulating PiT2 expression.

[0522] Example 3: ASO can correct abnormal splicing involving crpE2 or crpE10, thereby improving the translation and production efficiency of functional PiT2 proteins. To evaluate whether ASOs can suppress selective screening dependent on hidden exons, we transfected Neuro-2a cells with a construct of the SLC20A2 minigene and cotransfected them with ASO formulations targeting the splice sites of crpE2 and / or crpE10 to suppress the production levels of transcripts with abnormal splicing. Of the various ASO modifications, 2'-O-methoxyethyl (MOE) and phosphorodiamidate morpholino oligonucleotides (PMOs) are the most common modification forms used in commercially available neurological ASO drugs, and we analyzed ASOs with these two types of modifications separately. For minigenes containing the c.289+1007 C>G mutation, 4 μM PMO-ASO (PMO-1, PMO-2, PMO-3) (PMO-1: 65.38 ± 3.081%; PMO-2: 67.17 ± 5.045%; PMO-3: 63.27 ± 3.078%) and 100 nM MOE-ASO (MOE-A, MOE-B, MOE-C) (MOE-A: 99.64 ± 0.2714%; MOE-B: 99.34 ± 0.4810%; MOE-C: 99.76 ± 0.2441%) were detected to significantly reduce the degree of splicing incorporation of crpE2 (Figure 4a-c). For the c.1795-1698 minigenes with A>G mutations, splicing integration of crpE10 was similarly significantly reduced with 4 μM PMO-ASO (PMO-7, PMO-8, PMO-9) (PMO-7: 17.51 ​​± 2.599%; PMO-8: 18.58 ± 2.594%; PMO-9: 19.40 ± 2.619%) or 100 nM MOE-ASO (MOE-G, MOE-H, MOE-I) (MOE-G: 36.76 ± 1.073%; MOE-H: 36.76 ± 1.073%; MOE-I: 36.76 ± 1.073%) (Figure 4d-f). These results support the potential application value of ASO in cerebral calcification diseases by its involvement in regulating PiT2 expression.

[0523] Furthermore, to test the effect of ASO in correcting intrinsic splicing, MOE-ASO was transfused into human fibroblasts derived from PFBC patients with intronic mutations. First, epithelial fibroblasts (P1) derived from patients with heterozygous SLC20A2 c.289+1007 C>G mutations were cultured. Compared to the solvent group (vehicle) or the control group MOE-ASO (MOE-NC), MOE-A and MOE-B were able to significantly remove crpE2-integrated SLC20A2 mRNA (MOE-A: 14.35 ± 5.2645%; MOE-B: 15.62 ± 5.206%; MOE-C: 15.48 ± 5.200%) (Figure 5a,b), leading to increased PiT2 protein levels (MOE-A: 21.24 ± 7.787%; MOE-B: 29.53 ± 10.26%; MOE-C: 6.632 ± 12.11%) (Figure 5c). These findings support the idea that ASO-mediated suppression of abnormal splicing is a viable way to alleviate SLC20A2 expression deficiency in PFBC patients with intronic mutations.

[0524] ASO regulates nonproductive alternative splicing of wild-type alleles and increases the level of normal SLC20A2 transcripts. To investigate this issue from another angle, we cultured volunteer fibroblasts (C1, C2) from two healthy control groups. In ASO cell-involved experiments (Figure 6a-e), we observed that MOE-A and MOE-B also had a regulatory effect on SLC20A2 mRNA splicing in healthy control fibroblasts and were able to upregulate PiT2 protein expression levels (C1, C2). MOEs targeting crpE10 (MOE-J, MOE-K, MOE-L) also reduced crpE10 integration and increased PiT2 protein expression in wild-type fibroblasts (Figure 6f-g). These results indicate that, in autosomal dominant haploinsufficiency disorders, the expression of normal genes without pathogenic mutations on the other chromosome can be regulated by ASOs, thereby serving the purpose of altering disease progression. In light of the expanding practical application value, the screening range of ASO sequences in healthy control fibroblasts was broadened to include new ASO sequences targeting crpE2 splicing regulation (cr5E2-m1, cr5E2-5, cr5E2-6, cr5E2-8, 5crE2-9, cr5E2-10, cr5E2-12, 5crE2-13, cr5E2-13L, cr5E2-13S, cr5E2-14, cr5E2-16, cr5E2-17, cr5E2-18, cr5E2-19, cr5E2-20, cr5E2-21, cr5E2-22, cr5E2-23, cr5 This includes E2-24, cr3E2-5, cr3E2-8, cr3E2-9, cr3E2-10, cr3E2-11, cr3E2-12, cr3E2-13, cr3E2-14, cr3E2-15, cr3E2-16), and novel ASO sequences targeting crpE10 splicing regulation (cr5E10-9, cr5E10-11, cr5E10-12, cr5E10-13, cr5E10-15, cr5E10-16, cr5E10-19, cr3E10-9, cr3E10-14, cr3E10-15). These sequences and related information are shown in Table 1.First, minigenes were transfused into N2A cells, and the response of the minigene expression splicing products to ASOs was analyzed by PCR, excluding most sequences that showed poor effects (Figure 7a, c). Then, to evaluate the regulatory effect of endogenous expression of the remaining ASO sequences on SLC20A2 splicing, they were transfused into healthy control human fibroblasts, and PCR and protein WB detection were repeated several times (Figure 7b, c). Finally, the list was narrowed down to seven ASO sequences that showed a good effect in promoting SLC20A2 or PiT2 expression, including the ASO sequences cr5E2-10, MOE-B (cr5E2-11), cr5E2-12, cr5E2-13, cr5E2-14, MOE-A (cr5E2-15) that target crpE2 splicing regulation, and the ASO sequences cr5E10-12 that target crpE10 splicing regulation. Due to limitations in the detection conditions, this result cannot rule out the possibility that ASO sequences targeting regions described in other patents also have a good effect; therefore, this only illustrates the flow of the inventive process analysis.

[0525] Given that hetero-dominant mutations in the SLC20A2 gene account for 60% of PFBC disease, ASO involvement has been shown to be applicable to repair haploinsufficiency in the SLC20A2 gene, regardless of the mutation morphology (including intronic mutations, exonic mutations, and transcriptional regulatory region mutations). To test whether MOE-A and MOE-B can restore PiT2 expression in patients with SLC20A2 loss-of-function mutations due to non-intronic mutations, which account for a larger proportion, epithelial fibroblasts derived from patients with heterozygous deletions of genomic fragments covering SLC20A2 (P2) were cultured. Compared to the solvent or control group ASO, MOE-A and MOE-B significantly reduced the production of endogenous crpE2-integrated SLC20A2 mRNA (MOE-A: 2.024 ± 0.1775%; MOE-B: 2.267 ± 0.16915%; MOE-C: 2.386 ± 0.1278%) (Figure 7a, b) and promoted a clear increase in PiT2 protein (MOE-A: 15.99 ± 4.225%; MOE-B: 13.53 ± 4.951%; MOE-C: 11.25 ± 4.507%) (Figure 8c). In other words, these results indicate that ASO-mediated modifications to hidden exon-dependent selective screening can rescue SLC20A2 haploinsufficiency caused by SLC20A2 intronic mutations, and consequently other non-intronic mutations.

