Therapy based on intracerebral phosphate homeostasis promoter

Abstract

The present invention relates to the field of biomedicine, in particular, to the use of an intracerebral phosphate homeostasis promoter in cerebral calcification diseases. The intracerebral phosphate homeostasis promoter comprises an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene or an agent targeting astrocytes. In addition, further provided in the present invention are an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene, and the use thereof. Further provided in the present invention are a cerebral calcification model based on alternative splicing in an intron of the human SLC20A2 gene, and a preparation method therefor and the use thereof.

Description

Therapies based on promoters of brain phosphorus homeostasis

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310890680.8 filed on July 19, 2023 and Chinese patent application No. 202311370896.8 filed on October 20, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of biomedicine, and more specifically to the use of agents promoting brain phosphorus homeostasis in treating brain calcification diseases. The agents include agents capable of regulating alternative splicing within introns of the SLC20A2 gene or agents targeting astrocytes. Furthermore, the present invention provides agents capable of regulating alternative splicing within introns of the SLC20A2 gene and uses thereof. The present invention also provides a brain calcification model based on alternative splicing within introns of the human SLC20A2 gene, a method for preparing the model, and uses thereof. Background Art

[0004] Brain calcification is a disease related to age and aging, and is relatively common in clinical imaging. In cranial CT observations of elderly people over 60 years old, more than 20% will have punctate calcifications, and more than 2-4% will have larger patchy calcifications. Some neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), have more than 20% of patients with brain calcification, and the lesions expand with age. Therefore, brain calcification is a neurodegenerative disease with treatment needs that needs to be paid attention to clinically.

[0005] Hereditary brain calcifications are neurodegenerative diseases caused by gene mutations and include various types, such as primary familial brain calcification (PFBC) and Aicardi-Goutières syndrome (AGS). PFBC is a representative type, with an incidence of approximately 0.6 per 1,000 patients. PFBC is characterized by bilaterally symmetrical calcifications, often in the basal ganglia, thalamus, and cerebellar dentate nucleus. Calcifications are also occasionally found in the subcortical regions of some patients. As the disease progresses, the calcifications may connect to form large patches. High-resolution brain histopathological imaging studies have revealed that calcium salt particles are primarily deposited in the perivascular spaces surrounding terminal arterioles, veins, and capillaries. The clinical symptoms of hereditary brain calcifications vary, primarily manifesting as movement disorders, mental disorders, cognitive impairment, seizures, dizziness, and headaches. Severe cases can lead to personality and behavioral changes, ultimately leading to psychosis or dementia. Some patients become disabled and unable to care for themselves, while others remain asymptomatic throughout their lives.

[0006] Hereditary brain calcifications can be sporadic (mostly due to de novo mutations) or familial, with a high degree of genetic heterogeneity. Over the past decade, human genetic studies have identified pathogenic genes for PFBC, including SLC20A2, XPR1, PDGFB, PDGFRB, MYORG, and JAM2. These genes suggest two main mechanisms of brain calcification: first, impaired integrity of the blood-brain barrier (BBB) ​​or decreased pericyte coverage, leading to increased BBB permeability. Patients with this pathway may harbor mutations in genes such as PDGFRB, PDGFB, and JAM2, all of which are expressed in cells that comprise the neurovascular unit.

[0007] The second pathway involves impaired inorganic phosphate (Pi) transport in the brain and the resulting imbalance in brain phosphate homeostasis (BPH), often manifested by significantly elevated cerebrospinal fluid (CSF) Pi levels. Patients belonging to this pathway may harbor mutations in the SLC20A2, MYORG, or XPR1 genes, which encode the sodium-pi cotransporter PiT2 and the phosphate efflux transporter XPR1 and its regulator, MYORG, respectively. Furthermore, statistical analysis of clinical calcification and symptoms has shown that patients with mutations in Pi transport genes have larger calcifications and more severe clinical symptoms compared with patients with mutations in genes related to the BBB pathway. Furthermore, among all reported cases of familial calcification, mutations in SLC20A2 account for 61% of all genetic factors, XPR1 for 6%, and MYORG mutations for 13%.

[0008] Currently, clinical treatment for PFBC is primarily symptomatic, and there are no effective drugs to remove or reduce abnormal calcium deposits. In particular, targeting the imbalanced mechanisms of BPH may provide opportunities for the development of clinical treatments.

[0009] Therefore, there is an urgent need to develop a novel brain calcification intervention strategy that is convenient and effective and targets the restoration of phosphorus homeostasis.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides use of a brain phosphorus homeostasis promoter in the preparation of a medicament for preventing and / or treating brain calcification diseases.

[0012] In certain embodiments, the agent that promotes brain phosphorus homeostasis is an agent that can regulate alternative splicing in an intron of the SLC20A2 gene. In certain embodiments, the alternative splicing comprises a cryptic exon inclusion, and the agent regulates (e.g., inhibits or enhances) the splicing of the cryptic exon from the pre-mRNA of the SLC20A2 gene. In certain embodiments, the agent thereby regulates (e.g., inhibits or enhances) the level of mature mRNA processed from the pre-mRNA and / or regulates (e.g., inhibits or enhances) the expression of the PiT2 protein.

[0013] In certain embodiments, the alternative splicing comprises alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO: 2.

[0014] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10. In certain embodiments, the alternative splicing within intron 10 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO: 3.

[0015] In certain embodiments, the agent specifically binds to a target region in the SLC20A2 gene or its pre-mRNA, the target region being located in an intronic region between two canonical exon regions, and wherein the intronic region contains the cryptic exon. In certain embodiments, the target region is within a region that is up to about 100 nucleotides upstream of the cryptic exon and up to about 100 nucleotides downstream of the cryptic exon. In certain embodiments, the target region is selected from a region spanning a splice site of the cryptic exon, a region within the cryptic exon, a region within 100 nucleotides upstream of the cryptic exon, or a region within 100 nucleotides downstream of the cryptic exon. In certain embodiments, the target region is selected from a region upstream of the 5' splice 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 donor site (SD).

[0016] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its pre-mRNA. In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:8.

[0017] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its pre-mRNA. In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:9.

[0018] In certain embodiments, the reagent is selected from antisense oligonucleotides (ASOs) or gene editing tools (e.g., CRISPR / Cas, base editors, or non-Cas protein-dependent RNA editing tools). In certain embodiments, the reagent is antisense oligonucleotides (ASOs). In certain embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In certain embodiments, the reagent is an RNA editor, such as CRISPR / Cas13. In certain embodiments, the reagent is a base editor Adenine / Cytosine base editor. In certain embodiments, the reagent is a non-Cas protein-dependent RNA editing tool.

[0019] In certain embodiments, the agent is an antisense oligonucleotide (ASO) that specifically binds to the target region in SLC20A2 pre-mRNA. In certain 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, it consists of 18 to 25 consecutive nucleotides. In certain embodiments, the ASO comprises modifications of the backbone nucleotides and nucleotide linkages, modifications of the ribose sugar, modifications of the ribose side chain bases, or a combination thereof.

[0020] In certain embodiments, the ASO specifically binds to a target region located within the region c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA. In certain embodiments, 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 of SEQ ID NO: 8. In certain embodiments, the ASO comprises the nucleotide sequence of any one 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.

[0021] In certain embodiments, the ASO specifically binds to a target region located within the region c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA. In certain embodiments, 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 within SEQ ID NO: 9. In certain embodiments, the ASO comprises the nucleotide sequence set forth in any one 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 thereto.

[0022] In certain embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool. In certain embodiments, the gene editing tool is a CRISPR / Cas system, which comprises a Cas protein and a guide RNA (gRNA), wherein the gRNA is capable of binding to the target region in the SLC20A2 gene or its precursor mRNA. In certain embodiments, the gRNA binds to the target region, thereby recruiting the binding of the editor complex and exerting editing activity. In certain embodiments, the target region is located in the region between c.289+801 and c.289+1135 of the SLC20A2 precursor mRNA; or in the region between c.1795-1803 and c.1795-1527 of the SLC20A2 precursor mRNA. In certain embodiments, the gene editing tool mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon in the genome or pre-mRNA sequence, thereby destroying its SA or SD function and inhibiting the integration of the cryptic exon in the mRNA. In certain embodiments, the gene editing tool is a DNA base editor, which comprises (i) a fusion protein including a DNA base editing domain (e.g., a deaminase) and a DNA binding domain (e.g., a Cas protein, such as Cas9 or Cas12) and (ii) a guiding RNA (gRNA), which is capable of binding to the target region in the SLC20A2 gene. In certain embodiments, the gene editing tool is an RNA base editing tool. In certain embodiments, the RNA base editing tool comprises: an RNA base editor based on the CRISPR / Cas system, or an editor that does not rely on the CRISPR / Cas system, such as an oligonucleotide-based base editor.

[0023] In certain embodiments, the agent that promotes brain phosphorus homeostasis is an astrocyte phosphorus transport promoter.

[0024] In certain embodiments, the agent promoting brain phosphorus homeostasis is selected from the group consisting of:

[0025] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0026] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0027] (Z3) XRP1 protein, a nucleic acid molecule encoding an XRP1 protein, an expression vector expressing an XRP1 protein, an XRP1 promoter, or a combination thereof;

[0028] (Z4) Any combination of the above Z1 to Z3.

[0029] In certain embodiments, the PiT2 protein comprises the amino acid sequence set forth in SEQ ID NO: 155. In certain embodiments, the MYORG protein comprises the amino acid sequence set forth in SEQ ID NO: 154. In certain embodiments, the XRP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 153.

[0030] In certain embodiments, the promoters described in Z1 to Z3 include small molecule compounds, gene editing reagents, small nucleic acid drugs (such as antisense nucleotide drugs), or combinations thereof.

[0031] In certain embodiments, the astrocyte phosphate transport enhancer is an expression vector comprising an exogenous gene and an astrocyte-selective enhancer element and / or an astrocyte-selective promoter operably linked thereto. The exogenous gene is selected from a nucleic acid molecule encoding PiT2 protein, MYORG protein, or XRP1 protein, or a combination thereof. In certain embodiments, the exogenous gene is a nucleic acid molecule encoding PiT2 protein. In certain embodiments, the expression vector is a viral vector, such as AAV. In certain embodiments, the astrocyte-selective promoter is gfaABC1D. In certain embodiments, the gfaABC1D promoter comprises the sequence set forth in SEQ ID NO:156.

[0032] In certain embodiments, the astrocyte phosphate transport enhancer is a small nucleic acid drug, which is linked to a targeting molecule that has targeting properties for astrocytes. In certain embodiments, the small nucleic acid drug is an ASO. In certain embodiments, the small nucleic acid drug is fully or partially encapsulated within a targeting molecule or conjugated to the targeting molecule. In certain embodiments, the targeting molecule is selected from an antibody or antibody fragment thereof, a polysaccharide, an aptamer, or a nanoparticle.

[0033] In certain embodiments, the brain calcification disease includes primary familial brain calcification (PFBC), primary basal ganglia calcification (PBGC), or other types of brain calcification associated with defects in brain phosphorus homeostasis (such as upregulation of inorganic phosphate in cerebrospinal fluid). The other types of brain calcification diseases may include other types of brain calcification caused by abnormal glial cell phosphorus transport function, such as some sporadic brain calcification cases, brain calcification caused by tumors or inflammation, and Aicardi-Goutières syndrome (AGS).

[0034] In a second aspect, the present invention provides a method for preventing and / or treating brain calcification diseases, comprising administering an effective amount of a brain phosphorus homeostasis promoter to a subject in need thereof. In certain embodiments, the brain phosphorus homeostasis promoter is as defined in the first aspect above. In certain embodiments, the brain calcification diseases include other types of brain calcifications associated with primary basal ganglia calcification (PBGC), primary familial brain calcification (PFBC), or brain phosphorus homeostasis defects (such as cerebrospinal fluid inorganic phosphate upregulation). The other types of brain calcification diseases may include other types of brain calcifications caused by abnormal glial cell phosphorus transport function, such as some sporadic brain calcification cases, brain calcification caused by tumors or inflammation, and Aicardi-Goutières syndrome (AGS).

[0035] In a third aspect, the present invention provides a kit for treating brain calcification diseases, wherein the kit comprises:

[0036] (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect the presence of a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and

[0037] (b) a second container, and a pharmaceutical composition in the second container, wherein the pharmaceutical composition comprises the astrocyte phosphate transport enhancer defined in the first aspect and a pharmaceutically acceptable carrier and / or excipient.

[0038] In certain embodiments, optionally, the medicine kit may further include a third container, and a solution in the third container for assisting the dissolution or in vivo delivery of the drug in the second container.

[0039] In a fourth aspect, the present invention provides a method for screening candidate drugs for preventing and / or treating brain calcification diseases, wherein the method comprises the following steps:

[0040] (a) In a test group, a test drug is added to a culture system of astrocytes or a cell line derived therefrom, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group is measured; in a control group, no test drug is added to the culture system of the same cells, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group is measured;

[0041] Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test drug has a promoting effect on the expression and / or activity of the phosphorus transport level and is a candidate drug for preventing and / or treating brain calcification diseases.

[0042] In a fifth aspect, the present invention provides use of an agent capable of regulating alternative splicing within an intron of the SLC20A2 gene in the preparation of a medicament for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject. Also provided is a method for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject, comprising administering to a subject in need thereof an effective amount of an agent capable of regulating alternative splicing within an intron of the SLC20A2 gene.

[0043] In certain embodiments, the agent capable of regulating alternative splicing in an intron of the SLC20A2 gene is as defined in the first aspect.

[0044] In certain embodiments, the PiT2 functional deficiency involves a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation, or a structural variation. In certain embodiments, the mutation is located in an exonic region, and the subject is heterozygous for the mutation in the exonic region. In certain embodiments, the mutation is located in an intron of the target region, and the subject may be heterozygous, homozygous, or compound heterozygous for the intronic mutation located in the target region. In certain embodiments, the target region is located within the region c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA, or within the region c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA. In certain embodiments, the PiT2 functional deficiency is SLC20A2 haploinsufficiency. In certain embodiments, the disease associated with the PiT2 functional deficiency benefits from regulation of functional PiT2 protein or RNA expression.

[0045] In certain embodiments, the disease is brain calcification, such as primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0046] In certain embodiments, the disease is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone disease associated with imbalanced phosphorus homeostasis.

[0047] In a sixth aspect, the present invention provides an agent capable of specifically binding to a target region in the SLC20A2 gene or its pre-mRNA, wherein the target region is located in an intron region between two typical exon regions, and wherein the intron region contains a cryptic exon.

[0048] In certain embodiments, the cryptic exon is located within intron 2; preferably, the cryptic exon is located within c.289+901 to c.289+1035 of the SLC20A2 gene or its precursor mRNA; preferably, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA.

[0049] In certain embodiments, the cryptic exon is located within intron 10; preferably, the cryptic exon is located within c.1795-1703 to c.1795-1627 of the SLC20A2 gene or its precursor mRNA; preferably, the target region is located within the region of c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA.

[0050] In certain embodiments, the target area is as defined in the first aspect.

[0051] In certain embodiments, the agent is an ASO as defined in the first aspect.

[0052] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the agent or a combination thereof capable of specifically binding to the target region in the SLC20A2 gene or its pre-mRNA according to the sixth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0053] In an eighth aspect, the present invention provides use of the agent capable of specifically binding to a target region in the SLC20A2 gene or its precursor mRNA as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect, in the preparation of a medicament for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject. Also provided is a method for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject, comprising administering to a subject in need thereof the agent capable of specifically binding to a target region in the SLC20A2 gene or its precursor mRNA as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect.

[0054] In certain embodiments, the disease associated with PiT2 functional deficiency is brain calcification, such as primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0055] In certain embodiments, the disease associated with PiT2 functional deficiency is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone diseases associated with phosphorus homeostasis imbalance.

[0056] In certain embodiments, the medicament treats a disease by regulating the expression of PiT2 protein or functional RNA in cells of the subject.

[0057] In certain embodiments, the subject comprises a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation, or a structural variation. In certain embodiments, the mutation is located in an exon region, and the subject is heterozygous for the mutation in the exon region. In certain embodiments, the mutation is located in an intron of the target region, and the subject may be heterozygous, homozygous, or compound heterozygous for the intronic mutation located in the target region. In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA, or within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA. In certain embodiments, the PiT2 functional defect is SLC20A2 haploinsufficiency.

[0058] In a ninth aspect, the present invention provides a method for regulating PiT2 protein expression in a cell, comprising contacting the cell in vitro with an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene. In certain embodiments, the agent capable of regulating alternative splicing in an intron of the SLC20A2 gene is as defined in the first aspect.

[0059] In a ninth aspect, the present invention provides a method for regulating PiT2 protein expression in a cell, comprising contacting the cell with the agent of the sixth aspect in vitro. In certain embodiments, the cell comprises a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation, or a structural variation; preferably, the mutation is located in an exon region, and the cell is heterozygous for the mutation in the exon region; preferably, the mutation is located in an intron of the target region, and the cell can be heterozygous, homozygous, or compound heterozygous for the intronic mutation located in the target region.

[0060] In the tenth aspect, the present invention provides a method for identifying an agent capable of regulating PiT2 expression in a cell, the method comprising: i) identifying an agent that targets a target region in the PiT2 gene or its precursor mRNA, the target region being located in an intron region between two typical exon regions, and wherein the intron region contains a cryptic exon, and the target region being within a region from at most about 100 nucleotides upstream of the cryptic exon to at most about 100 nucleotides downstream of the cryptic exon; ii) delivering the agent identified in step i) to the cell; and iii) measuring the mRNA and protein expression levels of PiT2 in the cell of step ii).

[0061] In certain embodiments, the method further comprises: iv) comparing the expression level measured in step iii) with the expression level of PiT2 in cells treated with a control.

[0062] In certain embodiments, the expression level is measured by RT-PCR, qPCR, or by Western blot.

[0063] In certain embodiments, the target region is selected from a region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, a region spanning the 5' splicing site, a region within the cryptic exon, a region spanning the 3' splicing site, or a region downstream of the 3' splicing donor site (SD).

[0064] In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene; preferably, the target region is located at 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 SEQ ID NO:8.

[0065] In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene; preferably, the target region is located at c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA; preferably, the target region is located within the sequence shown in SEQ ID NO:9.

[0066] In an eleventh aspect, the present invention provides a method for preparing a brain calcification model, wherein the method comprises 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 by an intronic genomic fragment corresponding to 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 by an intronic genomic fragment corresponding to the human SLC20A2 gene, wherein the intronic genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene, thereby providing a brain calcification model.

[0067] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon.

[0068] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11.

[0069] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in the first aspect.

[0070] In certain embodiments, the replacement method comprises gene knockout, gene editing, RNA interference, epigenetic inhibition, or any combination thereof.

[0071] In certain embodiments, the model is an animal model, such as a mammalian model, such as a rodent model (eg, a mouse or rat model) or a non-human primate model.

[0072] In certain embodiments, the model is a mammalian cell model, for example, human dermal fibroblasts or brain glial cells.

[0073] In a twelfth aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises: an intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced by an intronic genomic segment corresponding to the human SLC20A2 gene, or an intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced by an intronic genomic segment corresponding to the human SLC20A2 gene, wherein the intronic genomic segment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0074] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in the first aspect.

[0075] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO:10.

[0076] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon 10 and exon 11 comprises the sequence shown in SEQ ID NO: 11.

[0077] In certain embodiments, the non-human animal is a rodent, such as a mouse or rat.

[0078] In certain embodiments, the non-human animal is a non-human primate.

[0079] In certain embodiments, the genetically modified non-human animal is an animal model for brain calcification.

[0080] In a thirteenth aspect, the present invention provides a genetically modified cell, the genome of which comprises: an intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to the human SLC20A2 gene, or an intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to the human SLC20A2 gene, wherein the intronic genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0081] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon.

[0082] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11.

[0083] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in the first aspect.

[0084] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene is exogenously introduced into the cell.

[0085] In certain embodiments, the cell is a mammalian cell.

[0086] In certain embodiments, the cells are human dermal fibroblasts or brain glial cells.

[0087] In certain embodiments, the genetically modified cell is a cell model of brain calcification.

[0088] In a fourteenth aspect, the present invention provides uses of genetically modified non-human animals or cells, wherein the uses include: (i) for indicating and analyzing the onset stage of brain calcifications, or for use as a model for studying brain phosphorus homeostasis imbalance or disease targets of brain calcifications; (ii) for use as a model for screening candidate drugs or treatment regimens for treating brain calcifications or diseases related to brain phosphorus homeostasis imbalance; and / or (iii) for use as a model for screening candidate drugs or treatment regimens for diseases related to phosphorus homeostasis imbalance;

[0089] The genome of the genetically modified non-human animal or cell comprises: an intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced by an intronic genomic segment corresponding to the human SLC20A2 gene, or an intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced by an intronic genomic segment corresponding to the human SLC20A2 gene, wherein the intronic genomic segment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0090] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon.

[0091] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11.

[0092] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in the first aspect.

[0093] In a fifteenth aspect, the present invention provides a nucleic acid construct comprising a minigene fragment sequence of the human SLC20A2 gene, wherein the minigene fragment sequence comprises exons exon2 and exon3 and an intron sequence therebetween, or comprises exons exon10 and exon11 and an intron sequence therebetween, wherein the intron sequence comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0094] In certain embodiments, the nucleic acid construct comprises a promoter operably linked to the minigene fragment sequence.

[0095] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in the first aspect.

[0096] In certain embodiments, the minigene fragment sequence comprises the sequence shown in SEQ ID NO:4 or 5.

[0097] In certain embodiments, the downstream of the minigene fragment sequence comprises a polyA signal sequence.

[0098] In certain embodiments, the nucleic acid construct further comprises a reporter gene, such as a fluorescent protein (eg, GFP, mCherry) or luciferase.

[0099] In a sixteenth aspect, the present invention provides a method for screening an agent capable of regulating SLC20A2 mRNA splicing, comprising: providing (i) a cell comprising the nucleic acid construct of the fifteenth aspect, (ii) the genetically modified non-human animal of the twelfth aspect, or the genetically modified cell of the thirteenth aspect, or (iii) a brain calcification model obtained by the method of the eleventh aspect; contacting a test agent with any one of (i) to (iii); and detecting alternative splicing of SLC20A2 mRNA.

[0100] In certain embodiments, the cell is from, for example, a mammal, such as a rodent (eg, a mouse or rat), a non-human primate, or a human.

[0101] In certain embodiments, a test agent is selected that inhibits alternative splicing (eg, reduces the amount of expression product containing cryptic exon insertions), thereby increasing the production of functional PiT2 protein.

[0102] In certain embodiments, a test agent is selected that enhances alternative splicing (eg, increases the amount of expression product containing a cryptic exon insertion), thereby reducing the production of functional PiT2 protein.

[0103] In certain embodiments, the detecting comprises analyzing the length of the SLC20A2 mRNA expression product.

[0104] In certain embodiments, the alternative splicing is alternative splicing in the intron 2 defined in the first aspect; preferably, the detection comprises PCR analysis using primers spanning exons exon2 and exon3; preferably, an amplicon comprising a cryptic exon insertion of approximately 135 nt in length is generated.

[0105] In certain embodiments, the alternative splicing is alternative splicing in the intron 10 defined in the first aspect; preferably, the detection comprises PCR analysis using primers spanning exons exon10 and exon11; preferably, an amplicon comprising a cryptic exon insertion of approximately 76 nt in length is generated.

[0106] In certain embodiments, the method comprises contacting a test agent with a cell comprising the nucleic acid construct in vitro and detecting alternative splicing of the nucleic acid construct; preferably, the detection comprises 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 DESCRIPTION OF THE DRAWINGS

[0107] Figure 1: SLC20A2 intronic variants detected by WGS in a PFBC family.

[0108] a, Kinship relationships of six PFBC families with SLC20A2 intronic mutations. Solid, open, gray, or slashed symbols indicate individuals affected by brain calcifications, individuals without brain calcifications, individuals who have not yet developed symptoms, and individuals who have died, respectively. Symbols with a question mark (?) indicate individuals for whom brain CT imaging data were unavailable. "#" and "&" indicate individuals selected for whole-exome sequencing (WES) or whole-genome sequencing (WGS), respectively. b, Representative brain CT images of patients in the six PFBC families. c, Distribution of five intronic mutations in the SLC20A2 locus. Light purple boxes indicate exons, and the numbers below indicate the exon number. Lines between exons indicate introns. In the intron mutation labeling, the mutations in italic bold font are predicted not to produce new splice acceptors or donors, suggesting that they may act by affecting potential cryptic exons; while the mutations in italic non-bold font may produce splice acceptor or donor mutations.

[0109] Figure 2: SLC20A2 cryptic exon mutations alter normal splicing of SLC20A2 mRNA and PiT2 protein expression.

[0110] ab, Schematic diagram of minigene construction and transcripts for the SLC20A2 intronic mutations c.289+937G>A and c.289+1007C>G (a). (b) PCR analysis of the minigenes to detect splicing alterations following intronic mutations. RT-PCR analysis of Neuro-2A cell samples transfected with SLC20A2 minigenes carrying the c.289+937G>A, c.289+1007C>G, and c.289+1021G>C mutations, respectively, revealed that the lower band represents the correctly spliced ​​exon, while the upper band represents the PCR product of a transcript containing a cryptic exon inserted between SLC20A2 exons 2 and 3. cd, Schematic diagram of the SLC20A2 minigene construct containing the c.1795-1698A>G mutation in human SLC20A2 intron 10 (c) and PCR analysis of the transcript (d). The lower band represents the correctly spliced ​​transcript, while the upper band represents the PCR product of the transcript containing a cryptic exon inserted between SLC20A2 exons 10 and 11. e, Schematic diagram of the SLC20A2 CDS construct (each with an N-terminal HA tag) with two intronic mutations causing aberrant splicing. f, Western blot analysis of the effects of endogenous SLC20A2 mutations on PiT2 protein production in Neuro-2A cells transfected with the HA-tagged SLC20A2 CDS construct. gh, Immunoblotting results of the SLC20A2 gene product PiT2 in fibroblast extracts from PFBC patients carrying the c.289+1007C>G mutation (P1), heterozygous deletion of the SLC20A2 gene (P2), or healthy controls (C1 and C2) showed that intronic mutations led to decreased PiT2 expression, similar to exonic mutations. (h) shows the statistical results.

[0111] Figure 3: Cryptic exons crpE2 and crpE10 are present in SLC20A2 transcripts produced by cells from human brain.

