Therapy that modulates splicing and expression of SLC20A2 gene

Abstract

The present invention provides an agent capable of modulating variable splicing in the intron of the SLC20A2 gene, and its use for modulating the expression of functional PiT2 protein to prevent and / or treat disease. The invention further provides a brain calcification model based on variable splicing in the intron of the human SLC20A2 gene, a preparation method of the brain calcification model, and application of the model to indication and analysis of the incidence stage of brain calcification, or to a model for researching phosphorus homeostasis imbalance in brain or a target of brain calcification, or to screening of drugs.

Description

Technical Field

[0001] The present invention provides an agent capable of regulating alternative splicing in the intron of the SLC20A2 gene, and its use for regulating the expression of functional PiT2 protein to prevent and / or treat diseases. The present invention also provides a brain calcification model based on alternative splicing in the intron of the human SLC20A2 gene and a preparation method thereof, as well as the use of the model 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, or for screening drugs. Background Art

[0002] Brain calcification is a hallmark of neurodegenerative diseases, and it is estimated that approximately 20-30% of the elderly (aged >60 years) have brain calcification. Primary familial brain calcification (PFBC) is an aging-related neurogenetic disease characterized by bilateral calcification deposits, mainly in the basal ganglia, thalamus, cerebellum, and sometimes in the subcortical white matter. PFBC occurs between the ages of 20 and 60 years with an incidence of 0.2%-0.6%, and may affect hundreds of millions of people, half of whom may show a wide range of neurological or psychological symptoms, such as headaches, Parkinson's disease-like movement deficits, cognitive decline, anxiety, and depression. Currently, PFBC patients are only treated symptomatically in clinical practice, such as anticonvulsants, antipsychotics, antidepressants, and drugs to control Parkinson's disease-like symptoms. Bisphosphonates, a pyrophosphate analog used primarily in patients with osteoporosis, have been hypothesized to mitigate brain calcification by inhibiting the nucleation process mediated by hydroxyapatite crystals. However, oral bisphosphonates have been reported to show only mild and transient symptom improvement in a small proportion of PFBC patients, and no calcification-suppressing effect has been observed. Therefore, there is an unmet need for new therapies in PFBC patients.

[0003] Antisense oligonucleotides (ASOs) are single-stranded polymers of chemically modified nucleic acids that have demonstrated significant reversal of disease phenotypes in many neurological diseases. The most common function of ASOs is to silence gene expression by recruiting RNase-H-mediated RNA degradation, such as in amyotrophic lateral sclerosis, Angelman syndrome, and Huntington's disease. Another important role of ASOs is to regulate splicing by forming stable ASO-RNA duplexes, sterically blocking the binding of the splicing complex to cis-regulatory sequences, such as ASO drugs used to treat spinal muscular atrophy (SMA) and ASO agents targeting alternatively spliced ​​exons of target genes in Duchenne muscular atrophy (DMD). Recently, ASO-mediated splicing regulation has received increasing attention because it can effectively regulate the expression levels of disease-related genes in autosomal dominant haploids, with recent examples such as ASO agents that upregulate SCN1A expression in Dravet syndrome. In addition, the ability to deliver ASOs to the whole brain with high efficiency, target-directed specificity based on base sequence complementarity, limited toxicity, and precise dosing make ASOs a promising therapeutic approach in clinical trials.

[0004] However, in the field of PFBC treatment, there have been no reports on inhibiting brain calcification by regulating the splicing regulation of the SLC20A2 gene. Summary of the invention

[0005] Genetic studies have found that PFBC may be caused by autosomal heterozygous mutations in SLC20A2, PDGFRB, PDGFB, or XPR1, or autosomal biallelic mutations in MYORG, JAM2, or CMPK2; in particular, dominant heterozygous mutations in the SLC20A2 gene account for 61% of all reported PFBC cases, indicating that PFBC can be treated by gene therapy targeting the corresponding gene. However, the lack of understanding of the pathogenic mechanism of PFBC and the lack of effective delivery tools have largely hindered the development of gene editing therapy for PFBC.

[0006] The inventors of the present application performed whole genome sequencing (WGS) on 135 PFBC patients who were confirmed to have exon mutations or CNV negative by whole exome sequencing (WES), and found intronic variations of 5 SLC20A2 genes from 6 autosomal dominant PFBC families. Subsequently, minigene detection and patient-derived cell experiments determined that there were cryptic exon sequences in the intronic sequences of SLC20A2 pre-mRNA, and the above-mentioned intronic mutations increased the integration probability of these cryptic exon sequences during mRNA processing; because these cryptic exon sequences contain premature termination codons, the abnormally spliced ​​mRNA sequences cannot be translated to produce functional protein products PiT2. Therefore, the inventors of the present application discovered a mechanism of premature termination of PiT2 translation caused by previously unreported cryptic exon insertion in abnormal splicing of SLC20A2 mRNA, and this mechanism provides a new strategy for regulating mRNA splicing and expression of SLC20A2 gene.

[0007] Based on the above findings, the inventors of the present application were able to enhance the expression of normal SLC20A2 mRNA and PiT2 protein in cells containing SLC20A2 intronic mutations and fibroblasts from patients with SLC20A2 haploinsufficiency through ASO-mediated splicing regulation. Further, in a humanized SLC20A2 mouse model that mimics SLC20A2-deficient PFBC patients, ASO administered intracerebroventricularly (ICV) was able to reduce abnormal SLC20A2 transcripts and inhibit the occurrence and progression of brain calcification.