[0526] Example 4: Construction of a humanized SLC20A2 intron mouse model (SLC20A2-KI) and analysis of progressive cerebral calcification. To evaluate whether ASO therapy is effective in the development and progression of cerebral calcification at the animal level, a humanized mouse model was subsequently developed. The entire human intron 2 and bilateral linker sequences with the SLC20A2 c.289+1007 C>G mutation were knocked in under design conditions where the reading frame was matched, so that the transcript of the chimeric SLC20A2 gene in the mouse model contained the crpE2 hidden exon and was bound and engaged by a human-compatible ASO formulation (Figure 9a). Specifically, using CRISPR / Cas9 technology, the genomic fragment from exon 2 to exon 3 of the mouse SLC20A2 gene was replaced with the genomic sequence of the human SLC20A2 gene with the c.289+1007 C>G mutation, with a code frame matched, in conjunction with a simultaneously introduced targeting DNA fragment. In short, a target sequence with bilateral 1.4 kb homology arms was microinjected into the uterus of C57BL / 6J mice along with sgRNAs and Cas9 RNA. Genomic DNA extracted from the mouse tails was used for genotyping to screen for founder (F0) mice with the precise gene knock-in sequence. F1 KI mice were generated by crossing with C57BL / 6J wild-type mice, and germline transmission of F0 KI mice was demonstrated. Long-chain sequencing and Southern blotting analysis were performed on further F1 mice to verify target mice with more precise homologous recombination. Heterozygous and homozygous SLC20A2-KI mice were selected and subjected to biochemical and phenotypic analysis.

[0527] In heterozygous and homozygous KI mice, RT-PCR and sequencing analysis revealed significantly increased crpE2-integrated SLC20A2 mRNA (Figure 9b), and consequently, the differentiation of SLC20A2 mRNA and PiT2 protein in these animals was significantly reduced (Figures 9c, d). Analysis of inorganic phosphate ions in cerebrospinal fluid (CSF) showed a clear increase in inorganic phosphate (Pi) levels in the CSF of heterozygous and homozygous SLC20A2-KI mice (Figure 9e), thus demonstrating a metabolic reproduction of the cerebral Pi imbalance seen in PFBC patients with SLC20A2 mutations. This suggests that this model can be successfully applied to the modeling of SLC20A2 haploinsufficiency caused by the SLC20A2 c.289+1007 C>G mutation.

[0528] Subsequently, the cerebral calcification status of SLC20A2-KI mice was checked using micro-CT, fluorescent RIS staining (Figure 10a, b) which can recognize calcification deposits, and histological staining (including alizarin red, fonkossa, Alcian blue, periodic acid-Schiff (PAS), and H&E). Micro-CT scans (Figure 10a) showed that approximately 71% of SLC20A2-KI mice developed clear symmetrical cerebral region calcification in the bilateral basal forebrain, hypothalamus, and dorsal midbrain regions by 5 months of age, which was 3 months later than in SLC20A2-KO mice. At 7 months of age, calcification deposits in homozygous SLC20A2-KI mice were widely distributed across many brain regions, such as the basal forebrain, thalamus, hypothalamus, midbrain, and dentate nucleus region of the cerebellum (Figure 10a). At 9.5 months of age, both the number and volume of calcified nodules continued to increase to some extent, and a prominent large calcification deposit appeared in the thalamus, closely resembling that of PFBC patients. Fluorescent RIS-AF647 staining, which allows for the recognition of calcification deposits, also showed similar brain region calcifications as observed on micro-CT (Figure 10b), but the fluorescently indicated calcifications were clearer and more suitable for quantification statistics. Furthermore, histological staining analysis (including alizarin red, fonkossa, Alcian blue, periodic acid-Schiff (PAS), and H&E) showed that the calcification deposits in the mouse model closely matched human brain calcifications morphologically (Figure 11), suggesting that they are driven by similar pathological mechanisms. These results demonstrate that our humanized SLC20A2-KI mouse model can reproduce the distribution patterns of cerebral calcification in PFBC patients with SLC20A2 haploinsufficiency, cerebral Pi impairment, and SLC20A2 mutations.

[0529] Example 5: ASO involvement effectively suppresses cerebral calcification in SLC20A2-KI mice Since mutations in the SLC20A2 gene are the most common cause of PFBC, our humanized SLC20A2-KI mouse model represents a valuable model and is used to test novel ASO-based therapies for treating cerebral calcification. We call this Calcification Repression by ASO Corrected Expression (CRACE) therapy. We designed four doses of MOE-ASO (50, 100, 200, and 400 μg) and evaluated whether splicing correction by single-dose ASO ventricular injection could suppress cerebral calcification in SLC20A2-KI mice before cerebral calcification occurred over three months (Figure 12a). MOE-A and MOE-B (collectively referred to as therapeutic MOEs) were selected for in vivo administration because they showed a significant effect in repairing PiT2 expression in cell experiments.

[0530] After injecting 200 μg of MOE-A into ICV for 4 months, in-situ hybridization with a probe that specifically recognizes MOE-A revealed ASO signals widely distributed throughout the brain (Figure 12b), indicating that MOE effectively diffuses in the cerebrum and can remain present and exert its effects over a long period. In terms of target gene expression, compared to SLC20A2-KI mice injected with control MOE, homozygous SLC20A2-KI mice injected with therapeutic MOE showed a significant decrease in abnormal SLC20A2 transcripts, including crpE2 integration, a clear increase in the expression of normal SLC20A2 transcripts, and a clear enhancement in the level of its translation product, PiT2 protein (Figures 12c, d, e). Simultaneously, after four months of treatment with 100 μg and 200 μg of therapeutic MOE-ASO in SLC20A2-KI mice, CSF Pi levels also decreased significantly (Figure 12f), suggesting that the abnormal phosphate ion homeostasis in the brain was restored.