[0112] a, RT-PCR analysis of SLC20A2 mRNAs in HEK293T and A172 cells for cryptic exon insertions between adjacent exons. CHX, cyclohexylamine (a widely used compound that inhibits degradation of aberrant transcripts). Typically, the bottom band represents normal transcripts; the upper band represents aberrant transcripts containing certain cryptic exons (marked with asterisks and designated crpE2 and crpE10). b, Sanger sequencing of the upper bands of the cryptic exons (crpE2 and crpE10) in HEK293T and A172 cells in panel a revealed sequences identical to those found in the minigene assay in Figure 2, indicating that these cryptic exons are endogenous, but that mutations significantly increase their probability of integration into mRNA. c, RT-PCR products of crpE2 and crpE10 from human fibroblasts derived from human cell lines including HEK293T, HeLa, HepG2, and A172, as well as healthy controls (C1, C2); a PFBC patient with a heterozygous SLC20A2 c.289+1007C>G mutation (P1); and a PFBC patient with a heterozygous SLC20A2 deletion (P2).

[0113] Figure 4: ASO inhibits integration of the cryptic exon of SLC20A2 in the minigene

[0114] a, Schematic diagram of the cryptic exon of the SLC20A2 genomic sequence carrying the c.289+1007C>G mutation targeted by PMO-modified ASOs (PMO-1, PMO-2, PMO-3) and MOE-modified ASOs (MOE-A, MOE-B, MOE-C) on the minigene. b, c, RT-PCR analysis and statistical results of the integration of crpE2 cryptic exon in Neuro-2A cells transfected with the SLC20A2 c.289+1007C>G minigene and treated with PMO-modified ASOs (PMO-1, PMO-2, PMO-3) (b) or MOE-modified ASOs (MOE-A, MOE-B, MOE-C) (c) compared with control ASOs (PMO-NC and MOE-NC). The upper band represents the non-productive transcript crpE2 (729 bp); the lower band represents the normal transcript (594 bp). d, Schematic diagram of the PMO-modified ASOs (PMO-7, PMO-8, PMO-9) and MOE-modified ASOs (MOE-G, MOE-H, MOE-I) targeting the cryptic exon of the SLC20A2 genomic sequence carrying the c.1795-1698 mutation on the minigene. e, f, RT-PCR analysis and statistical analysis of crpE10 cryptic exon integration in Neuro-2A cells transfected with the SLC20A2 c.1795-1698A>G mutation and treated with PMO-modified ASOs (PMO-7, PMO-8, PMO-9) (e) or MOE-modified ASOs (MOE-G, MOE-H, MOE-I) (f), compared with control ASOs (PMO-NC and MOE-NC). RNA extracted from treated cells was analyzed for cryptic crpE10 exon integration. The upper band represents the transcript with crpE10 insertion (261 bp), and the lower band represents the normal transcript (185 bp). The red line represents the MOE-modified ASO; the blue line represents the PMO-modified ASO. The quantification value (%) of splicing events incorporating cryptic exons was calculated based on the intensity of the RT-PCR bands as follows: (transcripts incorporating cryptic exons / (transcripts incorporating cryptic exons + normal transcripts)) × 100. Relative PiT2 protein was assessed using Tubulin as an internal control (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0115] Figure 5: ASO inhibits the integration of cryptic exons in SLC20A2 mRNA caused by intronic mutations and upregulates PiT2 protein expression

[0116] a, Schematic diagram showing that MOE-modified ASOs (MOE-A, MOE-B, MOE-C) can target cryptic exons in the pre-mRNA of the SLC20A2 gene on chromosomes carrying the SLC20A2 c.289+1007C>G intronic mutation and on normal chromosomes without the mutation, inhibiting aberrant splicing of SLC20A2. b, RNA was extracted from stably cultured fibroblasts derived from a PFBC patient (P1) carrying the SLC20A2 c.289+1007C>G intronic mutation and treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or the control MOE-NC). RT-PCR analysis and statistical analysis of the integration of the crpE2 cryptic exon into SLC20A2 mRNA were performed. c, Western blot results and statistical analysis of PiT2 protein levels in P1-derived fibroblasts treated with MOE-modified ASOs (MOE-A, MOE-B, and MOE-C) compared with MOE-NC. The red line represents the MOE-modified ASO. Quantification of splicing events incorporating cryptic exons (%) was calculated based on the intensity of the RT-PCR bands as follows: (transcripts incorporating cryptic exons / (transcripts incorporating cryptic exons + normal transcripts)) × 100. Relative PiT2 protein was assessed using Tubulin as an internal control (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0117] Figure 6: ASO can also increase PiT2 expression in normal human fibroblasts by regulating splicing events involving cryptic exons of SLC20A2.

[0118] a, Schematic diagram of the effects of MOE-modified ASOs (targeting crpE2: MOE-A, MOE-B, MOE-C; targeting crpE10: MOE-J, MOE-K, MOE-L) on the pre-mRNA of the SLC20A2 gene on normal chromosomes in cultured human fibroblasts. These ASOs modulate the splicing of the cryptic crpE2 or crpE10 exons, thereby increasing SLC20A2 mRNA and PiT2 protein expression. b, RT-PCR analysis and statistical analysis of the expression levels of SLC20A2 mRNA transcripts inserted into the cryptic crpE2 exon in human fibroblasts derived from a healthy control (C1) after treatment with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or the control group, MOE-NC). The upper band represents the transcript with crpE2 insertion (699 bp), and the lower band represents the normal transcript (564 bp). c, Western blot analysis and statistical analysis of PiT2 protein levels in human fibroblasts from healthy controls (C1) treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. d, RT-PCR analysis and statistical analysis of SLC20A2 mRNA transcript expression levels in human fibroblasts from healthy controls (C2) treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or the control group, MOE-NC). The upper band represents the transcript with crpE2 insertion (699 bp), and the lower band represents the normal transcript (564 bp). e, Western blot analysis and statistical analysis of PiT2 protein levels in human fibroblasts from healthy controls (C2) treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared to MOE-NC. f, RT-PCR analysis and statistical analysis of the expression levels of SLC20A2 mRNA transcripts in human fibroblasts derived from healthy controls (C1) after treatment with MOE-modified ASOs (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). g, PiT2 protein levels in human fibroblasts derived from healthy controls (C1) after treatment with MOE-modified ASOs (MOE-G, MOE-H, MOE-I) compared to MOE-NC. Quantification of splicing events incorporating the cryptic exon (%) was calculated based on the intensity of the RT-PCR bands as follows: (transcripts incorporating the cryptic exon / (transcripts incorporating the cryptic exon + normal transcript)) × 100.The relative expression of PiT2 protein was assessed using Tubulin as an internal control (i.e., PiT2 / Tubulin). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t-test.

[0119] Figure 7: Expanded Screening of ASO Sequences that Modulate SLC20A2 Splicing

[0120] a, Minigenes were transfected into N2A cells, and the responses of the minigene's expressed splicing products to ASOs were analyzed by PCR, including new ASO sequences targeting crpE2 splicing regulation (cr5E2-m1, cr5E2-5, cr5E2-6,

[0121] cr5E2-8,5crE2-9,cr5E2-10,cr5E2-12,5crE2-13,cr5E2-13L,cr5E2-13S,

[0122] cr5E2-14,cr5E2-16,cr5E2-17,cr5E2-18,cr5E2-19,cr5E2-20,cr5E2-21,

[0123] cr5E2-22,cr5E2-23,cr5E2-24,cr3E2-5,cr3E2-8,cr3E2-9,cr3E2-10,

[0124] cr3E2-11, cr3E2-12, cr3E2-13, cr3E2-14, cr3E2-15, cr3E2-16), and new ASO sequences targeting crpE10 splicing regulation (cr5E10-9, cr5E10-11, cr5E10-12, cr5E10-13,

[0125] cr5E10-15, cr5E10-16, cr5E10-19, cr3E10-9, cr3E10-14, cr3E10-15). Their sequences and related information are listed in the attached table. b. ASO sequences identified by minigene screening were further screened in healthy human fibroblasts. The splicing regulatory effects of ASOs on endogenously expressed SLC20A2 were evaluated by repeated PCR and Western blotting. The experimental results for only a subset of sequences are listed above. c. After comprehensive analysis of the two rounds of screening, several ASO sequences with promising effects were identified.

[0126] Figure 8: ASO regulates SLC20A2 mRNA splicing and upregulates PiT2 protein expression in patients with heterozygous SLC20A2 deletion

[0127] a. Schematic diagram of how MOE-modified ASOs (MOE-A, MOE-B, MOE-C) can be used to target the splicing event of the cryptic exon crpE2 in the pre-mRNA of the SLC20A2 gene on another normal chromosome after culturing fibroblasts from a patient (P2) carrying a large heterozygous deletion of SLC20A2. b. RNA was extracted from stably cultured fibroblasts from a patient (P2) carrying a large heterozygous deletion of SLC20A2 and treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) or the control MOE-NC. RT-PCR analysis and statistical analysis of the integration of the cryptic exon crpE2 into SLC20A2 mRNA were performed. c, Western blot results and statistical analysis of PiT2 protein levels in P2 fibroblasts treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared with MOE-NC. The red line represents the MOE-modified ASO. Quantification of splicing events incorporating cryptic exons (%) was calculated based on the intensity of the RT-PCR bands as follows: (transcripts incorporating cryptic exons / (transcripts incorporating cryptic exons + normal transcripts)) × 100. Relative PiT2 protein was assessed using Tubulin as an internal control (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0128] Figure 9: Construction of humanized SLC20A2 intron knock-in (SLC20A2-KI) model mice and analysis of SLC20A2 expression.

[0129] a, Schematic diagram of the construction of the SLC20A2-KI mouse model with humanized SLC20A2 intron 2. CRISPR / Cas9 was used to create a nick in the SLC20A2 intron 2 genomic region. Homologous recombination-based repair was then performed using an introduced targeted donor sequence. This replaced the mouse SLC20A2 intron 2 and the flanking exon 2 and exon 3 regions with sequences from human SLC20A2 intron 2 carrying the c.289+1007C>G mutation. The resulting transcripts were in-frame with the entire mouse SLC20a2 reading frame. b, RT-PCR analysis of the expression levels of SLC20A2 transcripts incorporating humanized crpE2 in brain tissue from heterozygous and homozygous SLC20A2-KI mice and wild-type mice. The upper band represents the aberrant transcript (crpE2) produced by the humanized intron 2 c.289+1007C>G mutation; the lower band corresponds to the normal SLC20A2 transcript. WT, wild type; HET, heterozygous; HOM, homozygous; Ctx, cerebral cortex; Hip, hippocampus; Tha, thalamus. c, d, qPCR analysis (c) and western blot analysis (d) of SLC20A2 transcript levels in the brains of WT, HET, and HOM SLC20A2-KI mice show defects in Slc20a2 mRNA and PiT2 protein expression similar to those in Slc20a2 KO mice. e, CSF Pi levels in heterozygous and homozygous SLC20A2-KI mice show a graded increase relative to wild-type mice, similar to those in Slc20a2 KO mice.

[0130] FIG10 : Micro-CT and RIS staining analysis of brain calcification in humanized SLC20A2 (SLC20A2-KI) model mice.

[0131] a, Micro-CT scans (voxel resolution: 9 μm x 9 μm x 9 μm) of the brains of homozygous SLC20A2-KI mice at 5, 7, and 9.5 months of age, following perfusion fixation with PFA, demonstrate calcification. From left to right, micro-CT images show the coronal section of the dorsal midbrain, the coronal section of the basal forebrain, and the sagittal section of one hemisphere. White dots represent calcified nodules. b, Brain calcification images were acquired using a VS120 microscope and stained with the fluorescent dye RIS-AF647 (an imaging reagent carrying the AlexaFluo-647 nm fluorophore that can bind to calcium bisphosphonates) in brain sections from homozygous SLC20A2-KI mice at 5, 7, and 9.5 months of age. Images are representative of analyses of three mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test. Scale bar, 1 mm.

[0132] FIG11 : Histochemical staining analysis of brain calcification in humanized SLC20A2 (SLC20A2-KI) model mice.

[0133] OCT frozen sections of fixed brains from homozygous SLC20A2-KI mice at 7 months (a), 10 months (b), and 12 months (c) were then stained with five different histochemical stains: alizarin red, Von Kossa, Alcian blue, H&E, and PAS. Alizarin red and Von Kossa stains primarily reveal calcifications, while Alcian blue and PAS stains primarily reveal glycosaminoglycans attached to calcifications, a common feature of calcifications. Scale bar, 500 μm.

[0134] Figure 12: Intraventricular ASO intervention restored SLC20A2 expression in the brain of SLC20A2-KI mice and downregulated phosphate levels in the cerebrospinal fluid.

[0135] a, Experimental design for intracerebroventricular (ICV) administration of an ASO to homozygous SLC20A2-KI mice. Mice were treated ICV with MOE-NC, MOE-A, or MOE-B at 3 months of age for 4 months. Doses of each MOE were 50 μg, 100 μg, 200 μg, or 400 μg. Brain tissue was collected from 7-month-old mice and analyzed for SLC20A2 transcripts containing cryptic exon inclusions, PiT2 protein levels, cerebrospinal fluid phosphate levels, and brain calcification. b, In situ hybridization analysis of MOE-A distribution throughout the brain (cortex, hippocampus, thalamus, and cerebellum) of 5-month-old SLC20A2-KI mice, 2 months after ICV administration. RNA probes were designed to bind specifically to the MOE-A sequence. MOE-A is stained red, and DAPI is stained blue. c, Quantitative PCR analysis of normal SLC20A2 mRNA transcript expression in SLC20A2-KI mice 4 months after MOE-NC, MOE-A, or MOE-B administration compared to age-matched WT mice and untreated homozygous SLC20A2-KI mice. d, RT-PCR analysis of transcripts containing the cryptic exon insertion crpE2 and normal SLC20A2 transcripts 4 months after ICV injection of MOE-NC, MOE-A, and MOE-B. The top band represents transcripts containing the cryptic exon insertion crpE2 (crpE2), while the bottom band represents normal SLC20A2 transcripts. e, PiT2 protein levels in the thalamus of SLC20A2-KI mice 4 months after ICV injection of MOE-A show significant restoration of PiT2 expression compared to control MOE and untreated mice, demonstrating the efficacy of the ASO. f, In addition to the expression of target genes, the level of inorganic phosphate in cerebrospinal fluid, a biomarker directly related to brain calcification, also showed a differential decrease, further demonstrating the effectiveness of ASO.

[0136] Figure 13: Intraventricular ASO treatment of SLC20A2-KI mice significantly inhibited the occurrence and progression of brain calcification

[0137] a, Sagittal micro-CT images of brain calcification in 3-month-old SLC20A2-KI mice 4 months after ICV injection of different doses (50, 100, 200, and 400 μg) of MOE-NC, MOE-A, and MOE-B; white highlights indicate brain calcified nodules. b, Quantification of the number and area of ​​calcified nodules distributed in the thalamus, hypothalamus, midbrain, and basal forebrain in 3-month-old SLC20A2-KI mice 4 months after ICV injection of different doses (50, 100, 200, and 400 μg) of MOE-NC, MOE-A, and MOE-B, at 7 months of age. Images are representative of analyses of two to three mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test. Scale bar, 1 mm.

[0138] Figure 14: ICV injection of ASO drugs can inhibit the further deterioration of brain calcification in SLC20A2-KI mice that already have brain calcification.

[0139] a, Experimental design for ICV administration of PMO-modified ASOs in homozygous SLC20A2-KI mice. Mice, already exhibiting significant calcification throughout multiple brain regions at 5 months of age, were treated with ICV injections of PMO-NC or PMO-1 for 2 months. Each PMO dose was 25 μg or 75 μg. Brain tissue was harvested from 7-month-old mice and analyzed for cryptic exon inclusion and brain calcification. CSF was extracted and analyzed before and at the end of PMO treatment (i.e., 5 and 7 months). b, Changes in CSF Pi levels in homozygous SLC20A2-KI mice before and after administration of PMO-NC and PMO-1. c, Sagittal micro-CT images of brain calcification in 7-month-old homozygous SLC20A2-KI mice after ICV administration of control and therapeutic ASOs at doses of 25 μg and 75 μg. White highlights indicate cerebral calcification nodules. Images are representative of analyses of two to three mice per group. ****P < 0.0001, Student's t-test. Scale bar, 1 mm.

[0140] Figure 15: Effects of global or conditional knockout of Pit2 (Slc20a2) on brain phosphorus homeostasis.

[0141] Figure A shows that the average level of inorganic phosphate (Pi) in the cerebrospinal fluid (CSF) of PiT2 knockout (Pit2 KO) mice is ~1.8mM, which is significantly increased compared to the CSF Pi concentration in the brain of WT mice (~0.5mM). Figure B shows that there is no significant change in inorganic phosphate (Pi) in the serum of PiT2 knockout mice. Figure C shows the changes in the concentration of CSF Pi in the brain of Pit2 conditional knockout (Pit2 cKO) mice in different cell types, among which astrocytes are conditionally knocked out (using Aldh1L1-CreER T2 The CSF Pi of mice induced by Tamoxifen (Achieved after induced by Tamoxifen) increased most significantly (increase of about 170%), with an average level of about 1.35mM. Other conditional knockout mice, including oligodendrocytes (Plp1-CreER T2 Changes in CSF Pi in mice expressing CSF Pi in excitatory neurons (Camk2a-iCre), inhibitory neurons (Vgat-Cre), and perivascular cells (Pdgfrb-Cre) were more modest (increases ranging from 40-60%), suggesting that astrocyte-mediated Pi transport is crucial for maintaining brain phosphate homeostasis. Figure D shows no significant differences in serum Pi concentrations between the different conditional knockout mouse strains.

[0142] Figure 16: Intravenous injection of AAV virus mediates the selective expression of PiT2 in brain astrocytes.

[0143] Panel A shows the AAV serotype (PHP.eB) and expression vector structure, as well as a schematic diagram of intravenous injection in mice. Panel B shows sagittal brain sections prepared from mice one month after intravenous injection, fixed with PFA. Immunofluorescence staining reveals widespread expression of PiT2-V5 signals (red) in astrocytes throughout the mouse brain, with blue Hoechst staining indicating cell nuclei.

[0144] Figure 17: The inhibitory effect of selective compensatory expression of AAV in astrocytes on calcification in the mouse brain.

[0145] MicroCT scans of the mouse brain after perfusion showed that Pit2 KO mice treated with the control virus (expressing only GFP) showed significant calcification in multiple brain regions on both coronal and sagittal planes at 5 and 7 months of age. Significant and large areas of calcification were found in the dorsal midbrain, basal forebrain, thalamus, and hypothalamus. The calcification area and granules were larger in 7-month-old mice than in 5-month-old mice. However, calcification was significantly suppressed in the brains of Pit2 KO mice that successfully received therapeutic AAV injections (Replenished) at both 5 and 7 months of age.

[0146] Detailed Description of the Invention

[0147] I. Definition

[0148] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0149] As used herein, the term "SLC20A2" refers to the gene encoding solute carrier family 20 member 2 (see, e.g., human gene NCBI ID: 6575), also known as PiT2, which encodes a 652-amino acid phosphate transporter, known as PiT2 protein, or PiT-2 protein (see, e.g., NP_006740). The mRNA sequence of SLC20A2 is well known to those skilled in the art and can be found, e.g., at NCBI: NM_006749, NM_001257181, or NM_001257180. When referring to the location of the mRNA sequence of the SLC20A2 gene herein, for example, to describe the location of cryptic exons, the sequence shown in NCBI: NM_006749 is used as a reference. An exemplary amino acid sequence of the PiT2 protein is shown in SEQ ID NO: 155.

[0150] As used herein, the term "XPR1" refers to the gene encoding the phosphate efflux transporter protein XPR1. The sequences of the XPR1 gene (e.g., gene ID: 9213) and the XPR1 protein (e.g., NP_004727.2) are known to those skilled in the art and can be found in public databases, such as HGNC: 12827. An exemplary amino acid sequence of the XPR1 protein is shown in SEQ ID NO: 153.

[0151] As used herein, the term "MYORG" refers to the gene encoding the regulatory factor MYORG of the phosphate efflux transporter protein XPR1. The sequences of the MYORG gene (e.g., gene ID: 57462) and the MYORG protein (e.g., NP_065753.2) are known to those skilled in the art and can be found in public databases, such as HGNC: 19918. An exemplary amino acid sequence of the MYORG protein is shown in SEQ ID NO: 154.

[0152] As used herein, the term "modulation of splicing" refers to changes that affect the levels of any RNA or mRNA variant produced from the natural precursor mRNA of SLC20A2. For example, modulation can mean, for example, causing an increase or decrease in the level of an abnormal SLC20A2 mRNA containing a cryptic exon insertion, causing an increase or decrease in the level of a normal full-length SLC20A2 mRNA, and / or causing an increase or decrease in the level of an abnormal SLC20A2 RNA or mRNA containing a premature stop codon (nonsense codon). It will be readily understood that any change in the ratio between SLC20A2 splice variants is also considered a result of splicing modulation. Each possibility represents a separate embodiment of the present invention. In certain embodiments, modulation means increasing the level of normal full-length SLC20A2 mRNA and / or decreasing the level of an abnormal (e.g., containing a cryptic exon insertion) SLC20A2 mRNA.

[0153] As used herein, the term "complementary" or "complementarity" refers to the ability of nucleic acids such as oligonucleotides, polynucleotides, etc. to form base pairs with each other. Base pairs are generally formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can carry out base pairing in a Watson-Crick manner (e.g., A to T, A to U, C to G) or in any other manner that allows the formation of a duplex. As known to those skilled in the art, when using RNA rather than DNA, the base that is considered to be complementary to adenosine is uracil rather than thymine. However, when representing U in the context of the present invention, unless otherwise indicated, the ability to replace T is implied. "At least partially complementary" refers to that two sequences can be fully complementary, or generally no more than 6, 5, 4, 3, 2 or 1 mismatched base pairings, while retaining the ability to hybridize under relevant conditions. Those skilled in the art can determine the conditions that are most suitable for testing the complementarity of two sequences according to the final application of the nucleotides of hybridization. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours in an in vitro assay system, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, can also be applied.

[0154] As used herein, unless otherwise specified, the left end of a single-stranded nucleic acid (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) sequence is 5' end, and the left direction of a single-stranded or double-stranded nucleic acid sequence is referred to as 5' direction. Similarly, the right end or right direction of a nucleic acid sequence (single-stranded or double-stranded) is 3' end or 3' direction. Generally speaking, the region or sequence 5' of a reference point in nucleic acid is referred to as "upstream," and the region or sequence 3' of a reference point in nucleic acid is referred to as "downstream." Generally speaking, the 5' direction or end of mRNA is the position where the initiation codon (initiation codon / start codon) is located, and the 3' end or direction is the position where the termination codon is located. In some aspects, the nucleotides upstream of a reference point in nucleic acid can be represented by a negative number, while the nucleotides downstream of a reference point can be represented by a positive number. For example, a reference point (e.g., an exon-exon junction in an mRNA) can be represented as the "zero" position, and the nucleotides immediately adjacent to and upstream of the reference point are represented as "minus one," e.g., "-1," while the nucleotides immediately adjacent to and downstream of the reference point are represented as "plus one," e.g., "+1."

[0155] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art. "Pharmaceutically acceptable carriers and / or excipients" do not exert or are not intended to exert a therapeutic effect at the intended dose. Such ingredients can play the following roles: a) assist in the processing of the drug delivery system during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of the active ingredient, c) assist in product identification, and / or d) enhance the overall safety, efficacy, delivery or any other properties of the active ingredient during storage and use. For example, the siRNA of the present invention can be encapsulated by a delivery vehicle. "Pharmaceutically acceptable carriers and / or excipients" include, but are not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic 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 agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents 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 polyols (e.g., glycerol).

[0156] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (if not receiving treatment).

[0157] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for treating a disease is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease, such as an amount necessary to improve at least one clinical parameter of the disease or reduce the severity of at least one clinical parameter of the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0158] II. Therapies that Regulate Alternative Splicing

[0159] In some aspects, the present invention relates to the regulation of alternative splicing and its application in disease treatment, disease model preparation, etc.

[0160] 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 SLC20A2 account for 61% of all reported PFBC cases, suggesting that PFBC could be treated with gene therapy targeting these genes. However, a lack of understanding of the pathogenic mechanisms of PFBC and a lack of effective delivery tools have significantly hindered the development of gene-editing therapies for PFBC.

[0161] The inventors of this application performed whole-genome sequencing (WGS) on 135 PFBC patients identified as exon-mutated or CNV-negative by whole-exome sequencing (WES). They discovered intronic variants in the SLC20A2 gene in five families with autosomal dominant PFBC. Subsequently, minigene testing and patient-derived cell experiments confirmed the presence of cryptic exon sequences within the intronic sequence of the SLC20A2 pre-mRNA. These intronic mutations increased the probability of integration of these cryptic exon sequences during mRNA processing. Because these cryptic exon sequences contain premature stop codons, the aberrantly spliced ​​mRNA sequence cannot be translated to produce the functional protein product, PiT2. Thus, the inventors of this application discovered a mechanism for premature termination of PiT2 translation caused by previously unreported cryptic exon insertions in aberrant SLC20A2 mRNA splicing. This mechanism provides a new strategy for regulating SLC20A2 mRNA splicing and expression.

[0162] Based on the above findings, the inventors of the present application were able to enhance the expression of normal SLC20A2 mRNA and PiT2 protein through ASO-mediated splicing regulation in cells containing SLC20A2 intronic mutations and fibroblasts from patients with SLC20A2 haploinsufficiency. Furthermore, in a humanized SLC20A2 mouse model that mimics SLC20A2-deficient PFBC patients, intracerebroventricular (ICV) administration of ASOs reduced aberrant SLC20A2 transcripts and inhibited the occurrence and progression of brain calcification.

[0163] Therefore, the inventors of the present application provide a therapy for inhibiting brain calcification by regulating the SLC20A2 alternative splicing process and reducing the integration of cryptic exons in introns, providing a promising treatment for diseases related to PiT2 functional deficiency (such as PFBC caused by SLC20A2 haploinsufficiency).

[0164] In addition, based on the discovered alternative splicing in the SLC20A2 intron, the inventors of the present application further provide cell and animal models of brain calcification and methods for preparing the same, which can be used to indicate and analyze the onset stage of brain calcification, as a model for studying phosphorus homeostasis imbalance in the brain or disease targets of brain calcification, and for screening drugs.

[0165] II-A. Application of Regulating Alternative Splicing in Disease Treatment

[0166] In some aspects, the present invention relates to reagents, methods and uses for regulating the splicing of SLC20A2 gene pre-mRNA to restore or enhance the expression and function of the protein product PiT2 of the SLC20A2 gene.

[0167] One aspect of the present invention provides the use of an agent capable of regulating alternative splicing within an intron of the SLC20A2 gene for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject; or the use of an agent in the preparation of a medicament for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject. The present invention also provides a method for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent capable of regulating alternative splicing within an intron of the SLC20A2 gene. The PiT2 functional deficiency includes a lack of expression or activity.

[0168] In certain embodiments, the alternative splicing comprises a cryptic exon inclusion, and the agent modulates (e.g., inhibits or enhances) the splicing of the cryptic exon from the pre-mRNA of the SLC20A2 gene. In certain embodiments, the agent thereby modulates (e.g., inhibits or enhances) the level of mature mRNA processed from the pre-mRNA, and / or modulates (e.g., inhibits or enhances) the expression of the PiT2 protein.

[0169] In certain embodiments, the agent inhibits the insertion of the cryptic exon in mature SLC20A2 mRNA, increases the level of correctly spliced ​​mature SLC20A2 mRNA, decreases the level of aberrantly spliced ​​mature SLC20A2 mRNA, and / or increases the production of functional PiT2 protein. As used herein, the phrase "increases the level of correctly spliced ​​mature SLC20A2 mRNA" refers to an increase in the level of correctly spliced ​​mature SLC20A2 mRNA after treatment with an agent provided herein, compared to the level before treatment or after control treatment.

[0170] In certain embodiments, the agent promotes insertion of the cryptic exon in mature SLC20A2 mRNA, inhibits the level of correctly spliced ​​mature SLC20A2 mRNA, and increases the level of aberrantly spliced ​​mature SLC20A2 mRNA; wherein the cryptic exon sequence carries a premature termination codon (PTC), and the insertion of the cryptic exon results in a reduction or inhibition of the production of functional PiT2 protein. Herein, the phrase "reducing the level of aberrantly spliced ​​mature SLC20A2 mRNA" refers to a reduction in the level of aberrantly spliced ​​mature SLC20A2 mRNA after treatment with the agent provided herein, compared to the level before treatment or after control treatment. The aberrantly spliced ​​mature SLC20A2 mRNA includes a cryptic exon insertion between exons 2 and 3 or between exons 10 and 11 as defined herein.

[0171] Alternative splicing in intron 2

[0172] In certain embodiments, the alternative splicing comprises alternative splicing within intron 2.

[0173] In certain embodiments, the alternative splicing in intron 2 is the insertion of a cryptic exon between exons 2 and 3 in the processed SLC20A2 mRNA sequence.

[0174] In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0175] In certain embodiments, the c.289+1007C>G mutation of the SLC20A2 gene promotes alternative splicing in intron 2.

[0176] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0177] In certain embodiments, the agent inhibits alternative splicing in intron 2. As used herein, the phrase "inhibiting alternative splicing in intron 2" refers to reducing the incidence of addition of a 135 nt long cryptic exon sequence (SEQ ID NO: 2) found in intron 2 of the SLC20A2 gene to mature SLC20A2 mRNA.

[0178] Alternative splicing in intron 10

[0179] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10.

[0180] In certain embodiments, the alternative splicing in intron 2 is the insertion of a cryptic exon between exons 10 and 11 in the processed SLC20A2 mRNA sequence.

[0181] In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0182] In certain embodiments, the c.1795-1698A>G mutation of the SLC20A2 gene promotes alternative splicing in intron 10.

[0183] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0184] In certain embodiments, the agent inhibits alternative splicing in intron 10. As used herein, the phrase "inhibiting alternative splicing in intron 10" refers to reducing the incidence of addition of a 76nt cryptic exon sequence (SEQ ID NO: 3) found in intron 10 of the SLC20A2 gene to mature SLC20A2 mRNA.

[0185] In certain embodiments, the alternative splicing comprises alternative splicing in intron 2 or alternative splicing in intron 10. In certain embodiments, the agent is an agent that inhibits alternative splicing in intron 2, or an agent that inhibits alternative splicing in intron 10.

[0186] Target area

[0187] In certain embodiments, the agent specifically binds to a target region in the SLC20A2 gene or its pre-mRNA, the target region being located in an intron region between two typical exon regions, and wherein the intron region contains the cryptic exon.

[0188] In certain embodiments, the target region is at most about 100 (e.g., at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) upstream of the cryptic exon. ) nucleotide to at most about 100 (e.g., at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides downstream of the cryptic exon.

[0189] In certain embodiments, the target region is selected from a region spanning the cryptic exon splice site, a region within the cryptic exon, a region within 100 nucleotides upstream of the cryptic exon, or a region within 100 nucleotides downstream of the cryptic exon.

[0190] In certain embodiments, the target region is selected from a region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, a region spanning the 5' splicing site, a region within the cryptic exon, a region spanning the 3' splicing site, or a region downstream of the 3' splicing donor site (SD).

[0191] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its pre-mRNA.

[0192] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:8.

[0193] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its pre-mRNA.

[0194] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:9.

[0195] In certain embodiments, the target region comprises a region spanning the 5' splicing acceptor site (SA) of the cryptic exon. In certain embodiments, the target region is within a region from at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides upstream relative to the 5' splice site of the cryptic exon to at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides downstream relative to the 5' splice site of the cryptic exon. In certain embodiments, the target region is within a region from up to about 20 nucleotides upstream of the 5' splice site relative to the cryptic exon to up to about 20 nucleotides downstream of the 5' splice site relative to the cryptic exon.

[0196] In certain embodiments, the target region comprises a region spanning the cryptic exon 3' splicing donor site (SD). In certain embodiments, the target region is within a region from at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides upstream of the 3' splice site of the cryptic exon to at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides downstream of the 3' splice site of the cryptic exon. In certain embodiments, the target region is within a region from up to about 20 nucleotides upstream of the 3' splice site relative to the cryptic exon to up to about 20 nucleotides downstream of the 3' splice site relative to the cryptic exon.

[0197] In certain embodiments, the target region is located within the cryptic exon. In certain embodiments, the target region or its vicinity comprises a splicing regulatory element, such as a splicing enhancer element or a splicing silencer element.

[0198] In certain embodiments, the target region is located in an intron region upstream or downstream of the cryptic exon. In certain embodiments, the target region or its vicinity comprises a splicing regulatory element, such as a splicing branch site, a splicing enhancer element, or a splicing silencer element.

[0199] Reagents

[0200] In certain embodiments, the reagent is an antisense oligonucleotide (ASO). In certain embodiments, the reagent is a gene editing tool, such as a DNA or RNA editing system, such as CRISPR / Cas, a base editor, or an RNA editing tool that is not dependent on Cas proteins. In certain embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In certain embodiments, the reagent is an RNA editor, such as CRISPR / Cas13. In certain embodiments, the reagent is a base editor Adenine / Cytosine base editor. In certain embodiments, the reagent is an RNA editing tool that is not dependent on CRISPR / Cas systems.

[0201] ASO

[0202] In certain embodiments, the agent is an antisense oligonucleotide (ASO), which specifically binds to the target region in the SLC20A2 pre-mRNA. The ASO specifically binds to the target region based on the principle of base complementarity, thereby regulating pre-mRNA splicing through a steric hindrance effect.

[0203] In certain embodiments, the ASO consists of 10-30 (e.g., 15-30, 15-25, 18-30, 18-25, 20-25) consecutive nucleotides; preferably, consists of 18-25 consecutive nucleotides.

[0204] As used herein, the term ASO includes any form of oligomeric molecule containing a nucleic acid base capable of complementary hybridization with a nucleic acid base on a target mRNA. ASOs may comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or more of the foregoing. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides having modified or substituted sugar groups and / or having modified backbones. Chemical modifications of ASOs will be apparent to those skilled in the art.

[0205] In certain embodiments, the ASO comprises modification of backbone nucleotides and nucleotide linking bonds, modification of ribose, modification of ribose side chain bases or a combination thereof. In certain embodiments, the backbone modification is selected from phosphorothioate linkages or phosphorodiamidate linkages. In certain embodiments, the sugar moiety modification includes but is not limited to: 2'-O-methoxyethyl (2'-MOE), morpholino (morpholino), 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), restricted ethyl (constrained ethyl, cEt), locked nucleic acid (LNA), peptide nucleic acid.

[0206] In certain embodiments, each monomer of ASO is modified in the same manner, for example, each linkage of the main chain of ASO comprises phosphorothioate linkage or each ribose moiety comprises 2'O-methyl modification. Such modification present in each of the monomer components of ASO is referred to as "uniform modification". In certain embodiments, a combination of different modifications may be desired, for example, ASO may comprise a combination of a diaminophosphorothioate linkage and a sugar moiety comprising a morpholine ring (morpholino). The combination of different modifications for ASO is referred to as "mixed modification" or "mixed chemical modification (mixed chemistries)".

[0207] In certain embodiments, the ASO comprises a 2'MOE modification. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification. In certain embodiments, the modification of the ASO comprises a combination of a 2'MOE and a locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0208] In certain embodiments, the ASO comprises a 2'-OMe modification. In certain embodiments, each nucleotide of the ASO has a 2'-OMe modification. In certain embodiments, the modifications of the ASO comprise a combination of 2'-OMe and locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0209] In certain embodiments, the ASO comprises a phosphorodiamidate linkage in combination with a sugar moiety comprising a morpholine ring (morpholino), such as a phosphorodiamidate morpholino oligomer (PMO).

[0210] In certain embodiments, the ASO specifically binds to a target region located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0211] In certain embodiments, the ASO has at least 80% complementarity to the nucleotide sequence within SEQ ID NO: 8. As used herein, complementarity (the degree to which one polynucleotide is complementary to another) can be quantified based on the proportion (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules. The sequence of an ASO need not be 100% complementary to the sequence of the target nucleic acid to which it hybridizes. In certain embodiments, an ASO may have 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% sequence complementarity to a target region within a target nucleic acid sequence to which it is targeted. For example, an ASO having 18 of the 20 nucleobases of an oligomeric compound complementary to the target region and thus specifically hybridizing would exhibit 90% complementarity.

[0212] In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof.

[0213] In some embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 phosphorothioate linkage. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification and has a phosphorothioate-linked backbone.

[0214] In other embodiments, the ASO comprises the nucleotide sequence shown in any one 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 thereto, and the ASO is a PMO.

[0215] In certain embodiments, the ASO specifically binds to a target region located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0216] In certain embodiments, the ASO has at least 80% (eg, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) complementarity to the nucleotide sequence within SEQ ID NO:9.

[0217] In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as set forth in 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 thereof.

[0218] In some embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 phosphorothioate linkage. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification and has a phosphorothioate-linked backbone.

[0219] In other embodiments, the ASO comprises the nucleotide sequence shown in any one 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 thereto, and the ASO is a PMO.

[0220] gene editing tools

[0221] In certain embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool, which typically comprises (i) a module with base editing activity and (ii) a module capable of binding to the target region in the SLC20A2 gene. The gene editing tool mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon in the genome or pre-mRNA sequence, thereby destroying its SA or SD function and inhibiting the integration of the cryptic exon into the mRNA.

[0222] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0223] CRISPR / Cas system

[0224] In certain embodiments, the agent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), wherein the gRNA is capable of binding to the target region in the SLC20A2 gene or its pre-mRNA.

[0225] In certain embodiments, the gRNA binds to the target region and recruits the binding of the Cas enzyme and exerts editing activity.

[0226] In certain embodiments, the CRISPR system mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon to destroy its SA or SD function, thereby inhibiting the integration of the cryptic exon into the mRNA.

[0227] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0228] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0229] DNA base editors

[0230] In certain embodiments, the agent is a DNA base editor. A DNA base editor typically comprises (i) a fusion protein comprising a DNA base editing domain (e.g., a deaminase) and a DNA binding domain (e.g., a Cas protein, such as Cas9 or Cas12) and (ii) a guiding RNA (gRNA) that is capable of binding to the target region in the SLC20A2 gene.

[0231] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0232] In certain embodiments, the DNA base editor performs base mutations in the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon, thereby destroying its SA or SD function and thereby inhibiting the integration of the cryptic exon in the mRNA.

[0233] In certain embodiments, the Cas protein is a catalytically inactive Cas protein or a Cas nickase protein.

[0234] In certain embodiments, the base editor comprises: ABE (Adenine base editor) or CBE (Cytosine base editor), or a new editor obtained by molecular evolution / mutation screening.

[0235] RNA editing tools

[0236] In certain embodiments, the reagent is an RNA editing tool. RNA editing tools include, for example, RNA base editors based on the CRISPR / Cas system and editors that do not rely on the CRISPR / Cas system, such as oligonucleotide-based base editors.

[0237] In certain embodiments, the RNA base editor based on the CRISPR / Cas system comprises (i) a fusion protein comprising an RNA base editing domain (e.g., an RNA deaminase) and an RNA binding domain (e.g., Cas13) and (ii) a guiding RNA (gRNA) that is capable of binding to the target region in the SLC20A2 pre-mRNA. The gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0238] In certain embodiments, the oligonucleotide-based base editor comprises (i) a sequence module that can recruit an endogenous RNA base editing protein (e.g., an RNA deaminase, such as ADAR) and (ii) a guide sequence module that can bind to the target region in the SLC20A2 pre-mRNA. The guide sequence module binds to the target region and thereby recruits the RNA base editing protein to bind and exert base editing activity.

[0239] In certain embodiments, the RNA editing tool performs base mutations in the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon in the pre-mRNA, thereby destroying its SA or SD function and inhibiting the integration of the cryptic exon in the mRNA.

[0240] application

[0241] In certain embodiments, the PiT2 functional defect involves a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation, or a structural variation.

[0242] In certain embodiments, the mutation is located in an exonic region and the subject is heterozygous for the mutation in the exonic region.

[0243] In certain embodiments, the mutation is an intronic mutation, such as a mutation within intron 2 and / or intron 10. In certain embodiments, the mutation is located in the target region within an intron, and the subject may be heterozygous, homozygous, or compound heterozygous for an intronic mutation located within the target region.

[0244] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0245] In certain embodiments, the PiT2 functional deficiency is SLC20A2 haploinsufficiency.

[0246] In certain embodiments, the medicament treats the disease by increasing the expression of PiT2 protein or functional RNA by cells of the subject. Therefore, in certain embodiments, the disease involved in the present invention can be any disease caused by or involving PiT2 functional deficiency, as long as the treatment of the disease will benefit from the regulation (e.g., increase) of functional PiT2 protein or RNA expression.

[0247] In certain embodiments, the disease associated with PiT2 functional deficiency is brain calcification. In certain embodiments, the brain calcification is primary familial brain calcification (PFBC).

[0248] In certain embodiments, the disease associated with PiT2 functional deficiency is a disease associated with abnormal Pi balance. In certain embodiments, the disease associated with abnormal Pi balance includes chronic kidney disease, heart valve calcification, arterial calcification, and / or bone diseases associated with phosphorus homeostasis imbalance.

[0249] In certain embodiments, the agent described in any of the above embodiments can be formulated into a dosage form compatible with its intended route of administration, for example, by local administration (such as direct injection or implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal or parenteral administration, including intracranial (such as intraventricular, intrameningeal or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal or topical (including buccal and sublingual) administration.

[0250] In certain embodiments, the reagent is administered by suction, intranasal administration, intratracheal administration or oropharyngeal inhalation. The active ingredient of the required amount can be mixed in a suitable solvent and then aseptically filtered to prepare a preparation suitable for inhalation administration. Generally, the preparation for inhalation administration is a sterile solution under physiological pH and has low viscosity. Salt can be added to the preparation to balance tension. In some cases, a surfactant or cosolvent can be added to increase the active ingredient solubility and improve aerosol properties. In some cases, an excipient can be added to control viscosity to ensure the size and distribution of the atomized droplets.

[0251] In certain embodiments, the agent can be administered by injection, such as intravenously, intramuscularly, subcutaneously, intradermally, intraarticularly, intraocularly, intraperitoneally, intraventricularly (e.g., by lumbar puncture) or topically. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS) and the like. Pharmaceutical compositions should remain stable under production and storage conditions and should prevent contamination by microorganisms such as bacteria and fungi. For example, sterile injection solutions can be prepared by the following method: the active ingredient of the required dose is mixed in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delayed absorption, preservatives, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use.

[0252] In certain embodiments, the agent is selected from ASO, and the pharmaceutical composition is preferably configured as a solution, emulsion, microemulsion, foam or a liposome-containing formulation (e.g., cationic liposomes or non-cationic liposomes). In certain embodiments, the pharmaceutical composition is preferably configured for intracerebroventricular injection (e.g., by lumbar puncture), intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation or intravenous injection.

[0253] In another aspect, the present invention provides a method for regulating PiT2 protein expression in a cell, comprising contacting the cell with the agent capable of regulating alternative splicing in an intron of the SLC20A2 gene as described above in vitro.

[0254] In certain embodiments, the cell comprises a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splice mutation, or a structural variation. In certain embodiments, the cell is heterozygous for the mutation. In certain embodiments, the cell is homozygous for the mutation, wherein the mutation is located within the target region.

[0255] II-B. Agents targeting the SLC20A2 target region and their applications

[0256] In some aspects, the present invention relates to reagents, methods, and uses for targeting a target region in SLC20A2 to restore or enhance the function of the SLC20A2 gene product.

[0257] One aspect of the present invention provides an agent capable of specifically binding to a target region in the SLC20A2 gene or its pre-mRNA, wherein the target region is located in an intron region between two typical exon regions, and wherein the intron region contains a cryptic exon.

[0258] In certain embodiments, the cryptic exon is located within intron 2.

[0259] In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene or its pre-mRNA.

[0260] In certain embodiments, the c.289+1007C>G mutation of the SLC20A2 gene promotes a cryptic exon insertion in said intron 2.

[0261] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0262] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its pre-mRNA.

[0263] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:8.

[0264] In certain embodiments, the cryptic exon is located within intron 10.

[0265] In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene or its pre-mRNA.

[0266] In certain embodiments, the c.1795-1698A>G mutation of the SLC20A2 gene promotes a cryptic exon insertion in said intron 10.

[0267] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0268] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its pre-mRNA.

[0269] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:9.

[0270] In certain embodiments, the target region is within a region from up to about 100 nucleotides upstream of the cryptic exon to up to about 100 nucleotides downstream of the cryptic exon.

[0271] In certain embodiments, the target region is selected from a region spanning the cryptic exon splice site, a region within the cryptic exon, a region within 100 nucleotides upstream of the cryptic exon, or a region within 100 nucleotides downstream of the cryptic exon.

[0272] In certain embodiments, the target region is selected from a region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, a region spanning the 5' splicing site, a region within the cryptic exon, a region spanning the 3' splicing site, or a region downstream of the 3' splicing donor site (SD).

[0273] In certain embodiments, the target region comprises a region spanning the 5' splicing acceptor site (SA) of the cryptic exon. In certain embodiments, the target region is within a region from at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides upstream relative to the 5' splice site of the cryptic exon to at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides downstream relative to the 5' splice site of the cryptic exon. In certain embodiments, the target region is within a region from up to about 20 nucleotides upstream of the 5' splice site relative to the cryptic exon to up to about 20 nucleotides downstream of the 5' splice site relative to the cryptic exon.

[0274] In certain embodiments, the target region comprises a region spanning the cryptic exon 3' splicing donor site (SD). Preferably, the target region is within a region from at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides upstream relative to the 3' splice site of the cryptic exon to at most about 25 (e.g., at most about 22, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1) nucleotides downstream relative to the 3' splice site of the cryptic exon. In certain embodiments, the target region is within a region from up to about 20 nucleotides upstream of the 3' splice site relative to the cryptic exon to up to about 20 nucleotides downstream of the 3' splice site relative to the cryptic exon.

[0275] In certain embodiments, the target region is located within the cryptic exon. Preferably, the target region or its vicinity comprises a splicing regulatory element, such as a splicing enhancer element or a splicing silencer element.

[0276] In certain embodiments, the target region is located in an intron region upstream or downstream of the cryptic exon. Preferably, the target region or its vicinity comprises a splicing regulatory element, such as a splicing branch site, a splicing enhancer element, or a splicing silencer element.

[0277] In certain embodiments, the target region is no more than 100 nucleotides in length, e.g., no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, or no more than 25 nucleotides in length.

[0278] In certain embodiments, the agent is selected from an antisense oligonucleotide (ASO) or a gene editing tool (e.g., CRISPR / Cas, a base editor, or a non-Cas protein-dependent RNA editing tool).

[0279] In certain embodiments, the reagent is antisense oligonucleotides (ASOs). In certain 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 non-Cas protein-dependent RNA editing tool. In certain embodiments, the reagent is a DNA editor, such as CRISPR / Cas9. In certain embodiments, the reagent is an RNA editor, such as CRISPR / Cas13. In certain embodiments, the reagent is a base editor Adenine / Cytosine base editor. In certain embodiments, the reagent is a non-Cas protein-dependent RNA editing tool.

[0280] ASO

[0281] In certain embodiments, the agent is an antisense oligonucleotide (ASO), which specifically binds to the target region in SLC20A2 pre-mRNA, thereby achieving splicing regulation and expression control of the SLC20A2 gene.

[0282] In certain embodiments, the ASO consists of 10-30 (e.g., 15-30, 15-25, 18-30, 18-25, 20-25) consecutive nucleotides; preferably, consists of 18-25 consecutive nucleotides.

[0283] In certain embodiments, the ASO comprises modification of backbone nucleotides and nucleotide linking bonds (e.g., phosphodiester bonds), modification of ribose, modification of ribose side chain bases, or a combination thereof. In certain embodiments, the backbone modification is selected from phosphorothioate linkages or phosphorodiamidate linkages. In certain embodiments, the sugar moiety modification is selected from, but is not limited to: 2'-O-methoxyethyl (2'-MOE), morpholino (morpholino), 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), constrained ethyl (constrained ethyl, cEt), locked nucleic acid (LNA), peptide nucleic acid.

[0284] In certain embodiments, each monomer of ASO is modified in the same manner, for example, each linkage of the main chain of ASO comprises phosphorothioate linkage or each ribose moiety comprises 2'O-methyl modification. Such modification present in each of the monomer components of ASO is referred to as "uniform modification". In certain embodiments, a combination of different modifications may be desired, for example, ASO may comprise a combination of a diaminophosphorothioate linkage and a sugar moiety comprising a morpholine ring (morpholino). The combination of different modifications for ASO is referred to as "mixed modification" or "mixed chemical modification (mixed chemistries)".

[0285] In certain embodiments, the ASO comprises a 2'MOE modification. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification. In certain embodiments, the modification of the ASO comprises a combination of a 2'MOE and a locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0286] In certain embodiments, the ASO comprises a 2'-OMe modification. In certain embodiments, each nucleotide of the ASO has a 2'-OMe modification. In certain embodiments, the modification of the ASO comprises a combination of 2'-OMe and locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0287] In certain embodiments, the ASO comprises a phosphorodiamidate linkage in combination with a sugar moiety comprising a morpholine ring (morpholino), such as a phosphorodiamidate morpholino oligomer (PMO).

[0288] In certain embodiments, the ASO specifically binds to a target region located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0289] In certain embodiments, the ASO has 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% sequence complementarity to the nucleotide sequence within SEQ ID NO: 8. In certain embodiments, the ASO is 100% complementary to the nucleotide sequence within SEQ ID NO: 8.

[0290] In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof.

[0291] In some embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 phosphorothioate linkage. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification and has a phosphorothioate-linked backbone.

[0292] In other embodiments, the ASO comprises the nucleotide sequence shown in any one 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 thereto, and the ASO is a PMO.

[0293] In certain embodiments, the ASO specifically binds to a target region located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0294] In certain embodiments, the ASO has 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% sequence complementarity to the nucleotide sequence within SEQ ID NO: 9. In certain embodiments, the ASO is 100% complementary to the nucleotide sequence within SEQ ID NO: 9.

[0295] In certain embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as set forth in 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 thereof.

[0296] In some embodiments, the ASO comprises a nucleotide sequence as set forth in any one 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 phosphorothioate linkage. In certain embodiments, each nucleotide of the ASO has a 2'MOE modification and has a phosphorothioate-linked backbone.

[0297] In other embodiments, the ASO comprises the nucleotide sequence shown in any one 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 thereto, and the ASO is a PMO.

[0298] gene editing tools

[0299] In certain embodiments, the reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool, which typically comprises (i) a module with base editing activity and (ii) a module capable of binding to the target region in the SLC20A2 gene. The gene editing tool mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon in the genome or pre-mRNA sequence, thereby destroying its SA or SD function and inhibiting the integration of the cryptic exon into the mRNA.

[0300] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0301] CRISPR / Cas system

[0302] In certain embodiments, the agent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), wherein the gRNA is capable of binding to the target region in the SLC20A2 gene or its pre-mRNA.

[0303] In certain embodiments, the gRNA binds to the target region and recruits the binding of the Cas enzyme and exerts editing activity.

[0304] In certain embodiments, the CRISPR system mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon to destroy its SA or SD function, thereby inhibiting the integration of the cryptic exon into the mRNA.

[0305] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0306] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0307] DNA base editors

[0308] In certain embodiments, the agent is a DNA base editor. A DNA base editor typically comprises (i) a fusion protein comprising a DNA base editing domain (e.g., a deaminase) and a DNA binding domain (e.g., a Cas protein, such as Cas9 or Cas12) and (ii) a guiding RNA (gRNA) that is capable of binding to the target region in the SLC20A2 gene.

[0309] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0310] In certain embodiments, the DNA base editor performs base mutations in the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon, thereby destroying its SA or SD function and thereby inhibiting the integration of the cryptic exon in the mRNA.

[0311] In certain embodiments, the Cas protein is a catalytically inactive Cas protein or a Cas nickase protein.

[0312] In certain embodiments, the base editor comprises: ABE (Adenine base editor) or CBE (Cytosine base editor), or a new editor obtained by molecular evolution / mutation screening.

[0313] RNA editing tools

[0314] In certain embodiments, the reagent is an RNA editing tool. RNA editing tools include, for example, RNA base editors based on the CRISPR / Cas system, oligonucleotide-based base editors, and the like.

[0315] In certain embodiments, the RNA base editor based on the CRISPR / Cas system comprises (i) a fusion protein comprising an RNA base editing domain (e.g., an RNA deaminase) and an RNA binding domain (e.g., Cas13) and (ii) a guiding RNA (gRNA) that is capable of binding to the target region in the SLC20A2 pre-mRNA. The gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0316] In certain embodiments, the oligonucleotide-based base editor comprises (i) a sequence module that can recruit an endogenous RNA base editing protein (e.g., an RNA deaminase, such as ADAR) and (ii) a guide sequence module that can bind to the target region in the SLC20A2 pre-mRNA. The guide sequence module binds to the target region and thereby recruits the RNA base editing protein to bind and exert base editing activity.