[0531] In terms of cerebral calcification, after administering 100-400 μg of therapeutic MOE-ASO via ICV to 3-month-old mice for 4 months, micro-CT scans of the brains of 7-month-old mice showed a significant decrease in both volume and number of calcification foci in the thalamus, hypothalamus, midbrain, and dorsal whole brain (Figure 13a, b). Therefore, splicing correction mediated by MOE-modified ASO can prevent the development of cerebral calcification in simulated PFBCs in humanized SLC20A2-KI mice.

[0532] Furthermore, to evaluate the effect of ventricular injection of ASO in mice that already had calcification, ASO was injected into the brains of 5-month-old homozygous SLC20A2-KI mice. As shown in Figure 9, calcification had already appeared in the brains of the mice at this time. Since previous cell experiments showed that both MOE-modified and PMO-modified ASO could repair SLC20A2 expression, 25 μg and 75 μg of PMO-ASO were selected and used in 5-month-old homozygous SLC20A2-KI mice in this animal experiment (Figure 14a). After the administration of PMO-1 was completed, a clear increase in the expression level of normal SLC20A2 transcript in the thalamus of SLC20A2-KI mice was detected (Figure 14b), and a clear decrease in Pi concentration in CSF was also detected (Figure 14c). Similarly, micro-CT showed that, compared to PMO-NC, the involvement of a single dose of 25 μg or 75 μg of PMO-1 significantly reduced the distribution of cerebral calcification in 7-month-old SLC20A2-KI mice (Figure 15d). In summary, these findings indicate that CRACE therapy can effectively enhance PiT2 expression, restore the balance of cerebral Pi, and inhibit or reduce cerebral calcification in SLC20A2-KI mice. Thus, these data and inventions support the use of ASO-mediated CRACE therapy for the treatment of cerebral calcification in PFBC patients.

[0533] Example 6: Characteristics of expression of hereditary cerebral calcification-related genes in the mouse cerebrum. Because abnormal brain Pi homeostasis is observed in PFBC patients with mutations in the Pi transport protein genes PIT2 or XPR1, we analyzed the expression patterns of the SLC20 sodium-phosphate cotransporter genes Pit1 (Slc20a1) and Pit2 (Slc20a2), and the Pi efflux protein gene Xpr1 in the mouse brain. We created cell type expression heatmaps of Pit1, Pit2, Xpr1, and the PFBC-associated glycosidase Myorg from publicly available mouse brain single-cell RNA-seq data (Source a: https: / / betsholtzlab.org / Publications / databases.html; Source b: http: / / celltypes.org / brain / ). In the single-cell expression heatmaps, both astrocytes and neurons showed abundant expression of Pit1, Pit2, and Xpr1, suggesting the presence of active phosphate transport in these two cell types.

[0534] To investigate the expression of Pi transporters in astrocytes and neurons, we studied the expression of Pit1 and Pit2 in the brains of adult mice using RNAscope in situ hybridization (ISH), focusing on areas known to cause calcification in PFBC patients, such as the thalamus, cerebellum, striatum, and hypothalamus. Astrocytes were labeled by immunostaining with the S100b marker protein. In the cerebellum, Bergmann cells (a specific subtype of astrocyte) did not show ISH signaling for Pit1, but showed strong Pit2 signaling. In the thalamus, ISH signaling for Pit2 was also highly expressed in all astrocytes, with only a small number (<20%) of astrocytes showing weak Pit1 signaling. This dominant expression of Pit2 over Pit1 in astrocytes was consistent across all four brain regions and was confirmed by dual in situ hybridization with another astrocyte marker gene, Aldh1l1. Furthermore, Olig1-positive oligodendrocytes and oligodendrocyte progenitor cells (OPCs) also express Pit2 but do not express Pit1.

[0535] In contrast, glutamatergic (Vglut2+) and GABAergic (Gad1+) neurons express strong Pit1 but relatively weak Pit2. Since oligodendrocytes express low levels of Xpr1, these results show a single binary expression pattern: Pit1 and Xpr1 are highly expressed in neurons, while Pit2 and Xpr1 are highly expressed in astrocytes. Combined with the fact that 61% of reported PFBC patients have PIT2 but no PIT1 mutations, the discovered dominant expression of Pit2 in astrocytes suggests its importance in the development of cerebral calcification due to astrocyte-mediated disruption of cerebral Pi homeostasis.

[0536] Example 7: Preparation of a mouse animal model Slc20a2 tm1aWtsi Mice (MGI:4431725) were mated with Actin-Flp mice (RRID:IMSR_JAX:005703), and the En2a-LacZ-PGK-Neo cassette was removed to produce Pit2 flox mice, or they were bred with Ella-Cre mice (RRID:IMSR_JAX:003724), and the PGK-Neo-flox-Exon3 cassette was removed to produce Pit2 KO mice. Pit1 (Slc20a1) flox mice were purchased from the KOAP Engineering Mouse Collection (T013241, GemPharmatech, China). These were then bred with wild-type C57BL / 6 mice (SLAC Animals, China) for at least three generations. The conditional Cre mouse varieties were from the following sources: Aldh1l1-CreER, manufactured in the Baljit S. Khakh1 laboratory. T2 (PMID: 27939582, RRID:IMSR_JAX:029655), Plp1-CreER provided by Jia-Wei Zhou Laboratory T2Pdgfrb-Cre (MMD: 21557454), produced in the Volkhard Lindner laboratory (RRID: IMSR_JAX: 005975), Camk2a-iCre (MMD: 11668676), produced in the G. Schutz laboratory (MMD: 11668676), and Vgat-Ires-Cre (MMD: IMSR_JAX: 016962), generated in the Jax mouse laboratory. Genotyping of mice was performed using Taq polymerase (Vazyme, China). Tamoxifen (20 mg / ml, corn oil solvent) was administered intraperitoneally once daily for 5 consecutive days, with a dose of 1 mg / 10g body weight.

[0537] The animal cores and experimental procedures were approved by the Animal Care and Use Committee of the Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences.

[0538] Example 8: Effects of PiT2 knockout in astrocytes on brain phosphate homeostasis 8.1 Preparation of serum and cerebrospinal fluid and measurement of Pi concentration After collecting blood from the orbital veins of adult mice, the serum was allowed to stand at room temperature for at least 1 hour, and then centrifuged (2000 × g, 15 min) to prepare the serum. Cerebrospinal fluid from anesthetized adult mice was collected from the cerebellar-medulla oblongata cistern and centrifuged (2000 × g, 4°C) for 5 minutes to remove cell debris. After detecting the concentration of inorganic phosphate ions in serum and cerebrospinal fluid using the PiColorLock free inorganic phosphate ion (Pi) detection kit (303-0030, Innova Biosciences), the values ​​were read at 635 nm using a SpectraMax (Molecular device) spectrophotometer, and the Pi concentration was calculated using a function obtained from a standard curve.