[0317] In certain embodiments, the RNA editing tool performs base mutations in the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon, thereby destroying its SA or SD function and inhibiting the integration of the cryptic exon in the mRNA.

[0318] In another aspect, the present invention provides a pharmaceutical composition comprising the agent or combination thereof as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0319] In certain embodiments, the pharmaceutical composition is formulated into a dosage form compatible with its intended route of administration, for example, local administration (such as direct injection or implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral administration, including intracranial (such as intraventricular, intrameningeal, or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0320] In certain embodiments, the pharmaceutical composition is administered by suction, intranasal administration, intratracheal administration or oropharyngeal inhalation. The active ingredient of the required amount can be mixed in a suitable solvent and then aseptically filtered to prepare a preparation suitable for inhalation administration. Generally, the preparation for inhalation administration is a sterile solution under physiological pH and has low viscosity. Salt can be added to the preparation to balance tension. In some cases, a surfactant or cosolvent can be added to increase the active ingredient solubility and improve aerosol properties. In some cases, an excipient can be added to control viscosity to ensure the size and distribution of the atomized droplets.

[0321] In certain embodiments, the pharmaceutical composition can be administered by injection, such as intravenously, intramuscularly, subcutaneously, intradermally, intraarticularly, intraocularly, intraperitoneally, intraventricularly (e.g., by lumbar puncture) or topically. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS) and the like. The pharmaceutical composition should remain stable under production and storage conditions and should prevent contamination by microorganisms such as bacteria and fungi. For example, a sterile injection solution can be prepared by the following method: the active ingredient of the required dose is mixed in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delayed absorption, preservatives, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use.

[0322] In certain embodiments, the agent is selected from ASO, and the pharmaceutical composition is preferably configured as a solution, emulsion, microemulsion, foam or a liposome-containing formulation (e.g., cationic liposomes or non-cationic liposomes). In certain embodiments, the pharmaceutical composition is preferably configured for intracerebroventricular injection (e.g., by lumbar puncture), intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation or intravenous injection.

[0323] In another aspect, the present invention provides the use of an agent or pharmaceutical composition as described above for preventing and / or treating a disease associated with a PiT2 functional deficiency (e.g., a lack of amount or activity) in a subject, or for preparing a medicament for preventing and / or treating a disease associated with a PiT2 functional deficiency (e.g., a lack of amount or activity) in a subject. The present invention also provides a method for preventing and / or treating a disease associated with a PiT2 functional deficiency in a subject, the method comprising administering an effective amount of an agent or pharmaceutical composition as described above to a subject in need thereof.

[0324] In certain embodiments, the disease associated with PiT2 functional deficiency is brain calcification, such as primary familial brain calcification (PFBC). In certain embodiments, the disease associated with PiT2 functional deficiency is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial calcification, and / or bone diseases associated with phosphorus homeostasis imbalance. In certain embodiments, the drug treats the disease by regulating (e.g., increasing) the expression of PiT2 protein or functional RNA by cells of the subject.

[0325] In certain embodiments, the subject comprises a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation, or a structural variation.

[0326] In certain embodiments, the mutation is located in an exonic region and the subject is heterozygous for the mutation in the exonic region.

[0327] In certain embodiments, the mutation is an intronic mutation, such as a mutation within intron 2 and / or intron 10. In certain embodiments, the mutation is located in the target region within an intron, and the subject may be heterozygous, homozygous, or compound heterozygous for an intronic mutation located within the target region.

[0328] In certain embodiments, the PiT2 functional deficiency is SLC20A2 haploinsufficiency.

[0329] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0330] On the other hand, the present invention also provides a method for regulating the expression of PiT2 protein in cells, which comprises contacting the above-mentioned agent or pharmaceutical composition with the cells in vitro.

[0331] In certain embodiments, the cell comprises a mutation in the SLC20A2 gene. In certain embodiments, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splice mutation, or a structural variation. In certain embodiments, the mutation is located in an exonic region, and the cell is heterozygous for the mutation in the exonic region. In certain embodiments, the mutation is an intronic mutation, such as a mutation within intron 2 and / or intron 10. In certain embodiments, the mutation is located in an intron of the target region, and the cell can be heterozygous, homozygous, or compound heterozygous for the intronic mutation located in the target region.

[0332] II-C. Screening Methods

[0333] In some aspects, the present invention relates to methods for screening for agents that modulate PiT2 expression.

[0334] One aspect of the present invention provides a method for identifying an agent capable of regulating PiT2 expression in a cell, the method comprising: i) identifying an agent that targets a target region in the PiT2 gene or its precursor mRNA, the target region being located in an intron region between two typical exon regions, and wherein the intron region contains a cryptic exon, and the target region being within a region from at most about 100 nucleotides upstream of the cryptic exon to at most about 100 nucleotides downstream of the cryptic exon; ii) delivering the agent identified in step i) to the cell; and iii) measuring the expression level of PiT2 in the cell of step ii).

[0335] In certain embodiments, the methods are performed in vitro.

[0336] In certain embodiments, the method further comprises: iv) comparing the expression level measured in step iii) with the expression level of PiT2 in cells treated with a control. If the expression level is increased compared to the control, the agent is an activator. If the expression level is decreased compared to the control, the agent is an inhibitor.

[0337] In certain embodiments, the expression level of PiT2 refers to the expression level of functional PiT2, such as the mRNA level of correctly spliced ​​products or the level of the protein encoded by it.

[0338] In certain embodiments, the expression level is measured by RT-PCR, qPCR, or by Western blot.

[0339] In certain embodiments, the target region is selected from a region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, a region spanning the 5' splicing site, a region within the cryptic exon, a region spanning the 3' splicing site, or a region downstream of the 3' splicing donor site (SD).

[0340] In certain embodiments, the cryptic exon is located between c.289+901 and c.289+1035 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as set forth in SEQ ID NO: 2. In certain embodiments, the target region is located within the region between c.289+801 and c.289+1135 of the SLC20A2 gene or its pre-mRNA. In certain embodiments, the target region is located within the sequence set forth in SEQ ID NO: 8.

[0341] In certain embodiments, the cryptic exon is located between c.1795-1703 and c.1795-1627 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as set forth in SEQ ID NO: 3. In certain embodiments, the target region is located within the region between c.1795-1803 and c.1795-1527 of the SLC20A2 gene or its pre-mRNA. In certain embodiments, the target region is located within the sequence set forth in SEQ ID NO: 9.

[0342] In certain embodiments, the agent targets the target region in the SLC20A2 pre-mRNA. In certain embodiments, the agent is an antisense oligonucleotide (ASO). In certain embodiments, the agent is an RNA editor, such as CRISPR / Cas13. In certain embodiments, the agent is a non-Cas protein-dependent RNA editing tool.

[0343] In certain embodiments, the agent targets the target region in the SLC20A2 gene. In certain embodiments, the agent is a DNA editor, such as CRISPR / Cas9. In certain embodiments, the agent is an Adenine / Cytosine base editor.

[0344] In some aspects, the invention relates to methods for screening for agents that modulate SLC20A2 mRNA splicing.

[0345] One aspect of the present invention provides a nucleic acid construct comprising a minigene fragment sequence of the human SLC20A2 gene, wherein the minigene fragment sequence comprises exons exon2 and exon3 and an intron sequence therebetween, or comprises exons exon10 and exon11 and an intron sequence therebetween, wherein the intron sequence comprises a mutation that can alter the splicing of the SLC20A2 gene.

[0346] In certain embodiments, the nucleic acid construct comprises a promoter operably linked to the minigene fragment sequence.

[0347] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0348] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0349] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0350] In certain embodiments, the minigene fragment sequence comprising exon2 and exon3 and the intron sequence therebetween comprises the sequence shown in SEQ ID NO:4.

[0351] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10.

[0352] In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0353] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0354] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0355] In certain embodiments, the minigene fragment sequence comprising exon10 and exon11 and the intron sequence therebetween comprises the sequence shown in SEQ ID NO:5.

[0356] In certain embodiments, the downstream of the minigene fragment sequence comprises a polyA signal sequence.

[0357] In certain embodiments, the nucleic acid construct further comprises a reporter gene, such as a fluorescent protein (eg, GFP, mCherry) or luciferase.

[0358] In another aspect, the present invention provides an expression vector comprising the nucleic acid construct described above. As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector is referred to as an expression vector when it is capable of expressing a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0359] In another aspect, the present invention provides a method for screening for an agent capable of regulating SLC20A2 mRNA splicing, comprising: providing (i) a cell comprising a nucleic acid construct as described above, (ii) a genetically modified non-human animal or cell of the present invention, or (iii) a brain calcification model obtained by the method for preparing a brain calcification model of the present invention; contacting a test agent with any one of (i) to (iii); and detecting alternative splicing of SLC20A2 mRNA. In certain embodiments, the method is used for non-diagnostic therapeutic purposes. In certain embodiments, the method is performed in vitro.

[0360] In certain embodiments, the cell is from, for example, a mammal, such as a rodent (eg, a mouse or rat), a non-human primate, or a human.

[0361] In certain embodiments, a test agent is selected that inhibits alternative splicing (eg, reduces the amount of expression product containing cryptic exon insertions), thereby increasing the production of functional PiT2 protein.

[0362] In certain embodiments, a test agent is selected that enhances alternative splicing (eg, increases the amount of expression product containing a cryptic exon insertion), thereby reducing the production of functional PiT2 protein.

[0363] In certain embodiments, the detecting comprises analyzing the length of the SLC20A2 mRNA expression product.

[0364] In certain embodiments, the alternative splicing is alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO: 2.

[0365] In certain embodiments, the detection comprises performing PCR analysis using primers spanning exons exon 2 and exon 3. In certain embodiments, an amplicon comprising a cryptic exon insertion of approximately 135 nt in length is generated. In certain embodiments, the primers comprise a forward primer as set forth in SEQ ID NO: 123 and a reverse primer as set forth in SEQ ID NO: 124.

[0366] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10. In certain embodiments, the alternative splicing within intron 2 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene. In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO: 3.

[0367] In certain embodiments, the detection comprises performing PCR analysis using primers spanning exons exon 10 and exon 11. In certain embodiments, an amplicon comprising a cryptic exonic insertion of approximately 76 nt in length is generated. In certain embodiments, the primers comprise a forward primer set forth in SEQ ID NO: 127 and a reverse primer set forth in SEQ ID NO: 128.

[0368] In certain embodiments, described method comprises contacting the test agent with the cell comprising the nucleic acid construct in vitro and detecting the alternative splicing of the nucleic acid construct. In certain embodiments, described method comprises introducing (such as transfection) cell by nucleic acid construct as above or the vector comprising this nucleic acid construct, to provide the cell comprising this nucleic acid construct. In certain embodiments, described detection comprises analyzing as above the length of the mRNA expression product of the nucleic acid construct. In certain embodiments, described detection comprises analyzing the expression of the reporter gene included in the nucleic acid construct. In certain embodiments, the expression of reporter gene indicates alternative splicing. In certain embodiments, the expression of reporter gene indicates correct splicing.

[0369] II-D. Brain calcification model and its preparation

[0370] In some aspects, the present invention relates to constructing a brain calcification model based on alternative splicing in introns of the SLC20A2 gene.

[0371] One aspect of the present invention provides a method for preparing a brain calcification model, wherein the method comprises modifying a target genome such that an intron sequence between exon 2 and exon 3 of the endogenous Slc20a2 gene is replaced with an intronic genomic fragment corresponding to the human SLC20A2 gene, or an intron sequence between exon 10 and exon 11 of the endogenous Slc20a2 gene is replaced with an intronic genomic fragment corresponding to the human SLC20A2 gene, wherein the intronic genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that alters SLC20A2 gene splicing, thereby providing a brain calcification model. In certain embodiments, the target genome is homozygous or heterozygous for the replacement.

[0372] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon. In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon.

[0373] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0374] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0375] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the human SLC20A2 gene.

[0376] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO:10.

[0377] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10.

[0378] In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the human SLC20A2 gene.

[0379] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0380] In certain embodiments, the mutation comprises the c.1795-1698A>G mutation of the human SLC20A2 gene.

[0381] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon 10 and exon 11 comprises the sequence shown in SEQ ID NO: 11.

[0382] In certain embodiments, the donor sequence for replacement has the following structure: Exon2 (3'-patial)-intron-crpE2 (or crpE10)-intron-exon3 (5'-partial).

[0383] In certain embodiments, the donor sequence for replacement has the following structure: Exon10 (3'-partial)-intron-crpE2 (or crpE10)-intron-exon11 (5'-partial).

[0384] In certain embodiments, expression of the modified endogenous Slc20a2 gene is under the control of a regulatory element at the endogenous Slc20a2 locus in the genome of interest.

[0385] The method of replacing the intron sequence of the endogenous Slc20a2 gene with the corresponding intron sequence of the human SLC20A2 gene can be performed by various suitable genome editing technologies known in the art. In certain embodiments, the replacement method includes: gene knockout, gene editing, RNA interference, epigenetic inhibition, or any combination thereof.

[0386] In certain embodiments, the gene knockout comprises: stem cell targeting technology based on homologous recombination, gene editing technology, chemical molecule mutagenesis genomic mutation, transposon site-directed inactivation or any combination thereof.

[0387] In certain embodiments, the gene editing comprises: CRISPR / Cas system (e.g., cas9, Cas12a (Cpf1)), TALEN, zinc finger nuclease, base editing, or any combination thereof.

[0388] In certain embodiments, the replacement method comprises introducing a targeting vector flanked by 5' and 3' homology arms carrying an insert nucleic acid (e.g., an intronic genomic fragment sequence corresponding to the human SLC20A2 gene to be introduced) into the cell. In certain embodiments, the insert nucleic acid further comprises a selectable marker gene (e.g., a self-deleting cassette containing a selectable marker gene), which may be flanked by or contain site-specific recombination sites (e.g., loxP, Frt, etc.). In certain embodiments, the insert nucleic acid further comprises a reporter gene.

[0389] In certain embodiments, the model is a non-human animal model, and the target genome is the genome of a non-human animal.

[0390] In certain embodiments, the model is a mammalian model. In certain embodiments, the animal model is a rodent model, such as a mouse or rat model. In certain embodiments, the animal model is a non-human primate model.

[0391] In certain embodiments, the method comprises: modifying the genome of a non-human animal embryonic stem (ES) cell so that the modified genome comprises the replacement in the endogenous Slc20a2 gene; and using the non-human animal ES cell comprising the modified genome to produce a non-human animal, thereby obtaining a humanized brain calcification animal model. In certain embodiments, the targeting vector can be introduced into the non-human animal ES cell by, for example, electroporation.

[0392] In certain embodiments, the model is a cell model, and the target genome is the endogenous genome of the target cell. In certain embodiments, the replaced intronic genomic fragment corresponding to the human SLC20A2 gene (with or without mutation) is exogenously introduced relative to the target cell.

[0393] In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human dermal fibroblast or brain glial cell.

[0394] In certain embodiments, the method comprises: modifying the genome of a target cell such that the modified genome comprises the replacement in the endogenous Slc20a2 gene; thereby obtaining a cell model comprising the modified genome.

[0395] In another aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises: an intronic sequence between exon 2 and exon 3 of an endogenous Slc20a2 gene replaced by an intronic genomic segment corresponding to a human SLC20A2 gene, or an intronic sequence between exon 10 and exon 11 of an endogenous Slc20a2 gene replaced by an intronic genomic segment corresponding to a human SLC20A2 gene, wherein the intronic genomic segment corresponding to the human SLC20A2 gene comprises a mutation that alters splicing of the SLC20A2 gene. In certain embodiments, the non-human animal is homozygous or heterozygous for the replacement.

[0396] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon. In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon.

[0397] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the human SLC20A2 gene.

[0398] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0399] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0400] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO:10.

[0401] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10.

[0402] In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0403] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0404] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0405] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon 10 and exon 11 comprises the sequence shown in SEQ ID NO: 11.

[0406] In certain embodiments, the non-human animal is a rodent, such as a mouse or rat.

[0407] In certain embodiments, the non-human animal is a non-human primate.

[0408] In certain embodiments, the genetically modified non-human animal is an animal model for brain calcification.

[0409] In another aspect, the present invention provides a genetically modified cell, the genome of which comprises: an intron sequence between exon 2 and exon 3 of the endogenous SLC20A2 gene is replaced with an intronic genomic segment corresponding to the human SLC20A2 gene, or an intron sequence between exon 10 and exon 11 of the endogenous SLC20A2 gene is replaced with an intronic genomic segment corresponding to the human SLC20A2 gene, wherein the intronic genomic segment corresponding to the human SLC20A2 gene comprises a mutation that alters splicing of the SLC20A2 gene. In certain embodiments, the cell is homozygous or heterozygous for the replacement.

[0410] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a cryptic exon. In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene comprises at least a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon.

[0411] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene is introduced exogenously relative to the target cell. "Exogenously introduced" refers to nucleic acid originating from outside the target cell. Exogenous nucleic acid can be artificially or recombinantly produced, or it can be isolated from a heterologous cell and introduced into the target cell.

[0412] In certain embodiments, the mutation that alters splicing of the SLC20A2 gene causes or enhances alternative splicing in intron 2. In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3. In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0413] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:2.

[0414] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0415] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO:10.

[0416] In certain embodiments, the alternative splicing comprises alternative splicing within intron 10.

[0417] In certain embodiments, the alternative splicing in intron 2 is a cryptic exon insertion between exons 10 and 11. In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0418] In certain embodiments, the cryptic exon has the sequence shown in SEQ ID NO:3.

[0419] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0420] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon 10 and exon 11 comprises the sequence shown in SEQ ID NO: 11.

[0421] In certain embodiments, the cell is a mammalian cell.

[0422] In certain embodiments, the cells are human dermal fibroblasts or brain glial cells.

[0423] In certain embodiments, the genetically modified cell is a cell model of brain calcification.

[0424] II-E. Application of brain calcification model

[0425] In some aspects, the present invention also relates to the application of the above-mentioned brain calcification model in disease research and drug screening.

[0426] One aspect of the present invention provides use of the brain calcification model of the present invention, or the genetically modified non-human animal or genetically modified cell for indicating and analyzing the onset stage of brain calcification, or as a model for studying phosphorus homeostasis imbalance in the brain or disease targets of brain calcification.

[0427] In another aspect, the present invention provides use of the brain calcification model of the present invention, or the genetically modified non-human animal or genetically modified cell as a model for screening candidate drugs or treatment regimens for treating brain calcification or diseases related to imbalance of phosphorus homeostasis in the brain.

[0428] In certain embodiments, the disease is a brain calcification disease.

[0429] In certain embodiments, the drug is a drug for preventing or treating brain calcification.

[0430] In certain embodiments, the drug is a drug that restores phosphorus homeostasis in the brain.

[0431] In another aspect, the present invention provides use of the brain calcification model, or the genetically modified non-human animal or genetically modified cell of the present invention as a model for screening candidate drugs or treatment options for diseases related to phosphorus homeostasis imbalance.

[0432] In certain embodiments, the diseases associated with phosphorus homeostasis imbalance include but are not limited to chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone diseases associated with phosphorus homeostasis imbalance.

[0433] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the expression of its encoded product PiT2.

[0434] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the function of its encoded product PiT2.

[0435] In certain embodiments, the agent inhibits alternative splicing of SLC20A2 mRNA and thereby increases the production of functional PiT2 protein.

[0436] In certain embodiments, the drug promotes alternative splicing of SLC20A2 mRNA and thereby inhibits the production of functional PiT2 protein.

[0437] In another aspect, the present invention provides a method for screening candidate drugs or treatment regimens for treating brain calcification or diseases related to imbalance of phosphorus homeostasis in the brain, the method comprising: administering the candidate drug or treatment regimen to the brain calcification model of the present invention, or a genetically modified non-human animal or genetically modified cell; and evaluating whether the candidate drug or treatment regimen can (i) increase the expression level of SLC20A2 full-length mRNA transcript and / or its encoded protein product PiT2, (ii) increase the phosphorus transport capacity of target cells (e.g., brain glial cells, such as astrocytes), and / or (iii) reduce the level of inorganic phosphate in cerebrospinal fluid.

[0438] In certain embodiments, the disease is a brain calcification disease.

[0439] In certain embodiments, the drug is a drug for preventing or treating brain calcification.

[0440] In certain embodiments, the drug is a drug that restores phosphorus homeostasis in the brain.

[0441] In certain embodiments, the candidate drug or treatment regimen can increase the expression level of the full-length SLC20A2 mRNA transcript and / or its encoded protein product PiT2, for example, by upregulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc. In certain embodiments, the candidate drug or treatment regimen can increase the expression level of the correctly spliced ​​SLC20A2 mRNA transcript and / or its encoded functional PiT2 protein product, for example, by upregulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0442] In certain embodiments, the candidate drug or treatment regimen is capable of increasing the phosphate transport capacity of target cells (such as brain glial cells, such as astrocytes), for example, by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0443] In certain embodiments, the candidate drug or treatment regimen is capable of reducing the level of inorganic phosphate in the cerebrospinal fluid, for example, reducing the abnormally elevated level of inorganic phosphate in the cerebrospinal fluid by at least 75%, at least 60%, at least 50%, at least 40%, at least 30%, at least 25%, for example, to a level close to a normal reference value (e.g., a healthy control).

[0444] In another aspect, the present invention provides a method for screening candidate drugs or treatment regimens for treating diseases associated with phosphorus homeostasis imbalance, the method comprising: administering the candidate drug or treatment regimen to the brain calcification model of the present invention, or a genetically modified non-human animal or genetically modified cell; and evaluating whether the candidate drug or treatment regimen can (i) increase the expression level of SLC20A2 full-length mRNA transcript and / or its encoded protein product PiT2, (ii) increase the phosphorus transport capacity of target cells (e.g., brain glial cells, such as astrocytes), and / or (iii) reduce the level of inorganic phosphate in cerebrospinal fluid.

[0445] In certain embodiments, the diseases associated with phosphorus homeostasis imbalance include but are not limited to chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone diseases associated with phosphorus homeostasis imbalance.

[0446] In certain embodiments, the candidate drug or treatment regimen can increase the expression level of the full-length SLC20A2 mRNA transcript and / or its encoded protein product PiT2, for example, by upregulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc. In certain embodiments, the candidate drug or treatment regimen can increase the expression level of the correctly spliced ​​SLC20A2 mRNA transcript and / or its encoded functional PiT2 protein product, for example, by upregulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0447] In certain embodiments, the candidate drug or treatment regimen is capable of increasing the phosphate transport capacity of target cells (such as brain glial cells, such as astrocytes), for example, by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0448] In certain embodiments, the candidate drug or treatment regimen is capable of reducing the level of inorganic phosphate in the cerebrospinal fluid, for example, reducing the abnormally elevated level of inorganic phosphate in the cerebrospinal fluid by at least 75%, at least 60%, at least 50%, at least 40%, at least 30%, at least 25%, for example, to a level close to a normal reference value (e.g., a healthy control).

[0449] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the expression of its encoded product PiT2.

[0450] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the function of its encoded product PiT2.

[0451] In certain embodiments, the agent inhibits alternative splicing of SLC20A2 mRNA and thereby increases the production of functional PiT2 protein.

[0452] In certain embodiments, the drug promotes alternative splicing of SLC20A2 mRNA, thereby inhibiting the production of functional PiT2 protein.

[0453] II-F, Beneficial Effects and Summary

[0454] The present invention first discovered and identified a previously unreported cryptic exon insertion in aberrant splicing of the SLC20A2 mRNA, and subsequently developed reagents and methods for modulating this aberrant splicing. Modulating this aberrant splicing upregulated expression of the SLC20A2 gene product, PiT2, in human cells from patients with brain calcification and in an SLC20A2-KI mouse model, reversing elevated CSF Pi levels and thereby inhibiting the progression of brain calcification.

[0455] In the embodiments provided herein, the method for regulating abnormal splicing of SLC20A2 and thereby upregulating expression of the SLC20A2 gene product, PiT2 protein, can be primarily achieved through the action of antisense oligonucleotides (ASOs). However, based on the method logic and implementation path described herein, those skilled in the art will appreciate that the present invention can also be achieved by permanently altering the splicing pattern of SLC20A2 mRNA at the genomic level through DNA base editing, or by altering the splicing pattern of SLC20A2 mRNA at the RNA level through RNA base editing.

[0456] In addition, since PiT2 is highly distributed in the brain, kidneys, cardiovascular system and bones, and is involved in the balance of Pi, splicing regulation of PiT2 expression can also be used to restore other syndromes related to abnormal Pi balance, including chronic kidney disease, cardiovascular and cerebrovascular arteriosclerosis and bone diseases.

[0457] In particular, the method for regulating abnormal splicing of SLC20A2 mRNA provided by the present invention can still increase the expression level of PiT2 protein in healthy cells, indicating that this method can rescue SLC20A2 haploinsufficiency that is not limited to a specific mutation form, thereby rescuing PiT2 expression in patients with SLC20A2 loss-of-function mutations caused by non-intronic mutations, which account for a larger proportion of the population.

[0458] Based on this, the present invention provides a novel strategy for treating SLC20A2 haploinsufficiency based on regulating SLC20A2 gene mRNA splicing, which has important clinical value for the treatment of brain calcifications or diseases related to phosphorus homeostasis imbalance.

[0459] III. Astrocyte-targeted therapies

[0460] In some aspects, the present invention relates to gene compensation therapy and applications targeting astrocytes to restore phosphorus homeostasis in the brain, including inhibiting the occurrence of brain calcification in individuals with PiT2 mutations, and delaying or curbing the progression of brain calcification in patients with PFBC or brain calcification associated with phosphorus homeostasis imbalance.

[0461] After extensive and in-depth research and a large amount of screening, the inventors have developed for the first time a gene compensation therapy targeting astrocytes that can effectively intervene in the progression of brain calcification. After in-depth experiments, the inventors have clarified for the first time the pathogenic mechanism of brain calcification caused by mutations and / or abnormalities in genes and / or molecules such as PiT2, MYORG, and XPR1. It is because these mutations and / or abnormalities cause abnormalities in the phosphorus transport function of astrocytes, thereby leading to an imbalance in phosphorus homeostasis in the brain, and then leading to brain calcification. At the method level, the present invention provides a strategy for repairing phosphorus homeostasis in the brain and curbing the occurrence of calcification or blocking its progression by increasing the expression of PiT2, MYORG, or XPR1 genes in brain astrocytes.