[0539] 8.2 Plasmid Construction, Virus and Delivery The ORFs for human MYORG, PIT2, or XPR1 expression plasmids are derived from expression plasmids from Origene. For in vivo expression, the gfaABC1D promoter was constructed by AAV2 expression, followed by the insertion of PiT2 and XPR1 expression cassettes, respectively. These expression plasmids were then combined with the PHP.eB serotype capsid protein plasmid (Taitools, Inc., Shanghai) to package the AAV virus. For intracerebral injection, a nanoinjector (NanojectIII, Drummond Scientific Company) was used at a rate of 60 nL / min, resulting in an initial titer of 1 x 10⁻¹⁰ e�¹⁰ e⁰¹⁰ e⁰¹⁰ e⁰¹⁰ e⁰¹⁰ e⁰¹� 13 Diluted AAV2 / 5 virus was injected into the thalamic region of adult mice. Additionally, to re-express astroglial cells throughout the brain, 100 μl of 2 × 10⁻¹⁰ e 11 A virus solution containing AAV5.PH.eB capsid was injected.

[0540] 8.3 Immunostaining, Antibodies, and Microscopic Imaging Mouse cerebral sections (20 / 25 μm) fixed with 4% PFA were first perforated with 1×PBS containing 0.5% Triton X-100, then blocked with PBS containing 0.1% Triton X-100 and 5% BSA, and incubated overnight with the corresponding primary antibody (4°C). The following day, the cerebral sections were washed three times in PBS for 10 minutes each time, incubated with secondary antibody at room temperature for 2 hours, stained with Hoechst, washed in PBS for 3×10 minutes, and finally fixed to glass 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), and rabbit anti-S100β (ab41548, ab52642, Abcam). Subsequently, fluorescence expression data was acquired from brain section samples using an FV3000 confocal laser microscope (Olympus). Parameters: 3-channel fluorescence at 488 / 568 / 647 nm, 40× or 60× objective lens, zoom 3-5, z-spacing 0.4 μm.

[0541] 8.4 Micro-CT Imaging Micro-CT scans were performed on mouse cerebral samples fixed with 4% paraformaldehyde using a high-resolution micro-CT scanner (Skyscan 1172, Bruker). The scan parameters were as follows: source power 40 kV, current 250 μA, exposure time 210 ms; rotation step 0.6°; camera pixel size 9 × 9 × 9 μm. Images were reconstructed using the NRecon program supported by the GPUReconServer engine, outputting BMP format images (pixel size, 5 μm) with parameters of beam hardening correction rate 33%, ring artifact reduction 8, and Gaussian smoothing kernel 2. Subsequently, the output BMP sequence images were imported into Imaris software and converted to a TIF sequence (pixel size, 20 μm). The TIF image stack was then imported into ImageJ software and its position was adjusted to output coronal and sagittal planes of the mouse micro-CT image cross-sections. Finally, using standard slice values ​​from a mouse cerebral atlas, the midline of the cerebrum was used as a reference, and sagittal plane images of cerebral calcification within a range of 0.1–1.6 mm lateral to the midline were superimposed (75 sections, 1.5 mm thick) to project images of cerebral calcification within that region. Coronal plane images of cerebral calcification were also projected from the hindbrain (75 sections, 1.5 mm thick) or the coronal plane covering the basal forebrain and hypothalamus (150 sections, 3 mm thick).

[0542] 8.5 Fluorescence imaging of calcified deposits Mouse brains were frozen with OCT reagent after micro-CT scans and then sectioned into 25 μm thick sections using a cryomicrotome. Brain sections at specific locations were selected, incubated with 0.2 μM RIS-AF647 dye (BV500101, Biovinc, USA) for 5 hours, then stained with Hoechst, and finally mounted on glass slides. In fluorescence mode, a VS120 section imaging system (Olympus) was used to acquire calcification images of the brain sections with a 20x objective lens. To perform quantitative analysis of brain calcification, the RIS stained channels of the VS120 images were batch-converted to black and white grayscale images in Tif format. The number and area of ​​calcified nodules were then analyzed in four designated regions of interest (ROIs) using the analyze particles function in ImageJ software. Each section was registered at the corresponding lateral coordinate position on the midline of the cerebrum, based on the topological features of the sagittal plane quantitative region in an adult mouse cerebral atlas. Finally, statistical analysis was performed on the number and area of ​​calcified nodules in each group using GraphPad Prism.

[0543] 8.6 Data Statistics All data are expressed as mean ± standard deviation, with some using mean ± SEM indicated in the footnotes. Between-group samples were statistically analyzed using between-group t-tests, and samples from multiple groups were analyzed using one-way ANOVA. * indicates p < 0.01, ** indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001. For all tests, p < 0.05 (**) is considered statistically significant.

[0544] 8.7 Results The effects of astrocyte PiT2 knockout on brain phosphate homeostasis are shown in Figure 15. Figure A shows that the level of inorganic phosphate ions (Pi) in cerebrospinal fluid (CSF) of PiT2 whole knockout mice was significantly elevated to approximately 1.8 mM, while the Pi concentration in CSF in the brain of wild-type mice was approximately 0.5 mM. Figure B shows that no significant change in inorganic phosphate ions (Pi) was observed in the serum of PiT2 whole knockout mice. Figure C shows the Pi concentrations in brain CSF of PiT2 conditionally knockout mice of different cell types, where astrocyte conditional knockout (Aldh1L1-CreER) is shown. T2 The most significant increase in Pi in mouse CSF was observed (170%), reaching 1.35 mM. Oligodendrocytes (Plp1-CreER) T2 Other conditional knockout mice, including excitatory neurons (Camk2a-iCre), inhibitory neurons (Vgat-Cre), and perivascular cells (Pdgfrb-Cre) mice, showed only a mild increase in CSF Pi. Figure D shows that there were no significant differences in serum Pi concentrations among different conditional knockout mouse breeds.

[0545] Figure 16 shows the results of intravenous injection of AAV virus mediating the selective expression of PiT2 in astrocytes in the brain. Immunofluorescence shows broad expression of the PiT2-V5 signal (red) in astrocytes throughout the mouse brain, with blue Hoechsts representing cell nuclei.

[0546] Figure 17 shows the results of the inhibitory effect of selective compensatory expression of AAV astrocytes on intracranial calcification in mice. In the control group (Pit2 KO mice), microCT images of intracranial calcification at 5 months and 7 months of age showed significant large-area calcification deposition in the dorsal midbrain, basal forebrain, thalamus, and hypothalamus. Furthermore, the 7-month-old mice showed a larger calcification coverage area and larger calcification granules than the 5-month-old mice. However, intracranial calcification was significantly suppressed in the Pit2 KO mice that received therapeutic AAV injections (replacement) at 5 months and 7 months of age.

[0547] 8.8 Consideration Examples 5-8 analyzed the expression characteristics of homologous genes in the mouse cerebrum of genes associated with specific hereditary cerebral calcification disorders, particularly PIT1, PIT2, XPR1, and MYORG, which are related to phosphate transport metabolism, by in-situ hybridization. The results showed that astrocytes selectively expressed specific gene combinations at a high level; specifically, astrocytes highly expressed the Na-Pi cotransporters Pit2(Slc20a2) and Xpr1, while Pit1(Slc20a1) was either not expressed in astrocytes or expressed only in very small amounts. Furthermore, in mice in which the Pit2 gene was conditionally knocked out in astrocytes, a significant increase in inorganic phosphate ion (Pi) levels in cerebrospinal fluid was observed. Therefore, glial cells, particularly astrocytes, were demonstrated to be the most important cell type for regulating phosphate transport metabolism and cerebral phosphate homeostasis, which are closely related to cerebral calcification disorders.

[0548] Based on the discovery of the cytological mechanism described above, we used a currently feasible method for high-proportion infection of specific types of cells throughout the entire brain, namely, intravenous injection of AAV virus. When these viruses cross the blood-brain barrier, they can selectively express the human PiT2 protein product, a compensatory gene, in astrocytes in the mouse cerebrum. Furthermore, mice in which compensatory expression of PIT2 in astrocytes in the brain were successfully induced showed a clear suppression of cerebral calcification, demonstrating that repair of cerebral phosphate homeostasis is an effective strategy for suppressing the development of cerebral calcification and has outstanding value for clinical application.

[0549] Based on the crucial physiological mechanism by which astrocytes, discovered for the first time worldwide, mediate the regulation of cerebral phosphate homeostasis, and the expression compensation by astrocytes discovered in a mouse model, we can effectively restore cerebral phosphate homeostasis in Pit2 KO mice and effectively inhibit the onset and further development of cerebral calcification. Furthermore, this can be used as an innovative therapy for cerebral calcification, bringing well-being to many PFBC patients.

[0550] [Table 1] JPEG2026534430000003.jpg255163JPEG2026534430000004.jpg255164JPEG2026534430000005.jpg255164JPEG2026534430000006.jpg255163 [Table 2] TIFF2026534430000008.tif244160 [Table 3] TIFF2026534430000010.tif255161TIFF2026534430000011.tif255161TIFF2026534430000012.tif255160TIFF2026534430000013.tif255160TIFF2026534430000014.tif255160 [Table 4] TIFF2026534430000016.tif255160TIFF2026534430000017.tif255160TIFF2026534430000018.tif255160TIFF2026534430000019.tif255161TIFF2026534430000020.tif255160TIFF2026534430000021.tif255160TIFF2026534430000022.tif255160TIFF2026534430000023.tif255160TIFF2026534430000024.tif255160TIFF2026534430000025.tif255160TIFF2026534430000026.tif255160TIFF2026534430000027.tif255160TIFF2026534430000028.tif255160TIFF2026534430000029.tif255160TIFF2026534430000030.tif255160TIFF2026534430000031.tif255160TIFF2026534430000032.tif255161TIFF2026534430000033.tif255161TIFF2026534430000034.tif255160TIFF2026534430000035.tif255159TIFF2026534430000036.tif255160TIFF2026534430000037.tif255159TIFF2026534430000038.tif255159TIFF2026534430000039.tif255159TIFF2026534430000040.tif255160TIFF2026534430000041.tif255159TIFF2026534430000042.tif255159TIFF2026534430000043.tif255159TIFF2026534430000044.tif255159TIFF2026534430000045.tif255159TIFF2026534430000046.tif255159TIFF2026534430000047.tif255159TIFF2026534430000048.tif255159TIFF2026534430000049.tif255159TIFF2026534430000050.tif255159TIFF2026534430000051.tif255160TIFF2026534430000052.tif255159TIFF2026534430000053.tif255159TIFF2026534430000054.tif255159TIFF2026534430000055.tif255159.

Claims

1. Use of cerebral phosphate homeostasis promoters in the manufacture of drugs for the prevention and / or treatment of cerebral calcification diseases.

2. The aforementioned cerebral phosphate homeostasis promoter is a reagent capable of regulating alternative splicing in the intron of the SLC20A2 gene. The use described in claim 1.

3. The alternative splicing involves cryptographic exon inclusion, and the reagent modulates (e.g., represses or enhances) the splicing of the cryptographic exon from the precursor mRNA of the SLC20A2 gene. Preferably, the reagent modulates (e.g., suppression or enhancement) the level of mature mRNA processed from the precursor mRNA, and / or modulates (e.g., suppression or enhancement) the expression of the PiT2 protein. The use described in claim 2.

4. The alternative splicing includes alternative splicing in intron 2, Preferably, the alternative splicing in intron 2 is a hidden exon incorporation between exons 2 and 3. Preferably, the hidden exons are located at c.289+901 to c.289+1035 of the SLC20A2 gene. Preferably, the hidden exon has the sequence shown in Sequence ID No.

2. The use described in claim 2 or 3.

5. The alternative splicing includes alternative splicing in intron 10. Preferably, the alternative splicing in the intron 10 involves the incorporation of a hidden exon between exons 10 and 11. Preferably, the hidden exons are located at c. 1795-1703 to c. 1795-1627 of the SLC20A2 gene. Preferably, the hidden exon has the sequence shown in Sequence ID No.

3. The use described in claim 2 or 3.

6. The reagent specifically binds to a target region in the SLC20A2 gene or its precursor mRNA, the target region is located in an intron region between two typical exon regions, and the intron region contains the hidden exon. Preferably, the target region is located within the region from approximately 100 nucleotides upstream of the hidden exon to approximately 100 nucleotides downstream of the hidden exon. Preferably, the target region is selected from a region that straddles the splice site of the hidden exon, a region within the hidden exon, a region within 100 nucleotides upstream of the hidden exon, or a region within 100 nucleotides downstream of the hidden exon. Preferably, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD). The use according to any one of claims 2 to 5.

7. The aforementioned target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA. Preferably, the target region is located within the sequence shown in Sequence ID 8. The use described in claim 6.

8. The aforementioned target region is located within the region c. 1795–1803 to c. 1795–1527 of the SLC20A2 gene or its precursor mRNA. Preferably, the target region is located within the sequence indicated by Sequence ID 9. The use described in claim 6.