[0462] In the embodiments provided by the present invention, a recombinant AAV virus is constructed that can achieve the selective expression of PiT2 protein in astrocytes. Experimental results show that after intravenous injection of a recombinant AAV virus that can cross the blood-brain barrier, the expression module for expressing PiT2 protein carried by it can selectively express the protein product PiT2 of the compensation gene carried by it in the astrocytes in the mouse brain. As a result, the astrocytes in the brain obtain expression compensation of Pit2-KO mice, which realizes the reconstruction of brain phosphorus homeostasis, thereby effectively curbing the occurrence and progression of brain calcification from the source. The strategy disclosed by the present invention also supports an alternative implementation form, that is, by delivering gene expression promoters, such as antisense nucleotide (ASO) drugs, or gene / base editing-based RNA splicing modification to astrocytes, thereby regulating the expression of PiT2, MYORG, or XPR1 in astrocytes, which can also achieve the purpose of repairing brain phosphorus homeostasis and intervening in brain calcification. These data indicate that the gene compensation therapy of the present invention can be used in humans to repair brain phosphorus homeostasis, inhibit the occurrence of brain calcification or prevent the further development of brain calcification, and improve related neurological symptoms, and has very outstanding clinical translational value.

[0463] III-(A), Astrocyte Phosphate Transport Promoter

[0464] One aspect of the present invention provides an astrocyte phosphate transport facilitator. In this article, the astrocyte phosphate transport facilitator refers to an agent that can promote phosphate transport in astrocytes. The agent can, for example, achieve astrocyte-specific expression, such as using an astrocyte-specific expression element (such as a promoter). Alternatively, astrocytes can be specifically targeted, such as by specifically binding to a specific receptor on the astrocyte membrane.

[0465] In certain embodiments, the agent promoting brain phosphorus homeostasis is selected from the group consisting of:

[0466] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0467] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0468] (Z3) XRP1 protein, a nucleic acid molecule encoding an XRP1 protein, an expression vector expressing an XRP1 protein, an XRP1 promoter, or a combination thereof;

[0469] (Z4) Any combination of the above Z1 to Z3.

[0470] In certain embodiments, the promoter described in any one of Z1 to Z3 is capable of activating or upregulating the expression of the gene, and / or activating or enhancing the activity of the protein product of the gene.

[0471] In certain embodiments, the enhancer can exert its activating effect by any mechanism, such as by activating gene expression at the RNA or protein level (e.g., enhancing transcription of the gene, and / or, enhancing translation of the mRNA product of the gene).

[0472] In certain embodiments, the promoter enhances its biological function by activating the expression level of the gene. In such embodiments, the mensuration of expression level can be implemented at the nucleic acid level or protein level. The method of measuring expression at the nucleic acid level includes but is not limited to Northern blotting, PCR, RT-PCR or real-time (real) RT-PCR. The method of measuring expression at the protein level includes but is not limited to Western blotting or polyacrylamide gel electrophoresis combined with protein staining techniques such as Coomassie brilliant blue or silver staining, mass spectrometry, ELISA etc.

[0473] In certain embodiments, the promoter described in any one of Z1 to Z3 includes 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 certain embodiments, the promoter is a small molecule compound. In certain embodiments, the promoter is a gene editing reagent (e.g., a base editing reagent). In certain embodiments, the promoter is a small nucleic acid drug (e.g., an antisense nucleotide drug).

[0474] In certain embodiments, the astrocyte phosphate transport promoter is selected from promoters that regulate the expression of PiT2, MYORG or XPR1 in astrocytes.

[0475] In some embodiments, the astrocyte phosphate transport facilitator described in any of the above embodiments can achieve astrocyte-specific expression, such as by an astrocyte-specific expression element (such as a promoter). In such embodiments, the astrocyte phosphate transport facilitator can be an expression vector comprising an exogenous gene and an astrocyte-selective expression enhancer element and / or an astrocyte-selective expression promoter operably linked thereto, wherein the exogenous gene is selected from a nucleic acid molecule encoding PiT2 protein, MYORG protein, or XRP1 protein, or a combination thereof.

[0476] In certain embodiments, the exogenous gene is a nucleic acid molecule encoding a PiT2 protein.

[0477] In certain embodiments, the astrocyte-selective promoter is gfaABC1D. In certain embodiments, the astrocyte-selective promoter is a promoter derived from genes such as ALDH1L1, AQP4, and S100B.

[0478] In certain embodiments, the astrocyte phosphate transport promoter is an expression vector (such as a viral vector, such as AAV) comprising a nucleic acid molecule encoding the PiT2 protein and an astrocyte-selective expression promoter (such as gfaABC1D) operably linked thereto.

[0479] In certain embodiments, the expression vector is a viral vector, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector (such as a vaccinia virus vector), or a herpes simplex virus vector.

[0480] In certain embodiments, the expression vector is AAV. Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependinovirus of the family Parvoviridae and is the simplest single-stranded DNA defective virus discovered so far. The wild-type AAV genome consists of a linear single-stranded DNA (ssDNA) of approximately 4.7 kb bases, flanked by inverted terminal repeats (ITRs), with the coding sequences of Rep and Cap proteins contained between the two ITR elements. Through regulation by optional promoters and splice variants at multiple locations, the Rep gene can express four different proteins that participate in viral genome replication, transcription, and integration. With the help of an important assembly activator protein AAP expressed by the open reading frame 2 (ORF2) of the Cap gene, the three structural capsid proteins VP1 / VP2 / VP3 expressed by the Cap gene are assembled into a 60-subunit icosahedral capsid at a ratio of 1:1:10. Adeno-associated virus (AAV) is considered an ideal delivery vector in gene therapy strategies because it is smaller than other viral vectors, has no or only weak immunogenicity, has an extremely low probability of genome integration, and can transfect both dividing and non-dividing cells.

[0481] The viral ITR elements contain cis-acting sequences required for genome replication and packaging. Therefore, AAV can be engineered into a viral delivery vector by removing all virally encoded gene sequences between the ITR elements and replacing them with exogenous DNA sequences encoding the gene of interest. These exogenous DNA sequences can encode conventional expression cassettes for expressing the selected target gene in target cells. These engineered recombinant adeno-associated viral vectors (rAAV) are considered one of the most promising gene transfer vectors due to their excellent safety profile, broad host cell range (dividing and non-dividing cells), low immunogenicity, and prolonged in vivo expression of exogenous genes. They are widely used in gene therapy and vaccine research worldwide. After more than a decade of research, the biological properties of rAAV have been deeply understood, and in particular, a wealth of data has been accumulated on their efficacy in various cell types, tissues, and in vivo experiments. In medical research, rAAV is being used in gene therapy studies for a variety of diseases (both in vitro and in vivo).

[0482] AAV vectors can be prepared using standard methods in the art. The production, purification, and use of adeno-associated viruses of various serotypes have all been tested in multiple laboratories, animals, and humans. Methods for purifying vectors can be found in various technical books and biotechnology method articles, and can also be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of recombinant AAV vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. Embodiments of using recombinant AAV vectors for in vitro and in vivo delivery of target genes have been described in literature and patents (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patents describe engineered AAV plasmids from various sources (strains, serotypes, mutant-optimized strains, etc.) that have been depleted of the native rep and / or cap genes in the AAV genome. These plasmids are then used to carry exogenous target genes for the production of recombinant AAV viruses and for delivery of the target genes in vitro (into cultured cells) or in vivo (directly into organisms). Due to safety concerns, AAV viruses used for therapeutic purposes must be replication-defective. These recombinant AAVs are typically produced by co-transfecting two plasmids into a stable cell line infected with a human helper virus (e.g., adenovirus): plasmid A carries a specific promoter and the target gene coding sequence, flanked by inverted terminal repeat (ITR) sequences; and plasmid B carries the AAV genome replication genes (rep) and capsid protein coding genes (cap). In certain production systems, for higher safety requirements, the replication genes (rep) and capsid protein coding genes (cap) can also be constructed on separate plasmids.

[0483] In some embodiments, in order to obtain better delivery and expression effects for targeted tissues and cell types, the expression vector of the recombinant AAV virus can be combined with viral capsid protein encoding plasmids of different serotypes. These serotypes or capsid protein encoding genes are derived from different wild-type serotypes (including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV capsid proteins), or they can be derived coding sequences of these capsid proteins that have been modified by mutation screening or molecular biology directed evolution. Therefore, the present disclosure may use recombinant AAV viruses containing any of the serotypes described above, or AAV serotypes with better effects developed in the future. The basic methods for developing, screening and producing such viral capsid protein serotypes are known in the art and are described in published academic literature and U.S. Patent No. 6,596,535.

[0484] In certain embodiments, the AAV (particularly the capsid protein) is selected from a serotype AAV that can penetrate the blood-brain barrier, such as PHP.eB or PHP.AX.

[0485] In certain embodiments, the astrocyte phosphate transport promoter is an AAV (such as PHP.eB or PHP.AX) comprising a nucleic acid molecule encoding the PiT2 protein and an astrocyte-selective expression promoter (such as gfaABC1D) operably linked thereto.

[0486] In other embodiments, the astrocyte phosphate transport facilitator described in any of the above embodiments can specifically target astrocytes, such as by specifically binding to a specific receptor on the astrocyte membrane. In such embodiments, the astrocyte phosphate transport facilitator is linked to a targeting molecule that has targeting properties for astrocytes. In certain embodiments, the astrocyte phosphate transport facilitator is fully or partially encapsulated within a targeting molecule, or conjugated to the targeting molecule. In certain embodiments, the targeting molecule is selected from an antibody or antibody fragment thereof, a polysaccharide, an aptamer, or a nanoparticle.

[0487] In certain embodiments, the astrocyte phosphate transport promoter is a small nucleic acid drug, which is linked to a targeting molecule, and the targeting molecule has targeting properties to astrocytes.

[0488] In certain embodiments, the small nucleic acid drug is an ASO. In certain embodiments, the ASO upregulates the expression of PiT2, MYORG or XPR1.

[0489] In certain embodiments, the astrocyte phosphate transport facilitator can be a gene editing or base editing agent that can regulate the splicing of target gene mRNA, which is connected to a targeting molecule with targeting to astrocytes as described above, thereby regulating the expression of PiT2, MYORG or XPR1 in astrocytes. In certain embodiments, the gene editing or base editing agent typically comprises (i) a module with base editing activity and (ii) a module that can bind to the target gene. Exemplary gene editing tools include but are not limited to CRISPR / Cas systems, DNA base editors, and RNA editing tools.

[0490] III-(B) Expression vector and host cells

[0491] One aspect of the present invention provides a plasmid construction and design strategy for expressing a phosphate transport facilitator (such as PiT2 protein) in astrocytes. The plasmid comprises a promoter for selectively driving transcription of a target gene in astrocytes and an open expression frame sequence encoding the phosphate transport facilitator (such as PiT2 protein). Using the provided sequence information, a skilled artisan can use publicly available molecular cloning techniques to construct a nucleic acid sequence or expression plasmid suitable for transduction into target cells.

[0492] 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, which can be used to introduce into astrocytes in the brain of humans or primates and express the target gene, thereby being used as a therapeutic vector. The vector can be viral or non-viral (e.g., a plasmid, or an mRNA sequence packaged in LNP). Viral vectors include viral vectors derived from the following types: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus, and SV40. Preferably, the viral vector is replication defective, although it is envisioned that it can be replication deficient, capable of replication, or conditionally replicating. The viral vector can generally maintain an extrachromosomal state without integrating into the genome of the host cell.

[0493] Preferred viral vectors for delivering PiT2 protein-encoding nucleic acid sequences to astrocytes are AAV vectors, such as AAV serotypes that can be injected intravenously and penetrate the blood-brain barrier, such as PHP.eB or PHP.AX, which have been shown to be effective in mice. Intravenous injection, which allows for blood-brain barrier penetration, can achieve efficient whole-brain viral delivery. Other approaches, such as intralymphatic injection or intraventricular / spinal lumbar puncture, are still under development. Some have achieved whole-brain delivery in mice and may eventually be able to deliver to the human brain. Using specific AAV serotypes (AAV2, AAV5, AAV9, etc.), or engineered versions of any of these serotypes (including AAV PHP.eB, AAV.CPP.16, and AAV.CPP.21 vectors), combined with cell-type-specific promoters (such as gfaABC1D), selective expression in targeted cell types can be achieved. Further advances in viral technology may allow for astrocyte-specific infection through serotype modification alone, reducing the reliance on selective promoters.

[0494] Viral vectors have the ability to enter cells, while non-viral vectors such as plasmids can be combined with other adjuvants to assemble virus-like or lipid vectors, which can also be taken up by target cells. Such technologies have been proven effective in some human tissues; in the near future, such technologies may be prepared for selective delivery to astrocytes throughout the brain. Such adjuvants include polycationic liposomes, lipid nanoparticles (LNPs), etc. The vectors used in the present invention are preferably suitable for use in vivo, and preferably suitable for use in humans.

[0495] The vector will preferably contain one or more regulatory sequences to direct the expression of the nucleic acid sequence in astrocytes. The vector can be connected to the regulatory sequence in an "operably connected" manner, which includes one or more sequence modules such as promoters, introns, enhancers, translation regulatory elements, RNA stability regulatory sequences, ligand-induced expression regulatory sequences, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites. The vector may also include a selective screening marker, for example, to support the effective assembly of the vector by the virus in a viral production system (such as 293 cells), or an element of inducible expression in astrocytes, or an element of negatively regulating the expression of the target gene in non-astrocytic cells.

[0496] "Operably linked" means that a nucleic acid sequence is linked to other sequence modules in a certain order or structure based on its functional role so that they regulate the expression or function of the target gene in a spatiotemporal manner according to the designed purpose. For example, a nucleic acid sequence operably linked to a promoter will have a transcriptional regulatory function that is affected by the promoter or affects the activity of the promoter.

[0497] The promoter mediates the transcriptional expression of the downstream open expression frame nucleic acid sequence encoding the target gene connected to it. The promoter can be constitutive or inducible. The promoter can direct selective expression in astrocytes, or simultaneously include oligodendrocytes and their precursor cells. In the latter case, the design and screening of the promoter needs to be tested for expression in both astrocytes and oligodendrocytes. A suitable promoter is the key to achieving astrocyte-specific expression in the present invention. For example, in the mouse model described in the present invention, the selective expression of astrocytes depends on gfaABC1D to drive the transcription of the PIT2 encoding gene; some studies have verified that the selectivity of the gfaABC1D promoter is conserved and available in human neural cells. Considering that single-cell research is driving the screening of more optional promoters, more promoters can be used in the future to achieve selective expression in astrocytes, such as promoters derived from genes such as ALDH1L1, AQP4, S100B and screened and modified.

[0498] From a technical feasibility perspective, a variety of expression vectors can be used to achieve expression of the PiT2 protein in astrocytes (preferably primates, more preferably human astrocytes). The present invention preferably uses adeno-associated virus as an expression vector. The present invention provides a method for constructing a recombinant adeno-associated virus expression vector carrying the PiT2 coding sequence. This method can quickly and stably construct an AAV expression vector carrying the PiT2 coding sequence, and use it to package and obtain therapeutic AAV viral preparations.

[0499] The present invention also provides a host cell comprising the vector described above.

[0500] The present invention also provides an AAV vector packaging system, comprising:

[0501] Exogenous gene expression plasmid: comprising a nucleic acid sequence encoding a phosphate transport facilitator (such as PiT2 protein) and an astrocyte-specific promoter (such as gfaABC1D) operably linked thereto; preferably, further comprising inverted terminal repeats (ITRs) at both ends;

[0502] Packaging plasmid: It contains the Rep and Cap genes.

[0503] In certain embodiments, the exogenous gene expression plasmid is AAV2. In certain embodiments, the packaging plasmid expresses PHP.eB serotype capsid protein.

[0504] In certain embodiments, the AAV vector packaging system further comprises a helper plasmid comprising genes that assist in the expression of Rep and Cap.

[0505] The present invention also provides a host cell comprising the AAV vector packaging system described above. In certain embodiments, the host cell is a genetically engineered cell for producing AAV viral particles. Preferably, the host cell is a mammalian cell (preferably a human, more preferably a human astrocyte), which increases the expression of the PiT2 protein.

[0506] III-(C), gene therapy vector

[0507] One aspect of the present invention provides a gene therapy vector, particularly a gene therapy vector for preventing and / or treating brain calcification diseases. The gene therapy vector is selected from the expression vectors described above that enable astrocytes to selectively express a phosphate transport facilitator (such as PiT2 protein).

[0508] 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. The technology for achieving serotype and promoter combinations that specifically express whole-brain astrocytes has been achieved in mice, such as using a combination of PHP.eB or PHP.AX plus the promoter gfaABC1D; however, the effectiveness of this combination in primates or humans has not been effectively demonstrated, and further engineering modification screening is still needed. However, it can be predicted that it may be modified from the AAV vectors in the group consisting of the serotypes AAV1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 listed below, or their derived chimeric AAVs such as AAV2-AAV3, AAVhu.14, AAV3a / 3b, AAVHSC15, AAV-HSC17, AAVhu.37, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6 (Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45, and some serotypes derived from non-human primates, such as AAVrh.10, AAVrh32.33, and AAVrh.8. The selection of more effective delivery serotypes relies on the directed evolution and screening of capsid protein coding sequences, especially based on in situ screening and testing in tissues and cell types of interest.

[0509] At present, gene therapy vectors are mainly delivered to astrocytes throughout the brain through intravenous injection. With technological advancement, other injection routes may also be able to achieve efficient infection and expression of astrocytes throughout the brain, including injection through the head and neck lymphatic system, sinuses, intraventricular injection, or lumbar puncture.

[0510] III-(D) Formulations and Compositions

[0511] One aspect of the present invention provides a preparation or composition comprising (a) an effective amount of a brain phosphorus homeostasis promoter as an active ingredient, and (b) a pharmaceutically acceptable carrier or excipient. In this context, the brain phosphorus homeostasis promoter comprises an astrocyte phosphorus transport promoter.

[0512] In certain embodiments, the formulation or composition is a pharmaceutical composition.

[0513] In certain embodiments, the preparation or composition can promote and / or repair the phosphorus transport function of astrocytes, thereby maintaining and / or restoring phosphorus homeostasis in the brain, and preventing and / or treating brain calcification caused by phosphorus homeostasis defects in the brain.

[0514] In certain embodiments, the formulation or composition is used to treat a hereditary brain calcification. The hereditary brain calcification is primary familial brain calcification (PFBC). The formulation of the present invention may be suitable for treating other types of brain calcification caused by abnormal glial cell phosphate transport function, such as some sporadic cases of brain calcification, brain calcification caused by tumors or inflammation, and Aicardi-Goutières syndrome (AGS).

[0515] For the convenience of clinical application, the preparation or composition can be contained in an injection container or a disposable injection device (such as an injection needle). The injection dispenser can contain a single dose of the preparation or composition. The injection dispenser can be contained in a medicine box for easy storage and use. During transportation, the small container containing the drug suspension needs to be placed in a refrigerated condition (such as dry ice or a refrigerated transport box). It should be stored in a -80°C refrigerator for a long time, and can be stored in a -20 degree or 4 degree refrigerator for a short time.

[0516] In certain embodiments, the "active ingredient" in the formulation or composition refers to a gene therapy vector as described above, such as a viral vector (including an adeno-associated viral vector). The "active ingredient," formulation, and / or composition can be used to treat brain calcification. An "effective amount" refers to an amount of the active ingredient sufficient to significantly improve key clinical observations (e.g., cerebrospinal fluid phosphate levels, other biomarkers of calcification), the progression of brain calcification, or the severity of key symptoms, preferably without causing serious side effects.

[0517] In certain embodiments, the formulation or composition can be a liquid or solid, such as a powder, gel, or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art, or have been demonstrated to be reliable and useful in clinical trials.

[0518] In certain embodiments, the preparation or composition can be administered intravenously, or other proven effective injection methods, to achieve delivery and expression of astrocytes throughout the brain. In any mode of administration, the preparation or composition is preferably provided as an injectable liquid. Preferably, the injection liquid can be diluted as a concentrate and then injected, or provided as a ready-to-use injection loaded in a syringe.

[0519] In certain embodiments, the formulation or composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0520] III-(E) Therapeutic Applications

[0521] The present invention provides a method for using astrocyte phosphorus transport promoter to curb the occurrence of brain calcification in advance, or to treat brain calcification, especially hereditary brain calcification. From an adaptability perspective, this method is applicable to all groups with imbalance of brain phosphorus homeostasis due to glial cell phosphorus transport defects, including patients with primary familial brain calcification (PFBC) associated with PiT2, MYORG or XPR1 gene mutations, and may also include non-PFBC types of brain calcification driven by imbalance of brain phosphorus homeostasis. The method includes introducing the astrocyte phosphorus transport promoter described in the present invention, such as a vector containing an optimized sequence encoding PiT2, MYORG or XPR1, into astrocytes for expression. The method may include delivering a nucleic acid expression vector administered to astrocytes in the brain by intravenous injection (or other technical means), or a promoter that regulates the expression of PiT2, MYORG or XPR1 in astrocytes, such as an antisense nucleotide (ASO) drug, or a gene editing or base editing agent that can regulate target gene mRNA splicing.

[0522] The astrocyte phosphorus transport promoter of the present invention can be administered alone or in combination with other therapeutic drugs (such as formulated in the same pharmaceutical composition or in separate pharmaceutical compositions).

[0523] In certain embodiments, the use of astrocyte phosphate transport enhancers can improve the pathological process of brain calcification or alleviate one or more symptoms of the disease, including rebuilding the phosphate transport capacity of astrocytes, repairing the imbalance of phosphate homeostasis in the brain, and reducing the occurrence of brain calcification.

[0524] In certain embodiments, the method comprises introducing a nucleic acid sequence encoding a PiT2, MYORG, or XPR1 protein into astrocytes in the brain. Preferably, the method comprises contacting the cells with a vector (preferably a virus, more preferably an adeno-associated virus) comprising a nucleic acid sequence encoding a PiT2 protein. Preferably, the cells are astrocytes.

[0525] In certain embodiments, in the method, an optimized nucleic acid sequence encoding PiT2, MYORG or XPR1, or a promoter that can regulate the expression of PiT2, MYORG or XPR1 genes is delivered to astrocytes and selectively expressed in astrocytes to reconstruct the phosphorus transport capacity of astrocytes, repair the imbalance of phosphorus homeostasis in the brain, and ultimately curb the further progression of brain calcification in the brain and alleviate related clinical symptoms.

[0526] In some aspects, the present invention provides a method for preventing and / or treating brain calcification diseases, comprising administering an effective amount of a brain phosphorus homeostasis promoter to a subject in need thereof. Also provided is the use of a brain phosphorus homeostasis promoter in the preparation of a medicament for preventing and / or treating brain calcification diseases.

[0527] In certain embodiments, the agent promoting brain phosphorus homeostasis is an astrocyte phosphorus transport promoter as described above.

[0528] In certain embodiments, the prevention comprises arresting calcification before it occurs in a subject carrying a genetic mutation. In certain embodiments, the mutation comprises a mutation in PiT2, MYORG, XRP1, or a combination thereof. In certain embodiments, the calcification comprises brain calcification.

[0529] In certain embodiments, the treatment includes arresting and / or delaying further progression of calcification in a subject.

[0530] In certain embodiments, the treatment includes reversing and / or repairing imbalances in brain phosphorus homeostasis and / or brain calcification.

[0531] In certain embodiments, the astrocyte phosphate transport enhancer is an expression vector expressing an exogenous gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof. In certain embodiments, the expression vector comprises a viral vector, such as AAV. In certain embodiments, the exogenous gene is operably linked to an astrocyte-selective enhancer element and / or an astrocyte-selective promoter. In certain embodiments, the promoter is gfaABC1D. In certain embodiments, the promoter is a screened and engineered promoter derived from genes such as ALDH1L1, AQP4, or S100B.

[0532] In certain embodiments, the astrocyte phosphate transport enhancer comprises: a small molecule compound, a gene editing reagent, a small nucleic acid drug, or a combination thereof. In certain embodiments, the enhancer is linked to a targeting molecule, and the targeting molecule has astrocyte targeting property. In certain embodiments, the enhancer is completely or partially encapsulated within the targeting molecule, or conjugated to the targeting molecule. In certain embodiments, the targeting molecule is selected from an antibody or antibody fragment thereof, a polysaccharide, an aptamer, or a nanoparticle.

[0533] In certain embodiments, the astrocyte phosphate transport enhancer is a small nucleic acid drug. In certain embodiments, the small nucleic acid drug includes antisense oligonucleotides (ASOs).

[0534] In certain embodiments, the astrocyte phosphate transport enhancer is a gene editing agent. In certain embodiments, the gene editing agent comprises a base editing agent.

[0535] In certain embodiments, the preventing or treating comprises introducing a cellular phosphate transport enhancer described herein into astrocytes of the subject.

[0536] In certain embodiments, the brain calcification disease includes brain calcification caused by abnormal phosphorus homeostasis in the brain. As used herein, "abnormal phosphorus homeostasis," "phosphorus homeostasis imbalance," "phosphorus homeostasis disorder," and "phosphorus homeostasis defect" are used interchangeably. As used herein, "phosphorus transport disorder" and "phosphorus transport defect" are used interchangeably.

[0537] In certain embodiments, the brain calcification disease comprises primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC).

[0538] In certain embodiments, the drug is administered to a subject selected from the group consisting of: PFBC patients carrying mutations in the PIT2, MYORG, or XPR1 genes, or a combination thereof, or patients with other types of brain calcification diseases associated with defects in brain phosphorus homeostasis (e.g., upregulation of inorganic phosphate in the cerebrospinal fluid). Such other types of brain calcification diseases may include other types of brain calcifications caused by abnormal glial cell phosphorus transport function, such as some sporadic cases of brain calcification, brain calcification caused by tumors or inflammation, and Aicardi-Goutières syndrome (AGS).

[0539] In certain embodiments, the method further comprises: detecting a genetic mutation in PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphorus transporters, or a combination thereof in the subject; and if the result of the detection indicates the presence of the mutation in the subject, administering the above-mentioned therapy to the subject.

[0540] In certain embodiments, the method further comprises: collecting cells from the subject, differentiating the cells into astrocytes, and testing the phosphate transport function of the astrocytes; if the test results indicate that the phosphate transport capacity C1 of the subject's astrocytes is significantly lower than the phosphate transport capacity C0 of astrocytes in a normal individual, administering the above-mentioned therapy to the subject. In certain embodiments, the term "significantly lower" means that the ratio of the phosphate transport capacity C1 of the astrocytes derived from the subject to the phosphate transport capacity C0 of astrocytes in a normal individual (C1 / C0) is ≤ 2 / 3, preferably ≤ 1 / 2, and more preferably ≤ 1 / 4.