9. The reagent is selected from antisense oligonucleotides (ASOs) or gene editing tools (e.g., CRISPR / Cas, base editors, or Cas protein-independent RNA editing tools). Preferably, the reagent is an antisense oligonucleotide (ASO), Preferably, the reagent is a DNA editor, for example, CRISPR / Cas9. Preferably, the reagent is an RNA editor, for example, CRISPR / Cas13. Preferably, the reagent is an adenine / cytosine base editor. Preferably, the reagent is a Cas protein-independent RNA editing tool. The use according to any one of claims 2 to 8.

10. The reagent is an antisense oligonucleotide (ASO) that specifically binds to the target region in the precursor mRNA of SLC20A2. Preferably, the ASO consists of 10 to 30 consecutive nucleotides (for example, 15 to 30, 15 to 25, 18 to 30, 18 to 25, 20 to 25), preferably consisting of 18 to 25 consecutive nucleotides. Preferably, the ASO includes modifications of the main chain nucleotide and nucleotide linkage, modifications of the ribose, modifications of the ribose side chain base, or a combination thereof. Preferably, the ASO specifically binds to a target region located within the region c.289+801 to c.289+1135 of the precursor mRNA of SLC20A2, and preferably, the ASO has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) complementarity with the nucleotide sequence in SEQ ID NO:

8. Preferably, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 13-46, 63-65 or a sequence having at least 80% (for example, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith. Preferably, the ASO specifically binds to a target region located within the region c. 1795-1803 to c. 1795-1527 of the precursor mRNA of SLC20A2, and preferably, the ASO has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) complementarity with the nucleotide sequence in SEQ ID NO:

9. Preferably, the ASO includes a nucleotide sequence represented by any of SEQ ID NOs: 47-61, 66-68, or a sequence having at least 80% (for example, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identity therewith. Use as described in claim 9.

11. The reagent is a gene editing tool, for example, a DNA gene editing system or an RNA base editing tool. Preferably, the gene editing tool is a CRISPR / Cas system comprising a Cas protein and a guide RNA (gRNA), wherein the gRNA can bind to the target region in the SLC20A2 gene or its precursor mRNA. Preferably, the gRNA binds to the target region and guides the binding of the editor complex to exert editing activity. Preferably, the target region is located within the region c. 289+801 to c. 289+1135 of the SLC20A2 precursor mRNA, or within the region c. 1795-1803 to c. 1795-1527 of the SLC20A2 precursor mRNA. Preferably, the gene editing tool disrupts the function of the splice acceptor (SA) or splice donor (SD) sequence of a hidden exon in the genome or precursor mRNA sequence by mutating the SA or SD sequence, and suppressing the integration of the hidden exon into mRNA. Preferably, the gene editing tool is a DNA base editor comprising (i) a fusion protein containing a DNA base editing domain (e.g., deaminase) and a DNA binding domain (e.g., a Cas protein, e.g., Cas9 or Cas12), and (ii) a guide RNA (gRNA), wherein the gRNA can bind to the target region in the SLC20A2 gene. Preferably, the gene editing tool is an RNA base editing tool, and preferably, the RNA base editing tool includes an RNA base editor based on the CRISPR / Cas system, or a CRISPR / Cas system-independent editor, such as an oligonucleotide-based base editor. Use as described in claim 9.

12. The use according to claim 1, wherein the cerebral phosphate homeostasis promoter is an astrocyte phosphate transport promoter.

13. The cerebral phosphate homeostasis promoter is selected from the following group for use according to claim 12: (Z1) PiT2 protein, nucleic acid molecule encoding PiT2 protein, expression vector expressing PiT2 protein, PiT2 promoter, or combination thereof; (Z2) MYORG protein, nucleic acid molecules encoding MYORG protein, expression vectors expressing MYORG protein, MYORG promoters, or combinations thereof; (Z3) XRP1 protein, nucleic acid molecules encoding the XRP1 protein, expression vectors expressing the XRP1 protein, XRP1 promoters, or combinations thereof; (Z4) Any combination of the above Z1 to Z3.

14. The accelerator includes small molecule compounds, gene editing reagents, small nucleic acid drugs (e.g., antisense nucleotide drugs), or combinations thereof. The use according to claim 12 or 13.

15. The astrocyte phosphate transport promoter is an expression vector comprising an exogenous gene and an enhancer element selectively expressed by astrocytes operably linked thereto, and / or a promoter selectively expressed by astrocytes, wherein the exogenous gene is selected from nucleic acid molecules encoding PiT2 protein, MYORG protein, or XRP1 protein, or a combination thereof. Preferably, the exogenous gene is a nucleic acid molecule encoding the PiT2 protein. Preferably, the expression vector is a viral vector, for example, AAV. Preferably, the promoter selectively expressed by the astrocytes is gfaABC1D. The use according to any one of claims 12 to 14.

16. The astrocyte phosphate transport promoter is a small nucleic acid drug, the small nucleic acid drug is linked to a targeting molecule, and the targeting molecule has targeting properties for astrocytes. Preferably, the small nucleic acid drug is an ASO. Preferably, the small nucleic acid drug is packaged entirely or partially within the targeting molecule, or is compounded with the targeting molecule. Preferably, the targeting molecule is selected from antibodies or antibody fragments, polysaccharides, aptamers, or nanoparticles. The use according to any one of claims 12 to 14.

17. The use according to any one of claims 1 to 16, wherein the cerebral calcification disorder includes primary familial cerebral calcification (PFBC), primary basal ganglia calcification (PBGC), or other types of cerebral calcification associated with cerebral phosphate homeostasis defects (e.g., upregulation of inorganic phosphate ions in cerebrospinal fluid).

18. A method for preventing and / or treating cerebral calcification disease, comprising the step of administering an effective amount of a cerebral phosphate homeostasis promoter to a required subject, Preferably, the cerebral phosphate homeostasis promoter is as defined in any one of claims 2 to 16, Preferably, the cerebral calcification disorders include primary basal ganglia calcification (PBGC), primary familial cerebral calcification (PFBC), or other types of cerebral calcification associated with cerebral phosphate homeostasis defects (e.g., upregulation of inorganic phosphate ions in cerebrospinal fluid). method.

19. A drug kit for treating cerebral calcification disorders, (a) A first container, and a first detection reagent for detecting gene mutations in the first container, the reagent for detecting the presence or absence of gene mutations in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof, and (b) A second container, and a drug composition contained in the second container, comprising a drug composition comprising an astrocyte phosphate transport promoter as defined in any one of claims 13 to 16 and a pharmaceutically acceptable carrier and / or excipient, Optionally, the third container may further include a solution that assists in the dissolution or delivery of the drug in the second container into the body. Drug kit.

20. A method for screening candidate drugs for the prevention and / or treatment of cerebral calcification disease, comprising the following steps: (a) In the test group, the test drug was added to a culture system of astrocytes or a derived cell line, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group was measured. In the control group, the test drug was not added to a culture system of the same cells, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group was measured. If the cellular phosphorus transport level (E1) and / or activity (A1) in the test group is significantly higher than in the control group, it indicates that the test compound has a promoting effect on the expression and / or activity of phosphorus transport levels and is a candidate drug for preventing and / or treating cerebral calcification disease.

21. Use of a reagent capable of modulating alternative splicing in the intron of the SLC20A2 gene in the manufacture of a drug, wherein the drug is for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject, and preferably the drug is as defined in any one of claims 3 to 11.

22. A method for preventing and / or treating a disease associated with PiT2 dysfunction in a subject, comprising the step of administering to the required subject an effective amount of a reagent capable of modulating alternative splicing in the intron of the SLC20A2 gene, Preferably, the reagent is as defined in any one of claims 3 to 11. method.

23. The aforementioned PiT2 functional defect is related to a mutation in the SLC20A2 gene. Preferably, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. Preferably, the mutation is located in the exon region, and the subject is heterozygous with respect to the mutation in the exon region. Preferably, the mutation is located in the target region of the intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region. Preferably, the target region is located within the region c. 289+801 to c. 289+1135 of the SLC20A2 precursor mRNA, or within the region c. 1795-1803 to c. 1795-1527 of the SLC20A2 precursor mRNA. Preferably, the PiT2 functional defect is a haploinsufficiency of SLC20A2. The use described in claim 21 or the method described in claim 22.

24. Regulation of functional PiT2 protein or RNA expression is beneficial for the aforementioned PiT2 dysfunction-related diseases. Preferably, the disease is cerebral calcification, for example, primary familial cerebral calcification (PFBC) or primary basal ganglia calcification (PBGC), Preferably, the disease is a disease associated with an abnormality in the Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis. The use described in claim 21 or the method described in claim 22.

25. A reagent capable of specifically binding to a target region in the SLC20A2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, and the intron region contains a hidden exon. Preferably, the hidden exon is located within intron 2, preferably located at c.289+901 to c.289+1035 of the SLC20A2 gene or its precursor mRNA, and preferably, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA. Preferably, the hidden exon is located within intron 10, preferably located at c. 1795-1703 to c. 1795-1627 of the SLC20A2 gene or its precursor mRNA, and preferably, the target region is located within the region c. 1795-1803 to c. 1795-1527 of the SLC20A2 gene or its precursor mRNA. Preferably, the target region has the features defined in any one of claims 6 to 8. reagent.

26. The reagent according to claim 25, having the features defined in claim 9 or 10.

27. A drug composition comprising the reagent or combination thereof according to claim 25 or 26, and a pharmaceutically acceptable carrier and / or excipient.

28. Use of the reagent according to claim 25 or 26, or the drug composition according to claim 27, in the manufacture of a drug, wherein the drug is for the prevention and / or treatment of a disease associated with PiT2 dysfunction in a subject. Preferably, the disease associated with the PiT2 dysfunction is cerebral calcification, for example, primary familial cerebral calcification (PFBC) or primary basal ganglia calcification (PBGC), Preferably, the diseases associated with the PiT2 dysfunction are diseases associated with Pi imbalance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis. Preferably, the drug treats the disease by regulating the expression of PiT2 protein or functional RNA in the subject's cells. use.

29. A method for preventing and / or treating a disease associated with PiT2 dysfunction in a subject, comprising the step of administering to a subject as required a reagent according to claim 25 or 26, or a drug composition according to claim 27, Preferably, the disease associated with the PiT2 dysfunction is cerebral calcification, for example, primary familial cerebral calcification (PFBC) or primary basal ganglia calcification (PBGC), Preferably, the diseases associated with the PiT2 dysfunction are diseases associated with Pi imbalance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or skeletal diseases associated with disruption of phosphorus homeostasis. method.

30. The aforementioned subject has a mutation in the SLC20A2 gene, Preferably, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms. Preferably, the mutation is located in the exon region, and the subject is heterozygous with respect to the mutation in the exon region. Preferably, the mutation is located in the target region of the intron, and the subject is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region. Preferably, the target region is located within the region c. 289+801 to c. 289+1135 of the SLC20A2 precursor mRNA, or within the region c. 1795-1803 to c. 1795-1527 of the SLC20A2 precursor mRNA. Preferably, the PiT2 functional defect is a haploinsufficiency of SLC20A2. The use described in claim 28 or the method described in claim 29.

31. A method for regulating the expression of PiT2 protein in cells, comprising the step of contacting the cells with a reagent capable of regulating alternative splicing in the introns of the SLC20A2 gene in vitro, Preferably, reagents capable of modulating alternative splicing in the intron of the SLC20A2 gene are as defined in any one of claims 2 to 11. method.

32. A method for regulating the expression of PiT2 protein in cells, comprising the step of contacting the cells with the reagent described in claim 25 or 26 in vitro, Preferably, the cells contain a mutation in the SLC20A2 gene, Preferably, the mutation is selected from nonsynonymous mutations, nonsense mutations, shift mutations, splicing mutations, or structural polymorphisms, preferably the mutation is located in an exon region and the cell is heterozygous with respect to the mutation in the exon region, preferably the mutation is located in a target region in an intron and the cell is heterozygous, homozygous, or compound heterozygous with respect to the mutation in the intron located in the target region. method.

33. A method for identifying a reagent capable of regulating the expression of PiT2 in cells, comprising: i) identifying a reagent that targets a target region in the PiT2 gene or its precursor mRNA, wherein the target region is located in an intron region between two typical exon regions, the intron region contains a hidden exon, and the target region is located in a region from approximately 100 nucleotides upstream of the hidden exon to approximately 100 nucleotides downstream of the hidden exon; ii) delivering the reagent identified in step i) to the cells; and iii) measuring the expression levels of PiT2 mRNA and protein in the cells of step ii). Preferably, the process further includes a step of comparing the expression level measured in step iii) with the expression level of PiT2 in cells treated with a control, Preferably, the expression level is measured by RT-PCR, qPCR, or Western blotting. Preferably, the target region is selected from the region upstream of the 5' splice site (splicing acceptor site, SA) of the hidden exon, the region spanning the 5' splice site, the region within the hidden exon, the region spanning the 3' splice site, or the region downstream of the 3' splice site (splicing donor site, SD). Preferably, the hidden exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene, preferably, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA, preferably, the target region is located within the sequence indicated by Sequence ID No.