[0541] In certain embodiments, the defect in brain phosphate homeostasis comprises a defect in astrocytic phosphate transport in the brain.

[0542] In certain embodiments, the brain calcification disease includes a brain calcification disease caused by an imbalance of phosphorus homeostasis in the brain.

[0543] In certain embodiments, for a subject (or patient) with a brain calcification disease characterized by an imbalance in brain phosphorus homeostasis, their astrocyte phosphate transport capacity C1 is significantly lower than the astrocyte phosphate transport capacity C0 of a normal individual. In certain embodiments, "significantly lower" means that the ratio of the astrocyte phosphate transport capacity C1 to the astrocyte phosphate transport capacity C0 of a normal individual (C1 / C0) is ≤ 2 / 3, preferably ≤ 1 / 2, and more preferably ≤ 1 / 4.

[0544] In certain 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 certain embodiments, the genetic mutation comprises an exon mutation, an intron mutation, a 5'-UTR mutation, a 3'-UTR mutation, or a combination thereof.

[0545] In certain embodiments, the subject comprises an animal model. In certain embodiments, the subject comprises a mammal. In certain embodiments, the subject comprises a human. In certain embodiments, the subject comprises a non-human mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject comprises a primate. In certain embodiments, the subject comprises a rodent. In certain embodiments, the subject comprises a human, a mouse, a rat, a monkey, or a combination thereof.

[0546] In certain embodiments, the subject is a subject for whom the administering comprises intravenous injection, subcutaneous injection, head and neck lymphatic injection, sinus administration, intraventricular injection, intraparenchymal injection, spinal lumbar puncture injection, or a combination thereof.

[0547] In some aspects, the present invention also provides a kit for preventing and / or treating brain calcification diseases. The kit comprises:

[0548] (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect the presence of a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and

[0549] (b) a second container, and a pharmaceutical composition in the second container, wherein the pharmaceutical composition comprises a pharmaceutical ingredient capable of regulating and repairing phosphorus homeostasis imbalance in the brain and a pharmaceutically acceptable carrier or excipient.

[0550] In certain embodiments, the pharmaceutical composition comprises an agent that promotes brain phosphorus homeostasis.

[0551] In certain embodiments, the pharmaceutical composition comprises an astrocyte phosphate transport enhancer as described herein. In certain embodiments, the pharmaceutical composition comprises an astrocyte phosphate transport enhancer as described herein.

[0552] In certain embodiments, the kit further comprises: (c) a third container, and a solution in the third container for assisting the dissolution or in vivo delivery of the pharmaceutical composition in the second container.

[0553] In certain embodiments, the kit may further include: (d) a fourth container, and a fourth detection reagent for detecting the phosphorus transport function of astrocytes located in the fourth container.

[0554] In certain embodiments, the pharmaceutical composition in the second container is administered to a subject selected from the group consisting of: PFBC patients with PIT2, MYORG, XPR1 gene mutations, or a combination thereof, or patients with other types of brain calcification diseases associated with impaired brain phosphate homeostasis (e.g., upregulation of inorganic phosphate in the cerebrospinal fluid). In certain embodiments, the impaired brain phosphate homeostasis includes a defect in astroglial phosphate transport in the brain. In certain embodiments, the brain calcification disease includes a brain calcification disease caused by an imbalance in brain phosphate homeostasis. In certain embodiments, for a subject (or patient) with a brain calcification disease with an imbalance in brain phosphate homeostasis, their astrocyte phosphate transport capacity, C1, is significantly lower than the astrocyte phosphate transport capacity, C0, of a normal individual. In certain embodiments, the term "significantly lower" means that the ratio of the astrocyte phosphate transport capacity, C1, to the astrocyte phosphate transport capacity, C0, of a normal individual (C1 / C0) is ≤ 2 / 3, preferably ≤ 1 / 2, and more preferably ≤ 1 / 4. In certain 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 XPR1 phosphate transporters, or a combination thereof. In certain embodiments, the genetic mutation comprises an exon mutation, an intron mutation, a 5'-UTR mutation, a 3'-UTR mutation, copy number variations (CNVs), or a combination thereof.

[0555] In certain embodiments, a first detection reagent is used to detect genetic mutations in PiT2, MYORG, XRP1, other pathogenic genes and / or molecules that regulate the expression and function of PiT2 and / or XPR1 phosphorus transporters, or a combination thereof in the desired subject. If the result of the detection indicates the presence of the mutation in the subject, the subject is considered suitable for treatment with the pharmaceutical composition in the kit.

[0556] In certain embodiments, cells of the desired subject are collected, the cells are differentiated into astrocytes, and the astrocyte phosphate transport function is detected using the fourth detection reagent. If the test result indicates that the astrocyte phosphate transport capacity C1 of the subject is significantly lower than the astrocyte phosphate transport capacity C0 of a normal individual, the subject is considered suitable for treatment with the pharmaceutical composition in the drug kit. In certain embodiments, the "significantly lower" refers to the ratio of the astrocyte phosphate transport capacity C1 derived from the subject to the astrocyte phosphate transport capacity C0 of a normal individual (C1 / C0) ≤ 2 / 3, preferably ≤ 1 / 2; more preferably ≤ 1 / 4. In certain embodiments, the cells include primary cells and passaged cells. In certain embodiments, the cells include somatic cells, germ cells, gametes, and stem cells. In certain embodiments, the stem cells include: totipotent stem cells, pluripotent stem cells, and unipotent stem cells. In certain embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In certain embodiments, the cells include: embryonic stem cells, adipose-derived stem cells, neural stem cells, mesenchymal stem cells, oligodendrocyte progenitor cells, hematopoietic stem cells, immune cells (such as T cells, B cells, NK cells). In certain embodiments, the cells include: healthy normal human cells, cancerous human cells. In certain embodiments, the cells include: suspension cells, adherent cells. In certain embodiments, the cells do not include embryonic stem cells. In certain embodiments, the cells do not include human embryonic stem cells.

[0557] In certain embodiments, the subject comprises an animal model. In certain embodiments, the subject comprises a mammal. In certain embodiments, the subject comprises a human. In certain embodiments, the subject comprises a non-human mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject comprises a primate. In certain embodiments, the subject comprises a rodent. In certain embodiments, the subject comprises a human, a mouse, a rat, a monkey, or a combination thereof.

[0558] In certain embodiments, the first container, the second container, the third container, and the fourth container are the same or different containers.

[0559] In certain embodiments, the pharmaceutical composition in the second container includes a single and / or compound preparation containing a guide RNA or its expression vector, and / or a gene editing protein or its expression vector. In certain embodiments, the gene editing protein includes a base editing protein. In certain embodiments, the single and / or compound preparation in the second container can be packaged in different pharmaceutical containers depending on the needs of drug transportation and storage.

[0560] In certain embodiments, the pharmaceutical composition in the second container is a single preparation containing other drugs that are helpful in treating brain calcification diseases.

[0561] In certain embodiments, the dosage form of the pharmaceutical composition in the second container is selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof.

[0562] In certain embodiments, the dosage form of the pharmaceutical composition in the second container includes an injection dosage form, a transdermal absorption dosage form, a nasal inhalation dosage form, or a combination thereof.

[0563] In certain embodiments, the kit further comprises instructions.

[0564] III-(F) Drug Screening

[0565] The present invention provides a method for screening candidate drugs for preventing and / or treating brain calcification diseases, comprising the steps of:

[0566] (a) In a test group, a test drug is added to the astrocyte culture system, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group is observed; in a control group, no test compound is added to the same cell culture system, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group is observed;

[0567] Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test drug is a candidate drug for preventing and / or treating brain calcification diseases that has a promoting effect on the expression and / or activity of the phosphorus transport level.

[0568] In certain embodiments, the test drug is a compound.

[0569] In certain embodiments, the “significantly higher” refers to E1 / E0 ≥ 1.5, preferably ≥ 2.

[0570] In certain embodiments, the “significantly higher” refers to A1 / A0 ≥ 1.5, preferably ≥ 2.

[0571] In certain embodiments, the cells comprise astrocytes.

[0572] In certain embodiments, the cells are cells cultured in vitro.

[0573] In certain embodiments, the method comprises step (b): administering the candidate drug identified in step (a) to a mammal model to determine its effect on the mammal. In certain embodiments, the mammal is a mammal suffering from a brain calcification disease.

[0574] In certain embodiments, the methods are non-diagnostic and non-therapeutic.

[0575] III-(G) Exemplary Scheme

[0576] The present invention relates to the following exemplary embodiments:

[0577] Embodiment 1. A use of a brain phosphorus homeostasis promoter, characterized in that it is used to prepare a drug for preventing and / or treating brain calcification diseases.

[0578] Embodiment 2. The use according to embodiment 1, characterized in that the brain phosphorus homeostasis promoter is selected from the following group:

[0579] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0580] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0581] (Z3) XRP1 protein, a nucleic acid molecule encoding an XRP1 protein, an expression vector expressing an XRP1 protein, an XRP1 promoter, or a combination thereof;

[0582] (Z4) Any combination of the above Z1 to Z3.

[0583] Embodiment 3. The use as described in Embodiment 1, characterized in that the promoter comprises a small nucleic acid drug.

[0584] Embodiment 4. The use according to embodiment 1, characterized in that the brain calcification disease includes primary familial brain calcification (PFBC).

[0585] Embodiment 5. A preparation and composition, characterized in that the preparation and composition contain a promoter of brain phosphorus homeostasis.

[0586] Embodiment 6. The preparation and composition according to embodiment 5, characterized in that the brain phosphorus homeostasis promoter is selected from the following group:

[0587] (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof;

[0588] (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof;

[0589] (Z3) XRP1 protein, a nucleic acid molecule encoding an XRP1 protein, an expression vector expressing an XRP1 protein, an XRP1 promoter, or a combination thereof;

[0590] (Z4) Any combination of the above Z1 to Z3.

[0591] Embodiment 7. A kit for treating brain calcification disease, characterized in that the kit comprises:

[0592] (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect the presence of a genetic mutation in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and

[0593] (b) a second container, and a pharmaceutical composition in the second container, wherein the pharmaceutical composition comprises a pharmaceutical ingredient capable of regulating and repairing phosphorus homeostasis imbalance in the brain and a pharmaceutically acceptable carrier.

[0594] Embodiment 8. The medicine kit according to Embodiment 7 is characterized in that the medicine kit further comprises: (c) a third container, and a solution located in the third container for assisting the dissolution or in vivo delivery of the drug in the second container.

[0595] Embodiment 9. A method for screening candidate compounds for preventing and / or treating brain calcification diseases, characterized in that the method comprises the steps of:

[0596] (a) In a test group, a test compound is added to the astrocyte culture system, and the phosphate transport level (E1) and / or activity (A1) in the cells of the test group is observed; in a control group, no test compound is added to the same cell culture system, and the phosphate transport level (E0) and / or activity (A0) in the cells of the control group is observed;

[0597] Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test compound is a candidate compound for preventing and / or treating brain calcification diseases that has a promoting effect on the expression and / or activity of the phosphorus transport level.

[0598] Embodiment 10. The method according to embodiment 9, characterized in that the method comprises step (b): administering the candidate compound determined in step (a) to a mammalian model to determine its effect on the mammal.

[0599] III-(H) Beneficial Effects and Summary

[0600] The present invention discovered for the first time that the astrocyte-mediated phosphate transport process, including the intake of inorganic phosphate (Pi) from the extracellular interstitial fluid by the PiT2 transporter expressed on the membrane of the glial leaflets, and the excretion of Pi taken in by glial cells through the outlet of the blood-brain barrier to the outside of the brain through the XPR1 transporter on the membrane of the endfoot structure, is an important link in the regulation of phosphorus homeostasis in the brain. Conditional knockout experiments in mouse animal models showed that after the astrocyte-mediated phosphate transport ability was blocked compared with other types of cells, the phosphate in the cerebrospinal fluid showed the most significant increase, that is, homeostasis imbalance. At the same time, expression studies based on in situ hybridization showed that astrocytes highly expressed the phosphate intake transporter PiT2 and the excretion transporter XPR1; while neurons highly expressed another intake transporter PiT1.

[0601] Based on the findings in (1), by delivering viruses into the astrocytes of Pit2-KO mice to specifically express PiT2 protein in astrocytes, the level of phosphate transport in the brain of Pit2-KO mice can be significantly reduced, and the occurrence and progression of brain calcification can be inhibited.

[0602] In terms of method, the present invention is based on the core cellular pathological mechanism that brain calcification occurs due to imbalance in phosphorus homeostasis. By selectively expressing PiT2 on astrocytes, the phosphorus transport capacity of astrocytes is rebuilt, the imbalanced phosphorus homeostasis in the brain is repaired, and the underlying driving factor of calcification is fundamentally eliminated.

[0603] It should be noted that this method is not only for treating the progression of brain calcification, but also for repairing phosphorus homeostasis in the brain. In the patient's brain, the imbalance of phosphorus homeostasis in the brain is first caused by genetic mutations or acquired factors, and then this imbalance induces the occurrence of brain calcification. Therefore, the most essential treatment of this method is to repair the imbalanced phosphorus homeostasis in the brain. In the future, when clinical experts recognize that phosphorus homeostasis imbalance is a disease, just as elevated blood sugar is diabetes, or elevated blood lipids are a pathology that requires immediate treatment, the treatment strategy of this method may benefit more people.

[0604] In addition, the core method strategy illustrated by this method is to target astrocytes to repair the imbalance of phosphorus homeostasis in the brain. Those skilled in the art will understand that this method can be based on different delivery strategies and can be further optimized in the future as technology advances. Example

[0605] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0606] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in this application. Those skilled in the art will appreciate that various modifications and changes may be made to the details based on all published teachings, and that these changes are within the scope of protection of the present invention. The present invention is fully encompassed by the appended claims and any equivalents thereof. The experimental methods in the embodiments are, unless otherwise specified, conventional methods. For those embodiments in which specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments used in which the manufacturer is not specified, conventional products that can generally be obtained commercially are available.

[0607] Materials and Methods

[0608] 1. Subject recruitment, whole genome sequencing, and genetic analysis

[0609] We recruited 778 patients suspected of PFBC from the Department of Neurology at the First Affiliated Hospital of Fujian Medical University between 2012 and 2023. The diagnostic criteria for PFBC were as follows: a) bilateral and symmetrical calcifications detected on CT scan in the basal ganglia and / or dentate nucleus; b) a total calcification score (TCS) exceeding previously reported age-specific thresholds; c) the absence of biochemical abnormalities, with normal serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations; and d) the absence of infectious, toxic, or traumatic causes. Neurologic examinations and clinical evaluations were performed by at least two senior neurologists. For all suspected patients, we first used whole-exome sequencing (WES) to exclude cases harboring mutations in the entire coding region and exon sequences of known PFBC-associated genes, including SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, JAM2, and CMPK2. We then identified cases and families with PFBC-associated intronic mutations in the remaining sample set. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Fujian Medical University, and all participants provided written informed consent before enrollment.

[0610] For whole-genome sequencing, genomic DNA was obtained from the blood of patients with brain calcification. Paired-end DNA libraries (Illumina Truseq library construction) were prepared from the human DNA samples according to the manufacturer's instructions for sequencing library construction kits. The constructed DNA libraries were sequenced using an Illumina NovaSeq 6000 with paired-end, 150-bp lengths. Average sequencing depth ranged from 31.35 to 57.77, with 90.1% to 99.2% of the genome covered by at least 20 reads. Sequencing results (without barcoding) were then aligned to the human reference genome hg19 using SpeedSeq. Single nucleotide variants and insertion / deletions (indels) were then called using the Genome Analysis Toolkit v4.1. Potential structural and copy number variations in the patient genomes were extracted using SpeedSeq, or annotated using ANNOVAR for single nucleotide variants, indels, structural variations, and copy number variations.

[0611] For parametric linkage analysis, DNA samples were isolated from whole blood of 45 family members of family 1, and genome-wide linkage analysis was performed using the HumanOmniZhongHua-8 BeadChip (Illumina). Genotype calling and quality control were performed using the GenomeStudio Genotyping Module (Illumina). SNPs with Mendelian errors or calling rates less than 100% were deleted. Parametric linkage analysis was performed using Merlin (v1.1.2), with a rare dominant model with a rare disease allele frequency of 0.0001.

[0612] 2. Construction of Minigene Plasmid

[0613] To rapidly analyze splicing events involving potential cryptic exons using RT-PCR, we constructed DNA fragments containing wild-type or mutant genomic sequences of exon 2-intron 2-exon 3, exon 6-intron 6-exon 7, and exon 10-intron 10-exon 11 of the human SLC20A2 gene in a minigene backbone plasmid driven by a CMV promoter. To test whether mutation-associated cryptic exon insertions could alter SLC20A2 mRNA translation, we cloned the corresponding SLC20A2 cDNAs containing cryptic exon insertions into the pCMV6-Entry plasmid (Origene) using the ClonExpress MultiS One Step Cloning Kit (Vazyme). Subsequently, patient-derived mutations were generated by overlapping PCR or using the Mut Express II Rapid Mutagenesis Kit (Vazyme). Primers for amplifying these fragments are listed in Table 3.

[0614] 3. Antisense Oligonucleotides (ASOs)

[0615] Antisense oligonucleotide technology is a drug development strategy based on chemically modified short oligonucleotides (12-24 nt) that bind to their RNA targets through Watson-Crick base pairing. The sequences of ASOs were designed according to ASO design guidelines. ASOs with MOE and PS modifications (2′-O-methoxyethyl sugars with a phosphorothioate backbone) were synthesized by Integrated DNA Technologies and purified by HPLC. ASOs with PMO modifications (phosphodiamidate morpholine backbone) were synthesized by Gen-Tools, LLC. All ASOs were dissolved in nuclease-free water for in vitro cell culture experiments and in artificial cerebrospinal fluid (aCSF) for in vivo mouse experiments. The detailed sequences are provided in Table 1.

[0616] 4. Construction of SLC20A2-KI mouse model

[0617] To generate a humanized SLC20A2 mouse model, a genomic fragment spanning exon 2 to exon 3 of the mouse SLC20A2 gene was replaced with a frame-matched human SLC20A2 sequence carrying the c.289+1007C>G mutation using CRISPR / Cas9 technology. Briefly, a targeting sequence flanked by 1.4 kb homology arms was co-injected into the uterus of C57BL / 6J mice along with sgRNAs and Cas9 RNA. To identify founder (F0) mice harboring the correct knock-in sequence, genomic DNA extracted from the mouse tails was used for genotyping. F1 KI mice were generated by crossing with wild-type C57BL / 6J mice, and germline transmission of the F0 KI mice was verified. To further verify the presence of the correct homologous recombination target, long-range sequencing and southern blot analysis were performed on the F1 mice. Heterozygous and homozygous SLC20A2-KI mice were selected for biochemical and phenotypic analysis. The sgRNA sequences and genotyping primers are listed in Table 3.

[0618] 5. Cell Culture and Transfection

[0619] HEK293T, Neuro-2A, A172, HepG2, and HeLa cells (Cell Bank, Chinese Academy of Sciences) and primary human fibroblasts were cultured in 6-well or 12-well plates (Corning) using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol / vol) fetal bovine serum (FBS). Human fibroblasts were derived from patients with PFBC harboring the SLC20A2 heterozygous c.289+1007C>G mutation and SLC20A2 heterozygous deletion, as well as from voluntary healthy human controls.

[0620] For plasmid transfection, approximately 3 μg of minigene or cDNA plasmid was transfected into HEK293T and Neuro-2A cells using Lipofectamine 3000 (Thermo Fisher). For western blot analysis, cells were harvested 24 hours after transfection. For splicing analysis using RT-PCR, cells were transfected with MOE- or PMO-modified ASOs after 24 hours of culture.

[0621] For MOE-modified ASOs, cells were transfected with Lipofectamine 3000 (Thermo Fisher) at a final concentration of 100 nM for 48 hours. For PMO-modified ASOs, cells were transfected with 4 μM PMO using 6 μL / mL Endo-Porter (Gene-Tools, LLC) for 48 hours. To inhibit NMD-mediated RNA degradation, cultured human fibroblasts were incubated with cyclohexanone (CHX, 50 μg / mL) for 6 hours before harvesting.

[0622] 6. RNA Extraction and Splicing Analysis

[0623] Total RNA was extracted from HEK293T, Neuro-2A, HepG2, HeLa, A172 cells, or human fibroblasts using TRIzol (Invitrogen) according to the manufacturer's instructions, and 1 μg of RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). For splicing analysis, the transcribed cDNA was used for PCR using primer pairs spanning the corresponding exons of the SLC20A2 gene. The primer sequences are listed in Table 3.

[0624] 7. Protein extraction and Western-blot analysis

[0625] Cells or mouse brains were harvested in RIPA lysis buffer containing protease inhibitors, and the lysate was centrifuged at 14,000 g for 15 minutes. To detect PiT2, protein extracts were incubated at 37°C for 10 minutes, separated on 9% SDS-PAGE gels, and blotted onto PVDF membranes. The membranes were blocked with 5% skim milk and probed with PiT2-7B5 (1:1000, a monoclonal antibody raised against the PiT2 loop region in mice), HA-tag (1:5000, #3724, Cell Signaling), GAPDH (1:3000, KC-5G5, KANGCHEN), α-Tubulin (1:5000, 66031-1-Ig, Proteintech), and the corresponding HRP-conjugated secondary antibodies (1:2000, Cell Signaling). Bands were visualized using ECL (TIANGEN) and measured using Image J software.

[0626] 8. Intracerebral (ICV) injection of ASO in mice

[0627] All animal surgeries were performed under standard aseptic conditions. Mice were anesthetized with isoflurane (5% for induction and 1.5% for maintenance) and placed in a small animal stereotaxic frame (Model 68528, RWD Life Science) with body temperature maintained on a heating pad. SLC20A2-KI mice were injected with 2% lidocaine at the incision site and shaved using a depilatory cream. After triple disinfection of the scalp at the surgical site with betaine and 75% alcohol, a midline incision was made and the scalp was removed to expose the skull. The periosteum and other soft tissues connected to the skull were cleaned using cotton swabs and a scalpel. After the skull was dried, the skin surrounding the skull was adhered to the exposed skull edge using tissue adhesive (3M Vetbond Tissue Adhesive). The skull plane was adjusted to stereotaxic level, and the bregma was determined. A layer of blue light-curing self-etching adhesive (3M ESPE Single Bond Universal, 3M Germany GmbH) was applied to and around the frontal bone to aid fixation. A custom head plate was then fixed flat on the frontal bone with gutta-percha. A small circular craniotomy (AP + 0.25 mm; ML - 0.75 mm) was carefully drilled while keeping the dura mater intact. The dura mater was covered with antibiotic ointment and a fast-curing silicone elastomer. The mouse was moved to a custom-made fixation plate with 1.5% isoflurane, and cerebrospinal fluid was collected from the tympanic cavity. The mouse was then allowed to recover for at least one week before ASO injection.

[0628] For ICV injections, mice were anesthetized with isoflurane (5% for induction and 1% for maintenance) and head-fixed on a stereotaxic machine 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 working concentration of 25 μg / μL. A total dose of 50 μg, 100 μg, 200 μg, or 400 μg of SLC20A2-ASO or control-ASO was injected into the right lateral cerebral ventricle (center coordinates: AP +0.25 mm; ML -0.75 mm; DV -1.85 mm) using a Nano-injector (Nanoject III, Drummond Scientific Company) at a rate of 1 nL / s. The glass pipette was withdrawn at least 10 minutes after injection. Seal the craniotomy with a flowable resin cured with blue light to avoid infection. Allow the mice to recover from anesthesia on a heating pad before returning them to their cages.

[0629] 9. CSF Collection and Pi Measurement

[0630] According to the established laboratory method, the mouse was fixed using a stereotaxic frame (Model 68528, RWD Life Science). Another ultra-microscale, three-way adjustable bracket was used to clamp the collection needle. The drawn glass needle was loaded into the needle tube and punctured into the ventricle from the medullary cisterna magna of the anesthetized adult mouse brain. Then, a slight negative pressure was applied to guide the CSF into the glass needle. Approximately 5-10 μL of CSF was collected and then centrifuged for 5 minutes (2000g, 4°C) to remove cell debris. The concentration of inorganic phosphate in CSF was detected using the PiColorLock gold phosphate detection kit (303-0030, Innova Biosciences), and then read at 635 nm using a SpectraMax (Molecular device).

[0631] 10. In situ hybridization (ISH) detection of ASO distribution in mouse brain

[0632] All reagents used in ISH analysis were prepared with diethyl carbonate (DEPC). 7-month-old SLC20A2-KI mice using MOE-A and MOE-NC were anesthetized with chloral hydrate, perfused with 4% PFA, and dehydrated with 15% and 30% sucrose successively. miRNAscope was used according to the manufacturer's instructions. TM ISH was performed on 18-μm thick brain sections using Assay Red (Advanced Cell Diagnostics, Cat. No. 324500) and a custom MOE-A probe (Advanced Cell Diagnostics, Cat. No. 1123181-S1). Whole-brain MOE-A expression was imaged using a VS120 microscope (Olympus, Japan) with a 40× objective.

[0633] 11. Analysis of brain calcification by micro-CT and histochemical staining

[0634] To assess brain calcification, SLC20A2-KI and WT mice were anesthetized with chloral hydrate and perfused with 4% PFA in PBS. For micro-CT scanning, three-dimensional images of the mouse brain were acquired using an X-ray micro-CT scanner (Bruker, Skyscan 1172); paraffin-embedded mouse brains were 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.

[0635] For histochemical staining of brain calcification, mouse brains were dehydrated through a series of graded ethanol treatments before paraffin embedding, and 4-μm sections were prepared. Brain calcification was assessed using standard, field-recognized staining methods, including alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E. Each analysis was performed in triplicate.

[0636] 13. Statistical analysis

[0637] Data are expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed by Student's t-test. Differences were considered statistically significant when the difference was < 0.05 and are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0638] Example 1: Identification of SLC20A2 intronic mutations in PFBC patients and analysis of their resulting abnormalities in SLC20A2 splicing and translation

[0639] After years of painstaking research, the inventors have compiled hundreds of cases of brain calcification and constructed pedigrees. Through genetic analysis using exon sequencing, they identified pathogenic mutations in the exons of known PFBC-causing genes (SLC20A2, PDGFB, PDGFRB, XPR1, MYORG, and JAM2) in 33 of 53 families (62.2%). Furthermore, CNV analysis identified exonic deletions flanking the SLC20A2 gene in three additional families. Among the remaining 17 families, linkage analysis was performed in one large family (Family 1) (Figure 1a), in which no exonic mutations were detected. Association mapping of genomic DNA samples from 45 family members using the Affymetrix 250K Nsp SNP array revealed a 32-Mb region on chromosome 8 (hg19 chr8:29042453-61341711) linked to the calcification phenotype in the family, with a LOD score of 4.5. Further WGS analysis identified a deep-intronic variant in SLC20A2, c.289+1007C>G. Sanger sequencing confirmed this variant's shared association with brain calcification in the family. In silico evaluation of this variant using SpliceAI yielded an acceptor gain score of 0.11 and a donor gain score of 0.11, suggesting that splicing from intron 2 of SLC20A2 may introduce an unreported cryptic exon (Table 2).