8. Preferably, the hidden exon is located at c. 1795-1703 to c. 1795-1627 of the SLC20A2 gene, preferably, the target region is located within the region c. 1795-1803 to c. 1795-1527 of the SLC20A2 gene or its precursor mRNA, and preferably, the target region is located within the sequence indicated by Sequence ID No.

9. method.

34. A method for producing a brain calcification model, comprising the steps of: modifying a target genome such that the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced in whole or in part with a corresponding intron genomic fragment of the human SLC20A2 gene, or the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced in whole or in part with a corresponding intron genomic fragment of the human SLC20A2 gene, wherein the corresponding intron genomic fragment of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene, thereby providing a brain calcification model; Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes at least a hidden exon. Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO:

11. Preferably, a mutation that can alter the splicing of the SLC20A2 gene induces or enhances alternative splicing in intron 2 or intron 10 as defined in any of the claims, Preferably, the substitution method includes gene knockout, gene editing, RNA interference, epigenetic repression, or any combination thereof. Preferably, the model is an animal model, for example, a mammal model, for example, a rodent model (for example, a mouse or rat model) or a non-human primate model. Preferably, the model is a mammalian cell model, for example, human epithelial fibroblasts or glial cells. method.

35. A genetically modified non-human animal in which the genome is modified in which the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene. Preferably, a mutation that can alter the splicing of the SLC20A2 gene induces or enhances alternative splicing in intron 2 or intron 10 as defined in any of the claims, Preferably, the genomic fragment of the intron of the human SLC20A2 gene substituted between exon 2 and exon 3 includes the sequence shown in SEQ ID NO:

10. Preferably, the genomic fragment of the human SLC20A2 gene intron substituted between exon 10 and exon 11 includes the sequence shown in SEQ ID NO:

11. Preferably in rodents, such as mice or rats. Preferably, non-human primates, Preferably, an animal model of cerebral calcification, animal.

36. A genetically modified cell in which the genome is replaced in either the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene. Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes at least a hidden exon. Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO:

11. Preferably, a mutation that can alter the splicing of the SLC20A2 gene induces or enhances alternative splicing in intron 2 or intron 10 as defined in any of the claims, Preferably, the genomic fragment of the corresponding intron of the human SLC20A2 gene is introduced exogenously into the cell. Preferably in mammalian cells, Preferably, human epithelial fibroblasts or glial cells, Preferably, a cellular model of brain calcification, cell.

37. The use of genetically modified non-human animals or cells, including (i) being used as a model to present or analyze the pathogenesis of cerebral calcification disease or to study the disruption of cerebral phosphate homeostasis or targets of cerebral calcification disease; (ii) being used as a model to screen candidate drugs or treatment plans for cerebral calcification disease or diseases associated with disruption of cerebral phosphate homeostasis; and / or (iii) being used as a model to screen candidate drugs or treatment plans for diseases associated with disruption of phosphate homeostasis. Here, the genome of the genetically modified non-human animal or cell includes a case in which the intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, or a case in which the intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced with a genomic fragment of the corresponding intron of the human SLC20A2 gene, wherein the genomic fragment of the corresponding intron of the human SLC20A2 gene includes a mutation that can alter the splicing of the SLC20A2 gene. Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes at least a hidden exon. Preferably, the corresponding intron genomic fragment of the human SLC20A2 gene includes a region from at least about 100 nucleotides upstream of the hidden exon to about 100 nucleotides downstream of the hidden exon, for example, the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 2 and exon 3 includes the sequence shown in SEQ ID NO: 10, or the intron genomic fragment of the human SLC20A2 gene that is replaced between exon 10 and exon 11 includes the sequence shown in SEQ ID NO:

11. Preferably, a mutation that can alter the splicing of the SLC20A2 gene induces or enhances alternative splicing in intron 2 or intron 10 as defined in any of the claims. use.

38. A nucleic acid construct comprising a minigene fragment sequence of the human SLC20A2 gene, wherein the minigene fragment sequence comprises exon 2 and exon 3 and the intron sequence between them, or exon 10 and exon 11 and the intron sequence between them, wherein the intron sequence comprises a mutation that can alter the splicing of the SLC20A2 gene. Preferably, it includes a promoter operably linked to the minigene fragment sequence, Preferably, a mutation that can alter the splicing of the SLC20A2 gene induces or enhances alternative splicing in intron 2 or intron 10 as defined in any of the claims, Preferably, the minigene fragment sequence includes the sequence shown in Sequence ID No. 4 or 5. Preferably, the polyA signal sequence is included downstream of the minigene fragment sequence. Preferably, the system further includes a reporter gene, for example, a fluorescent protein (e.g., GFP, mCherry) or luciferase. Nucleic acid constructs.

39. A method for screening reagents capable of modulating SLC20A2 mRNA splicing, comprising the steps of: (i) providing cells comprising the nucleic acid construct described in claim 38; (ii) providing a genetically modified non-human animal described in claim 35 or a genetically modified cell described in claim 36; or (iii) providing a brain calcification model obtained by the method described in claim 34; contacting a test reagent with any of (i)-(iii); and detecting alternative splicing of SLC20A2 mRNA. Preferably, the cells are of mammalian origin, such as rodents (e.g., mice or rats), non-human primates, or human origin. Preferably, a test reagent is selected that can suppress alternative splicing (for example, by reducing the amount of expression product containing hidden exon integration), thereby increasing the production of functional PiT2 protein. Preferably, a test reagent is selected that can enhance alternative splicing (e.g., increase the amount of expression product including hidden exon incorporation), thereby reducing the production of functional PiT2 protein. Preferably, the detection includes analyzing the length of the mRNA expression product of SLC20A2. Preferably, the alternative splicing is an alternative splicing in the intron 2 as defined in any of the claims, preferably, the detection comprises performing a PCR analysis using primers spanning exon 2 and exon 3, preferably, an amplicon is generated that includes a hidden exon incorporation of about 135 nt in length. Preferably, the alternative splicing is an alternative splicing in the intron 10 as defined in any of the claims, preferably, the detection comprises performing a PCR analysis using primers spanning exon 10 and exon 11, preferably, an amplicon is generated that includes a hidden exon incorporation of about 76 nt in length. Preferably, the method includes a step of contacting a test reagent with cells containing the nucleic acid construct in vitro to detect alternative splicing of the nucleic acid construct, and preferably, the detection includes analyzing the length of the mRNA expression product of the nucleic acid construct or analyzing the expression of a reporter gene contained in the nucleic acid construct. method.

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