[0640] Using similar WGS analysis and Sanger sequencing approaches to screen additional PFBC families without a definitive genetic diagnosis, we identified three additional genetic variants located deep within the SLC20A2 gene in four families (Figure 1a): c.289+937G>A in intron 2 (families 2 and 3), c.730+768C>T in intron 6 (family 4), and c.1795-1698A>G in intron 10 (family 5). None of these four intronic variants (Figure 1c) has been registered in major human population genetics databases and, according to SliceAI analysis, was predicted to affect splicing (Table 2). We also found a common association between these variants and brain calcification in all five families (Figure 1b). The subjects ranged in age from 30 to 60 years. Four of the patients (families 1, 3, 4, and 5) reported headaches, and one (family 2) experienced dizziness. CT imaging revealed moderate to severe brain calcifications (TCS, 16–39) in the basal ganglia and dentate nucleus (families 1–5), thalamus (families 1–3), and subcortical white matter (family 2) ( Figure 1b ). Serum calcium, phosphorus, and PTH levels were normal in all patients ( Table 2 ). These results support that intronic variants in SLC20A2 may be the genetic cause of PFBC.

[0641] In mammals, it is now clear that the 5' splice donor site and 3' splice acceptor site of introns are conserved. Intronic mutations can affect mRNA processing by disrupting splice sites and / or splicing regulatory elements, or by altering the secondary structure of the mRNA molecule. Further sequence prediction suggested that the c.289+937G>A mutation in intron 2 and the c.730+768C>T mutation in intron 6 may directly produce new splice acceptors or donors, thereby changing the original splicing mechanism; however, the c.289+1007C>G mutation in intron 2 and the c.1795-1698A>G mutation in intron 10 did not produce characteristic splice acceptors or donors, suggesting that they may be through other mechanisms, such as regulating the probability of splicing inclusion of cryptic exons.

[0642] To validate the genetic predictions, we first performed minigene analysis to explore how the intronic mutations found in the two families altered the splicing of the cryptic introns of SLC20A2. Specifically, we cloned the SLC20A2 genomic sequence (including the upstream and downstream exon 2-3 junctions of intron 2 and the junctions of exon 10 and exon 11 containing intron 10) and loaded it into a backbone plasmid driven by the CMV promoter to construct the wild-type plasmid of the minigene. We then used site-directed mutagenesis to generate point mutations corresponding to the c.289+937G>A and c.1795-1698A>G intronic mutations in the two PFBC families (Figure 2a, c).

[0643] Minigene testing confirmed that for families 1 and 5, although the c.289+1007C>G and c.1795-1698A>G mutations did not result in the creation of new splice sites, RT-PCR and Sanger sequencing showed increased splicing integration of 135-nt and 76-nt cryptic exons in intron 2 and intron 10, respectively (Figure 2b, d). In contrast, cells expressing the wild-type SLC20A2 minigene, which does not express these two mutations, did not show significant levels of abnormal transcripts in the mutant minigene assay, suggesting that these two mutations may alter the probability of splicing insertion of cryptic exons by affecting adjacent splicing regulatory elements.

[0644] We further analyzed the effects of these 135-nt and 76-nt cryptic exons on protein production by inserting them into the corresponding sequence positions of the CDS of N-terminally HA-tagged SLC20A2. Sequence prediction indicated that the insertion of these two cryptic exons introduced premature termination codons within the new coding frame (Figure 2g), thereby preventing translation of a functional PiT2 protein. Transfection of cultured Neuro-2A cells with HA-tagged SLC20A2 minigene constructs containing these two cryptic exons or with two other nascent exons generated by the new splice acceptor / donor revealed truncated PiT2 protein products consistent with the predictions (Figure 2h). Based on experience, these aberrant transcripts harbor PTC mutations in vivo, triggering NMD degradation, which may ultimately result in the production of a truncated protein. To this end, we compared fibroblasts from healthy controls and found that full-length PiT2 levels were significantly decreased in fibroblasts from patients carrying the c.289+1007C>G mutation (Figure 2i). These findings collectively support a new and important molecular genetic pathological mechanism, namely that intronic mutations in the SLC20A2 gene lead to increased splicing and integration of endogenous cryptic exons, resulting in SLC20A2 haploinsufficiency and causing brain calcification in mutation carriers of PFBC families.

[0645] Example 2: Endogenous cryptic exons exist in the human brain and are involved in the formation of alternative splicing isoforms of SLC20A2

[0646] The in vitro evidence in the aforementioned examples suggests that the c.289+1007C>G and c.1795-1698A>G mutations have the ability to introduce cryptic exons during mRNA splicing within introns 2 and 10, respectively. In silico analysis confirmed that these mutations do not generate predicted splice sites. An important question is whether these cryptic exons are inherently present in human cells, or whether these aberrant splice isoforms, produced at low levels under normal splicing conditions, are degraded.

[0647] We investigated the presence of potential splicing-regulated cryptic exons within each intron of SLC20A2 in HEK293T cells (a kidney-derived cell line) and A172 cells (a brain-derived cell line). RT-PCR analysis of intracellular RNA transcripts, which were treated with CHX to inhibit degradation of aberrantly spliced ​​transcripts, revealed a cryptic exon inclusion event (c.289+901 to c.289+1035, designated "crpE2") between exon 2 and exon 3 in both HEK293T and A172 cells, and a prominent cryptic exon inclusion event (c.1795-1703 to c.1795-1628, designated "crpE10") between exon 10 and exon 11 in A172 cells (Figure 3a,b). Notably, the expression levels of these aberrantly spliced ​​transcripts containing cryptic exons of crpE2 or crpE10 were sensitive to cyclohexylamine (CHX), a small molecule inhibitor used to assess potential involvement of the NMD pathway. This suggests that transcripts normally incorporating crpE2 or crpE10 may be degraded by the NMD pathway in the brain. Sanger sequencing of crpE2 and crpE10 confirmed that they are the 135-nt and 76-nt splice products of minigenes carrying the c.289+1007C>G and c.1795-1698A>G mutations (Figure 3b). These results indicate that the introduction of these two cryptic exons does occur naturally in human cells (and is significantly increased in the presence of the intronic mutations c.289+1007C>G and c.1795-1698A>G).

[0648] RT-PCR analysis of a wider range of human cells (HeLa cells, HepG2 cells, HEK293T cells, A172 cells, and human fibroblasts from the epidermis) further verified that the splicing integration of crpE2 and crpE10 in SLC20A2 mRNA transcripts can occur in different cell types from multiple tissue sources (Figure 3c), suggesting that this splicing mechanism can be used in scenarios aimed at intervening in diseases by regulating PiT2 expression.

[0649] Example 3: ASO can correct abnormal splicing involving crpE2 or crpE10 and improve the translation efficiency of functional PiT2 protein

[0650] To evaluate whether ASOs can inhibit cryptic exon-dependent alternative splicing, we transfected SLC20A2 minigenes into Neuro-2a cells and co-transfected ASOs targeting the crpE2 and / or crpE10 splice sites to suppress the production of aberrantly spliced ​​transcripts. Among various ASO modifications, 2′-O-methoxyethyl (MOE) and phosphorodiamidate morpholino oligomers (PMO) are the most common modifications used in marketed neurological ASOs, and we analyzed these two types of modified ASOs separately. For the minigene containing the c.289+1007C>G mutation, we detected that 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%) significantly decreased the splicing integration of crpE2 (Figures 4a-c). For the minigene with the c.1795-1698A>G mutation, the splicing integration of crpE10 was also greatly reduced by 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 of ASO intervention to regulate PiT2 expression for the treatment of brain calcification diseases.

[0651] To further test the effect of ASOs on correcting endogenous aberrant splicing, we transfected MOE-ASOs into human fibroblasts from PFBC patients carrying intronic mutations. We first cultured epidermal fibroblasts (P1) from a patient carrying a heterozygous SLC20A2 c.289+1007C>G mutation. Compared with the vehicle or control MOE-ASO group (MOE-NC), we found that MOE-A and MOE-B could largely eliminate crpE2 insertion into SLC20A2 mRNA (MOE-A: 14.35±5.2645%; MOE-B: 15.62±5.206%; MOE-C: 15.48±5.200%) (Figure 5a, b) and lead 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 ASO-mediated inhibition of aberrant splicing as a feasible approach to rescue insufficient SLC20A2 expression in PFBC patients with intronic mutations.

[0652] Considering that ASOs can modulate nonproductive alternative splicing of the wild-type allele and increase normal SLC20A2 transcript levels, we cultured fibroblasts from two healthy control volunteers (C1 and C2) to examine this question from another perspective. ASO cell intervention experiments revealed that MOE-A and MOE-B also modulated SLC20A2 mRNA splicing in healthy control fibroblasts (Figures 6a-e), and upregulated PiT2 protein expression (C1 and C2). MOEs targeting crpE10 (MOE-J, MOE-K, and MOE-L) also reduced crpE10 insertion and increased PiT2 protein expression in wild-type fibroblasts (Figures 6f-g). These results suggest that for target genes in autosomal dominant haploinsufficiency disorders, ASO intervention can modulate the expression of the target gene on the other chromosome, which is free of pathogenic mutations, and thus alter disease progression. In view of the expansion of practical application value, we expanded the screening range of ASO sequences in healthy control fibroblasts, including new ASO sequences targeting crpE2 splicing regulation (cr5E2-m1, cr5E2-5, cr5E2-6, cr5E2-8, cr5E2-9, cr5E2-10, cr5E2-12,

[0653] 5crE2-13,cr5E2-13L,cr5E2-13S,cr5E2-14,cr5E2-16,cr5E2-17,cr5E2-18,

[0654] cr5E2-19,cr5E2-20,cr5E2-21,cr5E2-22,cr5E2-23,cr5E2-24,cr3E2-5,

[0655] cr3E2-8,cr3E2-9,cr3E2-10,cr3E2-11,cr3E2-12,cr3E2-13,cr3E2-14,

[0656] cr3E2-15, cr3E2-16), and new ASO sequences targeting crpE10 splicing regulation (cr5E10-9, cr5E10-11, cr5E10-12, cr5E10-13, cr5E10-15, cr5E10-16, cr5E10-19, cr3E10-9,

[0657] cr3E10-14, cr3E10-15), their sequences and related information are listed in Table 1. First, the minigene was transfected into N2A cells, and the response of the minigene's expressed splicing products to ASO was analyzed by PCR, and most of the sequences with poor effects were filtered out (Figure 7a, c). Next, to evaluate the effects of the remaining ASO sequences on the splicing regulation of endogenously expressed SLC20A2, we transfected them into healthy control human fibroblasts. After repeated PCR and protein Western blotting (WB) assays (Figure 7b, c), we ultimately identified seven ASO sequences that exhibited significant effects in promoting SLC20A2 or PiT2 expression. These included the ASO sequences cr5E2-10, MOE-B (cr5E2-11), cr5E2-12, cr5E2-13, cr5E2-14 MOE-A (cr5E2-15) targeting crpE2 splicing regulation, and cr5E10-12 targeting crpE10 splicing regulation. Due to the limitations of the assay conditions, this result does not rule out the possibility that ASO sequences targeting regions described in other patents may also be effective. This is merely a demonstration of the analytical process of the invention.

[0658] In PFBC, heterozygous dominant mutations in the SLC20A2 gene account for up to 60% of all cases, highlighting the potential for ASO intervention to repair haploinsufficiency in SLC20A2 regardless of mutational form (including intronic mutations, exonic mutations, and mutations in transcriptional regulatory regions). To test whether MOE-A and MOE-B can also rescue PiT2 expression in patients with SLC20A2 loss-of-function mutations caused by non-intronic mutations, which account for a larger proportion of the population, we cultured dermal fibroblasts from a patient (P2) with heterozygous loss of a genomic region covering SLC20A2. Compared with the vehicle group or the control ASO group, MOE-A and MOE-B could greatly reduce the production of endogenous crpE2-inserted SLC20A2 mRNA (MOE-A: 2.024±0.1775%; MOE-B: 2.267±0.16915%; MOE-C: 2.386±0.1278%) (Figure 7a, b) and promote a significant increase in PiT2 protein (MOE-A: 15.99±4.225%; MOE-B: 13.53±4.951%; MOE-C: 11.25±4.507%) (Figure 8c). Taken together, these results indicate that ASO-mediated correction of cryptic exon-dependent alternative splicing can rescue SLC20A2 haploinsufficiency caused by SLC20A2 intronic mutations and even other non-intronic mutations.

[0659] Example 4: Construction of a humanized SLC20A2 intronic mouse model (SLC20A2-KI) and analysis of progressive brain calcification

[0660] To evaluate whether ASO therapy can effectively intervene in the development and progression of brain calcification in animals, we next developed a humanized mouse model. By knocking in the entire human intron 2 carrying the SLC20A2 c.289+1007C>G mutation and flanking linker sequences (Figure 9a), while maintaining frame alignment, the chimeric SLC20A2 gene transcript in this mouse model contains the crpE2 cryptic exon, making it accessible to human-compatible ASOs for targeting and intervention. Specifically, using CRISPR / Cas9 technology and co-introduced targeting DNA fragments, the genomic fragment spanning exons 2 to 3 of the mouse SLC20A2 gene was replaced with the frame-matched human SLC20A2 gene sequence carrying the c.289+1007C>G mutation. Briefly, the targeting sequence flanked by 1.4 kb homology arms was co-injected into the uterus of C57BL / 6J mice along with sgRNAs and Cas9 RNA. To identify founder (F0) mice with the correct knock-in sequence, genomic DNA extracted from their tails was used for genotyping. F1 KI mice were generated by crossing with C57BL / 6J wild-type mice, confirming germline transmission of the F0 KI mice. To further verify that the target mice had the correct homologous recombination, long-range sequencing and southern blot analysis were performed on the F1 mice. Heterozygous and homozygous SLC20A2-KI mice were selected for biochemical and phenotypic analysis.

[0661] In heterozygous and homozygous KI mice, RT-PCR and sequencing analysis revealed a significant increase in crpE2 insertion into SLC20A2 mRNA (Figure 9b), accompanied by a significant decrease in SLC20A2 mRNA and PiT2 protein levels in these animals (Figures 9c, d). Analysis of inorganic phosphate in the cerebrospinal fluid (CSF) showed a significant increase in inorganic phosphate (Pi) levels in the cerebrospinal fluid (CSF) of heterozygous and homozygous SLC20A2-KI mice (Figure 9e), metabolically recapitulating the brain Pi homeostasis disorder seen in PFBC patients with SLC20A2 mutations. This suggests that SLC20A2 c.289+1007C>G mutations can be successfully used to model SLC20A2 haploinsufficiency.

[0662] We subsequently examined brain calcification in SLC20A2-KI mice using micro-CT, fluorescent RIS staining (Figures 10a, b), and histological staining (including alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E). MicroCT scans revealed (Figure 10a) that approximately 71% of SLC20A2-KI mice began to develop prominent, symmetrical brain calcifications in the bilateral basal forebrain, hypothalamus, and dorsal midbrain at 5 months of age, 3 months later than in SLC20A2-KO mice. By 7 months of age, calcifications in homozygous SLC20A2-KI mice were widespread in numerous brain regions, including the basal forebrain, thalamus, hypothalamus, midbrain, and dentate nucleus of the cerebellum (Figure 10a). Furthermore, at 9.5 months of age, the number and size of calcified nodules continued to increase, with prominent, larger calcified foci appearing in the thalamus, highly similar to those in patients with PFBC. Using fluorescent RIS-AF647 staining, which can identify calcification deposits, we also observed calcification deposits in brain regions similar to those seen on microCT (Figure 10b), but the fluorescent calcifications were clearer and more suitable for quantification. Furthermore, histological staining analysis, including alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E, further demonstrated that the calcifications in the mouse model were morphologically highly consistent with those in humans (Figure 11), indicating that they are driven by the same pathological mechanism. These results demonstrate that our humanized SLC20A2-KI mouse model can recapitulate the brain calcification distribution patterns seen in patients with SLC20A2 haploinsufficiency, brain Pi disorders, and PFBC with SLC20A2 mutations.

[0663] Example 5: ASO intervention effectively inhibits brain calcification in SLC20A2-KI mice

[0664] Given that mutations in the SLC20A2 gene are the most common cause of PFBC, our humanized SLC20A2-KI mouse model represents a valuable model for testing novel ASO-based therapies to treat brain calcification. We term this therapy Calcification Repression by ASO Corrected Expression (CRACE). We designed four doses of MOE-ASOs (50, 100, 200, and 400 μg) to evaluate whether splicing correction mediated by a single intraventricular injection of ASOs could inhibit brain calcification in SLC20A2-KI mice before the development of brain calcification at 3 months (Figure 12a). MOE-A and MOE-B (collectively referred to as therapeutic MOE) were selected for in vivo administration because they demonstrated significant effects on restoring PiT2 expression in cell-based experiments.

[0665] Four months after a single 200 μg ICV injection of MOE-A, in situ hybridization using a probe specifically recognizing MOE-A revealed widespread ASO signal throughout the brain (Figure 12b), demonstrating that MOEs can effectively spread and persist in the brain. Regarding target gene expression, compared with SLC20A2-KI mice injected with control MOE, aberrant SLC20A2 transcripts containing crpE2 insertions were significantly reduced in the thalamus of homozygous SLC20A2-KI mice injected with therapeutic MOEs, while normal SLC20A2 transcripts showed a significant increase in expression, and the levels of its translation product, PiT2, were significantly enhanced (Figure 12c, d, e). Furthermore, CSF Pi levels were significantly decreased in SLC20A2-KI mice after four months of treatment with 100 μg and 200 μg of therapeutic MOE-ASOs (Figure 12f), suggesting that abnormal phosphate homeostasis in the brain is restored.

[0666] In terms of brain calcification, four months after ICV administration of 100-400 μg of therapeutic MOE-ASOs to 3-month-old mice, micro-CT scans of the brains of 7-month-old mice showed a significant decrease in the volume and number of calcification foci in the thalamus, hypothalamus, midbrain, and dorsal forebrain (Figure 13a, b). Therefore, splicing correction mediated by MOE-modified ASOs can prevent the occurrence of brain calcification in PFBC simulated by humanized SLC20A2-KI mice.

[0667] To further evaluate the efficacy of intraventricular (ICV) injection of ASOs in mice already calcified, we injected ASOs into the brains of 5-month-old homozygous SLC20A2-KI mice. As shown in Figure 9, calcification was already present in the brains of these mice at this time. Previous cell-based experiments have shown that both MOE-modified and PMO-modified ASOs can restore SLC20A2 expression. For this study, we administered 25 μg and 75 μg of PMO-ASOs to 5-month-old homozygous SLC20A2-KI mice (Figure 14a). At the end of PMO-1 administration, a significant increase in normal SLC20A2 transcript expression was detected in the thalamus of SLC20A2-KI mice (Figure 14b), and a significant decrease in CSF Pi concentration (Figure 14c). Similarly, micro-CT revealed that a single dose of 25 μg or 75 μg of PMO-1 significantly reduced brain calcification in 7-month-old SLC20A2-KI mice compared to PMO-NC mice (Figure 15d). Collectively, these findings indicate that CRACE therapy effectively increases PiT2 expression, restores brain Pi homeostasis, and suppresses or reduces brain calcification in SLC20A2-KI mice. Therefore, these data and findings support the potential of ASO-mediated CRACE therapy for the treatment of brain calcification in PFBC patients.

[0668] Example 6 Expression Characteristics of Genes Related to Hereditary Brain Calcification in Mouse Brain

[0669] Given the abnormal brain Pi homeostasis found in PFBC patients with mutations in the Pi transporter genes PIT2 or XPR1, we analyzed the expression patterns of the SLC20 sodium-phosphate cotransporter genes Pit1 (Slc20a1) and Pit2 (Slc20a2), as well as the Pi exporter gene Xpr1, in the mouse brain. Using publicly available mouse brain single-cell RNA-seq datasets (Source a: https: / / betsholtzlab.org / Publications / databases.html; Source b: http: / / celltypes.org / brain / ), we generated cell type expression heatmaps for Pit1, Pit2, Xpr1, and the PFBC-associated glycosidase Myorg. The single-cell expression heatmaps revealed that both astrocytes and neurons showed enriched expression of Pit1, Pit2, and Xpr1, suggesting active phosphate transport in these two cell types.

[0670] To validate the expression of Pi transporters in astrocytes and neurons, we used RNAscope in situ hybridization (ISH) to investigate the expression of Pit1 and Pit2 in the adult mouse brain, focusing on the thalamus, cerebellum, striatum, and hypothalamus, regions known to harbor calcifications in PFBC patients. Astrocytes were labeled by immunostaining for the S100b marker protein. In the cerebellum, Bergmann cells (a specific astrocyte subtype) showed no ISH signal for Pit1 but a strong signal for Pit2. In the thalamus, Pit2 was also highly expressed in all astrocytes, while only a minority (<20%) of astrocytes expressed a less intense Pit1 signal. This dominant expression of Pit2 over Pit1 in astrocytes was consistent across all four brain regions and was confirmed by double in situ hybridization with another astrocyte marker gene, Aldh1l1. In addition, Olig1-positive oligodendrocytes and oligodendrocyte precursor cells (OPCs) also express Pit2 but not Pit1.

[0671] In contrast, glutamatergic (Vglut2+) and GABAergic (Gad1+) neurons expressed strong Pit1 and relatively weak Pit2 expression. Considering that oligodendrocytes express mild levels of Xpr1, these results suggest a binary expression pattern, with high expression of Pit1 and Xpr1 in neurons and high expression of Pit2 and Xpr1 in astrocytes. Combined with the fact that 61% of reported PFBC patients have PIT2 but not PIT1 mutations, our finding of preferential Pit2 expression in astrocytes suggests the importance of astrocyte-mediated imbalance of brain Pi homeostasis in the development of brain calcification.

[0672] Example 7 Preparation of Mouse Animal Model

[0673] Slc20a2 tm1aWtsiMice (MGI:4431725) were mated with Actin-Flp mice (RRID:IMSR_JAX:005703) to remove the En2a-LacZ-PGK-Neo cassette to generate Pit2 flox mice, or bred with Ella-Cre mice (RRID:IMSR_JAX:003724) to remove the PGK-Neo-flox-Exon3 cassette to generate Pit2 KO mice. Pit1 (Slc20a1) flox mice were purchased from the KOAP engineered mouse collection (T013241, GemPharmatech, China). Subsequently, they were bred with wild-type C57BL / 6 mice (SLAC Animals, China) for at least three generations. The source of the conditional Cre mouse strains are: Aldh1l1-CreER generated in the laboratory of Baljit S. Khakh1 T2 (PMID:27939582,RRID:IMSR_JAX:029655), Plp1-CreER provided by Jia-Wei Zhou laboratory T2 (RRID:IMSR_JAX:005975), Pdgfrb-Cre generated by Volkhard Lindner's laboratory (PMID:21557454), Camk2a-iCre generated by G. Schutz's laboratory (PMID:11668676), and Vgat-Ires-Cre generated by Jax Mouse Laboratory (IMSR_JAX:016962). Mice were genotyped using Taq polymerase (Vazyme, China). Tamoxifen (20 mg / ml, corn oil as solvent) was administered intraperitoneally once daily for 5 consecutive days at a dose of 1 mg / 10 g body weight.

[0674] The animal care and experimental procedures were approved by the Animal Care and Use Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

[0675] Example 8 Effect of PiT2 knockout in astrocytes on brain phosphorus homeostasis

[0676] 8.1 Preparation of serum and cerebrospinal fluid and determination of Pi concentration

[0677] Blood was collected from the orbital vein of adult mice and allowed to stand at room temperature for at least 1 hour before centrifugation (2000 x g, 15 minutes) to prepare serum. Cerebrospinal fluid (CSF) was collected from the cisterna magna of anesthetized adult mice and centrifuged for 5 minutes (2000 x g, 4°C) to remove cellular debris. The concentration of inorganic phosphate in serum and CSF was determined using the PiColorLock Free Inorganic Phosphate (Pi) Assay Kit (303-0030, Innova Biosciences). The values ​​were read at 635 nm using a SpectraMax (Molecular Devices) spectrophotometer, and the Pi concentration was calculated using the function obtained from the standard curve.

[0678] 8.2 Plasmid Construction, Viruses, and Transmission

[0679] The ORFs of human MYORG, PIT2, or XPR1 expression plasmids were derived from Origene's expression plasmids. For in vivo expression, the PiT2 and XPR1 expression cassettes were inserted after the gfaABC1D promoter of the AAV2 expression construct, and the expression plasmids were combined with the plasmid of the PHP.eB serotype capsid protein (Taitools, Inc, Shanghai) for AAV virus packaging. For intraparenchymal injection, a Nano-injector (Nanoject III, Drummond Scientific Company) was used to inject the initial titer of approximately 1x10e at a rate of 60nL / min. 13 The diluted AAV2 / 5 virus was injected into the thalamus of adult mice. To achieve whole-brain re-expression of astrocytes, each 8-week-old (50-56 days) mouse was injected with 100ul of 2x10e 11 Virus solution containing AAV5.PH.eB capsid.

[0680] 8.3 Immunostaining, Antibodies, and Microscopy

[0681] Mouse brain sections (20 / 25 μm) fixed with 4% PFA were first perforated with 1X PBS containing 0.5% Triton X-100, then blocked with PBS containing 0.1% Triton X-100 and 5% BSA, and incubated with the corresponding primary antibody overnight at 4°C. The next day, brain sections were washed three times in PBS for 10 minutes each, then incubated with secondary antibodies for 2 hours at room temperature. Following Hoechst staining, sections were washed three times in PBS for 10 minutes each, and mounted on slides. The antibodies used were: mouse anti-V5 (R96025, Invitrogen), rat anti-HA (11867423001, Roche), mouse anti-Calnexin (ab10286, Abcam), chicken anti-GFP (ab13970, Abcam), rabbit anti-AQP4 (AB3594, Millipore), rabbit anti-Col4 (ab6586, Abcam), and rabbit anti-S100β (ab41548, ab52642, Abcam). Fluorescence expression data were captured on brain sections using an FV3000 laser confocal microscope (Olympus). The parameters were: three fluorescence channels at 488 / 568 / 647 nm, a 40× or 60× objective lens, a zoom factor of 3–5, and a z-spacing of 0.4 μm.

[0682] 8.4 Micro-CT Imaging

[0683] Micro-CT scanning of mouse brain samples fixed with 4% paraformaldehyde was performed using a high-resolution microCT scanner (Skyscan 1172, Bruker) with the following scanning parameters: source voltage 40 kV, current 250 μA, exposure time 210 ms; rotation step size 0.6°; camera pixel size 9 × 9 × 9 μm. Images were reconstructed using the NRecon program, powered by the GPUReconServer engine, and exported in BMP format (pixel size 5 μm). The following parameters were used: beam hardening correction factor 33%, ring artifact reduction 8, and Gaussian smoothing kernel 2. The exported BMP image series were then imported into Imaris software and converted into TIF series (pixel size 20 μm). Subsequently, the TIF image stack was imported into ImageJ software and repositioned to export coronal and sagittal sections of the mouse microCT images. Finally, based on the standard slice values ​​of the mouse brain atlas, sagittal planes within the range of 0.1-1.6 mm lateral to the midline were superimposed (75 slices, 1.5 mm thickness) with the midline as the reference to project sagittal images of brain calcification in that area. In addition, coronal plane images of brain calcification were projected from coronal planes covering the hindbrain (75 slices, 1.5 mm thickness) or the basal forebrain and hypothalamus (150 slices, 3 mm thickness).

[0684] 8.5 Fluorescence Imaging of Calcified Deposits

[0685] After microCT scanning, mouse brains were frozen in OCT reagent and then cut into 25 μm thick slices using a cryostat. Brain slices from specific locations were incubated with 0.2 μM RIS-AF647 dye (BV500101, Biovinc, USA) for 5 hours, stained with Hoechst, and mounted on glass slides. Calcification images of brain slices were acquired using a VS120 Slice Imaging System (Olympus) in fluorescence mode with a 20x objective. For quantitative analysis of brain calcification, the RIS staining channel of the VS120 images was batch-converted to monochrome grayscale images in Tif format. Next, the number and area of ​​calcified nodules within four defined regions of interest (ROIs) were analyzed using the analyze particles function in ImageJ software. Each slice was registered to the corresponding midline-to-lateral coordinates based on the sagittal topological features of the adult mouse brain atlas. Statistical analysis of the number and area of ​​calcified nodules within each group was performed using GraphPad Prism.

[0686] 8.6 Statistics

[0687] All data are presented as mean ± standard deviation (SD). Figure legends indicate where mean ± SEM is used. Statistical analysis was performed using t-tests for comparisons between two groups, and one-way ANOVA for multiple groups. * indicates p < 0.01, ** indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001. For all tests, p < 0.05 (**) was considered statistically significant.

[0688] 8.7 Results

[0689] The results of the effect of astrocyte knockout of PiT2 on phosphorus homeostasis in the brain are shown in Figure 15. Among them, Figure A shows that the level of inorganic phosphate (Pi) in the cerebrospinal fluid (CSF) of PiT2 knockout mice increased significantly, which was about 1.8mM, while the CSF Pi concentration in the brain of WT mice was about 0.5mM. Figure B shows that there was no significant change in inorganic phosphate (Pi) in the serum of PiT2 knockout mice. Figure C shows the concentration of CSF Pi in the brain of PiT2 conditional knockout mice of different cell types, among which astrocyte conditional knockout (Aldh1L1-CreER T2 ) mice showed the most significant increase in CSF Pi (170%), reaching 1.35 mM. Other conditional knockout mice, including oligodendrocytes (Plp1-CreER T2 CSF Pi levels in mice expressing Camk2a-iCre, excitatory neurons (Camk2a-iCre), inhibitory neurons (Vgat-Cre), and perivascular cells (Pdgfrb-Cre) were only modestly increased (40-60%). Figure D shows no significant differences in serum Pi concentrations between the different conditional knockout mouse strains.

[0690] The results of intravenous injection of AAV virus-mediated selective expression of PiT2 in brain astrocytes are shown in Figure 16. Immunofluorescence analysis showed widespread expression of PiT2-V5 signals (red) in astrocytes throughout the mouse brain, with blue Hoechst marking the cell nuclei.

[0691] Figure 17 shows the inhibitory effect of selectively complementing AAV expression in astrocytes on calcification in the mouse brain. Coronal and sagittal microCT images of brain calcification in Pit2 KO mice at 5 and 7 months of age, which received the control treatment (Ctrl), showed significant, extensive calcification deposits in the dorsal midbrain, basal forebrain, thalamus, and hypothalamus. The calcifications in 7-month-old mice were larger and covered a larger area than those in 5-month-old mice. However, brain calcification in Pit2 KO mice receiving the therapeutic AAV injection (Replenished) was significantly suppressed at both 5 and 7 months of age.

[0692] 8.8 Discussion

[0693] Example 5-8 used in situ hybridization to analyze the expression characteristics of the homologous genes of specific hereditary brain calcification-related genes, especially PIT1, PIT2, XPR1, MYORG and other genes related to phosphorus transport metabolism, in the mouse brain. It was found that astrocytes showed the characteristics of selectively high expression of specific gene combinations, that is, astrocytes highly expressed Na-Pi co-transporters Pit2 (Slc20a2) and Xpr1, but Pit1 (Slc20a1) was not expressed in astrocytes, or only a very small number of cells expressed it. Moreover, in mice with conditional knockout of the Pit2 gene in astrocytes, a significant increase in the level of inorganic phosphate Pi in the cerebrospinal fluid was observed. Therefore, glial cells, especially astrocytes, have been confirmed to be the most critical cell type for phosphorus transport metabolism closely related to brain calcification and the regulation of phosphorus homeostasis in the brain.

[0694] Based on the aforementioned cellular mechanism, researchers employed intravenous AAV injection, a method currently available to achieve large-scale infection of specific cell types throughout the brain. After penetrating the blood-brain barrier, these viruses selectively expressed the protein product of the compensatory gene, human PIT2, in astrocytes within the mouse brain. Furthermore, they found that mice in which PIT2 expression was successfully compensated in the brain's astrocytes showed significantly suppressed brain calcification, demonstrating that restoring brain phosphate homeostasis is an effective strategy for inhibiting brain calcification and possesses significant clinical translational value.

[0695] Based on the world's first discovery of the important physiological mechanism of astrocyte-mediated regulation of phosphorus homeostasis in the brain, and the discovery in mouse models that compensation through astrocyte expression can effectively restore phosphorus homeostasis in the brain of Pit2 KO mice, and effectively inhibit the occurrence and further progression of brain calcification, it can be used as an innovative therapy for the intervention of brain calcification, bringing good news to the majority of PFBC patients.

Claims

1. Use of a brain phosphorus homeostasis promoter in the preparation of a medicament for preventing and / or treating brain calcification diseases.

2. The use according to claim 1, wherein The brain phosphorus homeostasis promoter is an agent capable of regulating alternative splicing in the intron of the SLC20A2 gene.

3. The use according to claim 2, wherein The alternative splicing includes cryptic exon inclusion, and the agent regulates (e.g., inhibits or enhances) the splicing of the cryptic exon from the pre-mRNA of the SLC20A2 gene; Preferably, the agent thereby modulates (eg, inhibits or enhances) the level of mature mRNA processed from the pre-mRNA, and / or modulates (eg, inhibits or enhances) the expression of the PiT2 protein.

4. The use according to any one of claims 2 to 3, wherein The alternative splicing includes alternative splicing in intron 2; Preferably, the alternative splicing in intron 2 is a cryptic exon insertion between exons 2 and 3; Preferably, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene; Preferably, the cryptic exon has a sequence as shown in SEQ ID NO:

2.

5. The use according to any one of claims 2 to 3, wherein The alternative splicing includes alternative splicing in intron 10; Preferably, the alternative splicing in intron 10 is a cryptic exon insertion between exons 10 and 11; Preferably, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene; Preferably, the cryptic exon has a sequence as shown in SEQ ID NO:

3.

6. The use according to any one of claims 2 to 5, wherein The agent specifically binds to a target region in the SLC20A2 gene or its pre-mRNA, wherein the target region is located in an intron region between two typical exon regions, and wherein the intron region contains the cryptic exon; Preferably, the target region is within a region from at most about 100 nucleotides upstream of the cryptic exon to at most about 100 nucleotides downstream of the cryptic exon; Preferably, the target region is selected from the region spanning the cryptic exon splice site, the cryptic exon a region within 100 nucleotides upstream of the cryptic exon, or a region within 100 nucleotides downstream of the cryptic exon; Preferably, the target region is selected from the region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, the region across the 5' splicing site, the region within the cryptic exon, the region across the 3' splicing site or the region downstream of the 3' splicing donor site (SD).

7. The use according to claim 6, wherein The target region is located in the region from 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 SEQ ID NO:

8.

8. The use according to claim 6, wherein The target region is located in the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its pre-mRNA; Preferably, the target region is located within the sequence shown in SEQ ID NO:

9.

9. The use according to any one of claims 2 to 8, wherein The agent is selected from antisense oligonucleotides (ASOs) or gene editing tools (e.g., CRISPR / Cas, base editors, or non-Cas protein-dependent RNA editing tools); Preferably, the reagent is antisense oligonucleotides (ASOs); Preferably, the agent is a DNA editor, such as CRISPR / Cas9; Preferably, the agent is an RNA editor, such as CRISPR / Cas13; Preferably, the reagent is a base editor Adenine / Cytosine base editor; Preferably, the reagent is a Cas protein-independent RNA editing tool.

10. The use according to claim 9, wherein The agent is an antisense oligonucleotide (ASO) that specifically binds to the target region in SLC20A2 pre-mRNA; Preferably, 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, consists of 18 to 25 consecutive nucleotides; Preferably, the ASO comprises modifications of backbone nucleotides and nucleotide linkages, modifications of ribose, modifications of ribose side chain bases, or a combination thereof; Preferably, the ASO specifically binds to a target region located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA; preferably, the ASO has at least 80% affinity with the nucleotide sequence in SEQ ID NO:

8. (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) complementarity; Preferably, the ASO comprises a nucleotide sequence as shown in any one 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; Preferably, the ASO specifically binds to a target region located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA; 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 within SEQ ID NO:9; Preferably, the ASO comprises the nucleotide sequence shown in any one 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 thereto.

11. The use according to claim 9, wherein The reagent is a gene editing tool, such as a DNA gene editing system or an RNA base editing tool; Preferably, the gene editing tool is a CRISPR / Cas system, which comprises a Cas protein and a guiding RNA (gRNA), wherein the gRNA is capable of binding to the target region in the SLC20A2 gene or its precursor mRNA; Preferably, the gRNA binds to the target region and thereby recruits the binding of the editor complex and exerts editing activity; Preferably, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 precursor mRNA; or within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA; Preferably, the gene editing tool mutates the splicing acceptor (SA) or splicing donor (SD) sequence of the cryptic exon in the genome or pre-mRNA sequence to destroy its SA or SD function, thereby inhibiting the integration of the cryptic exon in the mRNA; Preferably, the gene editing tool is a DNA base editor, which comprises (i) a fusion protein including a DNA base editing domain (e.g., a deaminase) and a DNA binding domain (e.g., a Cas protein, such as Cas9 or Cas12) and (ii) a guiding RNA (gRNA), wherein the gRNA is capable of binding to the target region in the SLC20A2 gene; Preferably, the gene editing tool is an RNA base editing tool; preferably, the RNA base editing tool includes: an RNA base editor based on the CRISPR / Cas system, or an editor that is not dependent on the CRISPR / Cas system, such as an oligonucleotide-based base editor, etc.

12. The use according to claim 1, wherein The brain phosphorus homeostasis promoter is an astrocyte phosphorus transport promoter.

13. The use according to claim 12, wherein The brain phosphorus homeostasis promoter is selected from the following group: (Z1) PiT2 protein, a nucleic acid molecule encoding PiT2 protein, an expression vector expressing PiT2 protein, a PiT2 promoter, or a combination thereof; (Z2) MYORG protein, a nucleic acid molecule encoding a MYORG protein, an expression vector expressing a MYORG protein, a MYORG promoter, or a combination thereof; (Z3) XRP1 protein, a nucleic acid molecule encoding XRP1 protein, an expression vector expressing XRP1 protein, an XRP1 promoter, or a combination thereof; (Z4) Any combination of the above Z1 to Z3.

14. The use according to claim 12 or 13, wherein The promoter includes small molecule compounds, gene editing reagents, small nucleic acid drugs (such as antisense nucleotide drugs), or a combination thereof.

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

16. The use according to any one of claims 12 to 14, wherein The astrocyte phosphorus transport promoter is a small nucleic acid drug, and the small nucleic acid drug is connected to a targeting molecule, and the targeting molecule has targeting property to astrocytes; Preferably, the small nucleic acid drug is ASO; Preferably, the small nucleic acid drug is completely or partially encapsulated in the targeting molecule, or conjugated with the targeting molecule; Preferably, the targeting molecule is selected from antibodies or antibody fragments thereof, polysaccharides, aptamers, or nanoparticles.

17. The use according to any one of claims 1 to 16, wherein The brain calcification diseases include primary familial brain calcification (PFBC), primary basal ganglia calcification (PBGC), or other types of brain calcification associated with defects in phosphorus homeostasis in the brain (such as upregulation of inorganic phosphate in cerebrospinal fluid).

18. A method for preventing and / or treating brain calcification diseases, comprising administering an effective amount of a brain phosphorus homeostasis promoter to a subject in need thereof; Preferably, the brain phosphorus homeostasis promoter is as defined in any one of claims 2 to 16; Preferably, the brain calcification disease includes primary basal ganglia calcification (PBGC), primary familial brain calcification (PFBC), or other types of brain calcification associated with defective phosphorus homeostasis in the brain (such as upregulation of inorganic phosphate in cerebrospinal fluid).

19. A kit for treating brain calcification diseases, wherein: The medicine kit comprises: (a) a first container, and a first detection reagent for detecting a genetic mutation located in the first container, wherein the first detection reagent is used to detect whether a genetic mutation exists in a gene selected from the group consisting of PiT2, MYORG, XRP1, or a combination thereof; and (b) a second container, and a pharmaceutical composition in the second container, the pharmaceutical composition comprising the astrocyte phosphate transport promoter as defined in any one of claims 13 to 16 and a pharmaceutically acceptable carrier and / or excipient; Optionally, the medicine kit may further include a third container, and a solution in the third container for assisting the dissolution or in vivo delivery of the drug in the second container.

20. A method for screening candidate drugs for preventing and / or treating brain calcification diseases, wherein: The method comprises the following steps: (a) in a test group, a test drug is added to the culture system of astrocytes or a cell line derived therefrom, and the phosphorus transport level (E1) and / or activity (A1) in the cells of the test group is measured; in a control group, no test drug is added to the culture system of the same cells, and the phosphorus transport level (E0) and / or activity (A0) in the cells of the control group is measured; Among them, if the phosphorus transport level (E1) and / or activity (A1) of the cells in the test group is significantly higher than that in the control group, it indicates that the test drug has a promoting effect on the expression and / or activity of the phosphorus transport level and is a candidate drug for preventing and / or treating brain calcification diseases.

21. Use of an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene in the preparation of a medicament for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject; preferably, the agent is as defined in any one of claims 3 to 11.

22. A method for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject, comprising administering to a subject in need thereof an effective amount of an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene; Preferably, the agent is as defined in any one of claims 3-11.

23. The use according to claim 21 or the method according to claim 22, wherein The PiT2 functional defect involves mutations in the SLC20A2 gene; Preferably, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation or a structural variation; Preferably, the mutation is located in an exon region, and the subject is heterozygous for the mutation in the exon region; Preferably, the mutation is located in the target region in an intron, and the subject may be heterozygous, homozygous or compound heterozygous for the intronic mutation located in the target region; Preferably, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 precursor mRNA, or within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA; Preferably, the PiT2 functional defect is SLC20A2 haploinsufficiency.

24. The use according to claim 21 or the method according to claim 22, wherein The diseases associated with PiT2 functional deficiency benefit from the regulation of functional PiT2 protein or RNA expression; Preferably, the disease is brain calcification, such as primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC); Preferably, the disease is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone diseases associated with imbalance of phosphorus homeostasis.

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

26. The reagent according to claim 25, wherein The reagent has the characteristics defined in any one of claims 9-10.

27. A pharmaceutical composition comprising the agent according to any one of claims 25 to 26 or a combination thereof, and a pharmaceutically acceptable carrier and / or excipient.

28. Use of the agent according to any one of claims 25 to 26 or the pharmaceutical composition according to claim 27 in the preparation of a medicament for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject; Preferably, the disease associated with PiT2 functional deficiency is brain calcification, such as primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC); Preferably, the disease associated with PiT2 functional defect is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone disease associated with phosphorus homeostasis imbalance; Preferably, the drug treats a disease by regulating the expression of PiT2 protein or functional RNA in cells of the subject.

29. A method for preventing and / or treating a disease associated with PiT2 functional deficiency in a subject, comprising administering the agent of any one of claims 25-26 or the pharmaceutical composition of claim 27 to a subject in need thereof; Preferably, the disease associated with PiT2 functional deficiency is brain calcification, such as primary familial brain calcification (PFBC) or primary basal ganglia calcification (PBGC); Preferably, the disease associated with PiT2 functional defect is a disease associated with abnormal Pi balance, such as chronic kidney disease, heart valve calcification, arterial vascular calcification, and / or bone diseases associated with phosphorus homeostasis imbalance.

30. The use according to claim 28 or the method according to claim 29, wherein The subject comprises a mutation in the SLC20A2 gene; Preferably, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation or a structural variation; Preferably, the mutation is located in an exon region, and the subject is heterozygous for the mutation in the exon region; Preferably, the mutation is located in the target region in an intron, and the subject may be heterozygous, homozygous or compound heterozygous for the intronic mutation located in the target region; Preferably, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 precursor mRNA, or within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 precursor mRNA; Preferably, the PiT2 functional defect is SLC20A2 haploinsufficiency.

31. A method for regulating the expression of PiT2 protein in a cell, comprising contacting the cell in vitro with an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene; Preferably, the agent capable of regulating alternative splicing in an intron of the SLC20A2 gene is as defined in any one of claims 2-11.

32. A method for regulating the expression of PiT2 protein in a cell, comprising contacting the agent of any one of claims 25-26 with the cell in vitro; Preferably, the cell comprises a mutation in the SLC20A2 gene; Preferably, the mutation is selected from a non-synonymous mutation, a nonsense mutation, a frameshift mutation, a splicing mutation or a structural variation; preferably, the mutation is located in an exon region, and the cell is heterozygous for the mutation in the exon region; preferably, the mutation is located in the target region in an intron, and the cell may be heterozygous, homozygous or compound heterozygous for the intronic mutation located in the target region.

33. A method for identifying an agent capable of modulating PiT2 expression in a cell, the method comprising: i) identifying an agent that targets a target region in the PiT2 gene or its pre-mRNA, wherein the target region is located in an intron region between two typical exon regions, and wherein the intron region contains a cryptic exon, and the target region is within a region from at most about 100 nucleotides upstream of the cryptic exon to at most about 100 nucleotides downstream of the cryptic exon; ii) delivering the agent identified in step i) to the cell; and iii) measuring the mRNA and protein expression levels of PiT2 in the cells of step ii); Preferably, the method further comprises: iv) 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 by Western blot; Preferably, the target region is selected from a region upstream of the 5' splicing acceptor site (SA) of the cryptic exon, a region spanning the 5' splicing site, a region within the cryptic exon, a region spanning the 3' splicing site, or a region downstream of the 3' splicing donor site (SD); Preferably, the cryptic exon is located in the region from c.289+901 to c.289+1035 of the SLC20A2 gene; preferably, the target region is located in the region from c.289+801 to c.289+1135 of the SLC20A2 gene or its precursor mRNA; preferably, the target region is located in the sequence shown in SEQ ID NO:8; Preferably, the cryptic exon is located in the region from c.1795-1703 to c.1795-1627 of the SLC20A2 gene; preferably, the target region is located in the region from c.1795-1803 to c.1795-1527 of the SLC20A2 gene or its precursor mRNA; preferably, the target region is located in the sequence shown in SEQ ID NO:

9.

34. A method for preparing a brain calcification model, wherein: The method comprises modifying the target genome so that the intron sequence between exon2 and exon3 of the endogenous SLC20A2 gene is completely or partially replaced by the intron genomic fragment corresponding to the human SLC20A2 gene, or the intron sequence between exon10 and exon11 of the endogenous SLC20A2 gene is completely or partially replaced by the intron genomic fragment corresponding to the human SLC20A2 gene, wherein the intron genomic fragment corresponding to the human SLC20A2 gene contains a mutation that can change the splicing of the SLC20A2 gene, thereby providing a brain calcification model; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene contains at least a cryptic exon; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11; Preferably, the mutation that can change the splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in any of the preceding claims; Preferably, the replacement method comprises: gene knockout, gene editing, RNA interference, epigenetic inhibition or any combination thereof; Preferably, the model is an animal model, such as a mammalian model, such as a rodent model (such as a mouse or rat model) or a non-human primate model; Preferably, the model is a mammalian cell model, for example, human epidermal fibroblasts or brain glial cells.

35. A genetically modified non-human animal, wherein the genome of the animal comprises: an intron sequence between exon2 and exon3 of an endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to a human SLC20A2 gene, or an intron sequence between exon10 and exon11 of an endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to a human SLC20A2 gene, wherein the intronic genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene; Preferably, the mutation that can change the splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in any of the preceding claims; Preferably, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; Preferably, the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO:11; Preferably, the non-human animal is a rodent, such as a mouse or a rat; Preferably, the non-human animal is a non-human primate; Preferably, the genetically modified non-human animal is an animal model for brain calcification.

36. A genetically modified cell, wherein the genome of the cell comprises: an intron sequence between exon2 and exon3 of an endogenous SLC20A2 gene is replaced by an intron genomic fragment corresponding to a human SLC20A2 gene, or an intron sequence between exon10 and exon11 of an endogenous SLC20A2 gene is replaced by an intron genomic fragment corresponding to a human SLC20A2 gene, wherein the intron genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that can alter the splicing of the SLC20A2 gene; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene contains at least a cryptic exon; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11; Preferably, the mutation that can change the splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in any of the preceding claims; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene is exogenous to the cell. Introduction; Preferably, the cell is a mammalian cell; Preferably, the cells are human epidermal fibroblasts or brain glial cells; Preferably, the genetically modified cells are a cell model of brain calcification.

37. Use of a genetically modified non-human animal or cell, wherein: The uses include: (i) for indicating and analyzing the onset stage of brain calcification, or for being used as a model for studying the target of brain phosphorus homeostasis imbalance or brain calcification; (ii) for being used as a model for screening candidate drugs or treatment plans for treating brain calcification or diseases related to brain phosphorus homeostasis imbalance; and / or (iii) for being used as a model for screening candidate drugs or treatment plans for diseases related to phosphorus homeostasis imbalance; Wherein, the genome of the genetically modified non-human animal or cell comprises: an intron sequence between exon2 and exon3 of the endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to the human SLC20A2 gene, or an intron sequence between exon10 and exon11 of the endogenous SLC20A2 gene is replaced by an intronic genomic fragment corresponding to the human SLC20A2 gene, wherein the intronic genomic fragment corresponding to the human SLC20A2 gene comprises a mutation that can change the splicing of the SLC20A2 gene; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene contains at least a cryptic exon; Preferably, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a region from about 100 nucleotides upstream of the cryptic exon to about 100 nucleotides downstream of the cryptic exon; for example, the intronic genomic fragment of the human SLC20A2 gene replaced between exon2 and exon3 comprises the sequence shown in SEQ ID NO: 10; or the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO: 11; Preferably, the mutation that alters splicing of the SLC20A2 gene causes or enhances alternative splicing in intron 2 or alternative splicing in intron 10 as defined in any one of the preceding claims.

38. A nucleic acid construct comprising a minigene fragment sequence of a human SLC20A2 gene, wherein the minigene fragment sequence comprises exons exon2 and exon3 and an intron sequence therebetween, or comprises exons exon10 and exon11 and an intron sequence therebetween, wherein the intron sequence comprises a mutation that can alter the splicing of the SLC20A2 gene; Preferably, the nucleic acid construct comprises a promoter operably linked to the minigene fragment sequence; Preferably, the mutation that can change the splicing of the SLC20A2 gene causes or enhances the alternative splicing in intron 2 or the alternative splicing in intron 10 as defined in any of the preceding claims; Preferably, the minigene fragment sequence comprises the sequence shown in SEQ ID NO: 4 or 5; Preferably, the downstream of the minigene fragment sequence contains a polyA signal sequence; Preferably, the nucleic acid construct further comprises a reporter gene, such as a fluorescent protein (such as GFP, mCherry) or luciferase.

39. A method for screening an agent capable of modulating SLC20A2 mRNA splicing, comprising: Providing (i) a cell comprising the nucleic acid construct of claim 38, (ii) the genetically modified non-human animal of claim 35 or the genetically modified cell of claim 36, or (iii) a brain calcification model obtained by the method of claim 34; contacting a test agent with any one of (i)-(iii); detecting alternative splicing of SLC20A2 mRNA; Preferably, the cell is from, for example, a mammal, such as a rodent (e.g., a mouse or a rat), a non-human primate or a human; Preferably, a test agent is selected that is capable of inhibiting alternative splicing (e.g., reducing the amount of expression product containing cryptic exon insertions), thereby increasing the production of functional PiT2 protein; Preferably, a test agent is selected that is capable of enhancing alternative splicing (e.g., increasing the amount of expression product containing cryptic exon insertions), thereby reducing the production of functional PiT2 protein; Preferably, the detection comprises analyzing the length of the SLC20A2 mRNA expression product; Preferably, the alternative splicing is alternative splicing in intron 2 as defined in any of the preceding claims; preferably, the detection comprises PCR analysis using primers spanning exons exon2 and exon3; preferably, an amplicon comprising a cryptic exon insertion of approximately 135 nt in length is generated; Preferably, the alternative splicing is alternative splicing in intron 10 as defined in any of the preceding claims; preferably, the detection comprises PCR analysis using primers spanning exons exon 10 and exon 11; preferably, an amplicon comprising a cryptic exon insertion of approximately 76 nt in length is generated; Preferably, the method comprises contacting a test agent with cells containing the nucleic acid construct in vitro and detecting alternative splicing of the nucleic acid construct; preferably, the detection comprises 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.

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