[0008] 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 associated with PiT2 functional deficiency (e.g., PFBC caused by SLC20A2 haploinsufficiency).

[0009] In addition, based on the discovered alternative splicing in the SLC20A2 intron, the inventors of the present application further provide a cell and animal model of brain calcification and a method for preparing the same, which can be used to indicate and analyze the onset stage of brain calcification, as a model for studying brain phosphorus homeostasis imbalance or disease targets of brain calcification, and for screening drugs.

[0010] I. Application of regulating alternative splicing in disease treatment

[0011] In a first aspect, 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.

[0012] One aspect of the present invention provides the use of an agent capable of regulating alternative splicing in an intron of the SLC20A2 gene for preventing and / or treating a disease associated with a PiT2 functional defect in a subject; or the use in preparing a medicament for preventing and / or treating a disease associated with a PiT2 functional defect in a subject. The present invention also provides a method for preventing and / or treating a disease associated with a PiT2 functional defect in a subject, the method 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. The PiT2 functional defect comprises a lack of expression or activity.

[0013] In certain embodiments, the alternative splicing includes a cryptic exon insertion, 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.

[0014] 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 abnormally spliced ​​mature SLC20A2 mRNA, and / or increases the production of functional PiT2 protein. 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 by the present invention compared to the level before treatment or after control treatment.

[0015] In certain embodiments, the agent promotes the insertion of the cryptic exon in mature SLC20A2 mRNA, inhibits the level of correctly spliced ​​mature SLC20A2SLC20A2 mRNA, and increases the level of abnormally spliced ​​mature SLC20A2 mRNA; wherein the cryptic exon sequence carries a premature termination codon (PTC), and the insertion of the cryptic exon leads to a reduction or inhibition of the production of functional PiT2 protein. In this article, the phrase "reducing the level of abnormally spliced ​​mature SLC20A2 mRNA" refers to the reduction of the level of abnormally spliced ​​mature SLC20A2 mRNA after treatment with the agent provided by the present invention compared to the level before treatment or after control treatment. The abnormally spliced ​​mature SLC20A2 mRNA includes a cryptic exon insertion between exons 2 and 3 or between exons 10 and 11 as defined herein.

[0016] Alternative splicing in intron 2

[0017] In certain embodiments, the alternative splicing comprises alternative splicing in intron 2.

[0018] 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.

[0019] In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene.

[0020] In certain embodiments, the c.289+1007C>G mutation of the SLC20A2 gene promotes alternative splicing in said intron 2.

[0021] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0022] 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 a 135 nt long cryptic exon sequence (SEQ ID NO: 2) found in intron 2 of the SLC20A2 gene being added to mature SLC20A2 mRNA.

[0023] Alternative splicing in intron 10

[0024] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10.

[0025] 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.

[0026] In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene.

[0027] In certain embodiments, the c.1795-1698A>G mutation of the SLC20A2 gene promotes alternative splicing in intron 10.

[0028] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0029] 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 a 76 nt long cryptic exon sequence (SEQ ID NO: 3) found in intron 10 of the SLC20A2 gene being added to mature SLC20A2 mRNA.

[0030] In certain embodiments, the alternative splicing includes alternative splicing in the 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.

[0031] Target area

[0032] In certain embodiments, the agent specifically binds to a target region in the SLC20A2 gene or a pre-mRNA thereof, the target region being located in an intron region between two typical exon regions, and wherein the intron region contains the cryptic exon.

[0033] 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 ) 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.

[0034] 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.

[0035] In certain embodiments, the target region is selected from a region upstream of the 5' splicing site (splicing acceptor site, SA) of the cryptic exon, a region across the 5' splicing site, a region within the cryptic exon, a region across the 3' splicing site, or a region downstream of the 3' splicing donor site (splicing donor site, SD).

[0036] 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.

[0037] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:8.

[0038] 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.

[0039] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:9.

[0040] 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 of 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 of 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Reagents

[0045] 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 the CRISPR / Cas system.

[0046] ASO

[0047] In certain embodiments, the agent is an antisense oligonucleotide (ASO), which specifically binds to the target region in 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.

[0048] In certain embodiments, the ASO consists of 10-30 (eg, 15-30, 15-25, 18-30, 18-25, 20-25) consecutive nucleotides; preferably, consists of 18-25 consecutive nucleotides.

[0049] In this article, the term ASO includes any form of oligomeric molecules containing nucleic acid bases that can complementarily hybridize with nucleic acid bases on the target mRNA. ASOs may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of the foregoing two or three. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or with modified backbones. ASO chemical modifications will be apparent to those skilled in the art.

[0050] 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), constrained ethyl (constrained ethyl, cEt), locked nucleic acid (LNA), peptide nucleic acid.

[0051] 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 required, for example, ASO may comprise a combination of 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)".

[0052] 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 2'MOE and locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0053] 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.

[0054] In certain embodiments, the ASO comprises a phosphorodiamidate linkage in combination with a sugar moiety comprising a morpholine ring (morpholino), such as a phosphorodiamidate morpholinooligomer (PMO).

[0055] 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.

[0056] In certain embodiments, the ASO has at least 80% complementarity with the nucleotide sequence within SEQ ID NO: 8. In this context, complementarity (the degree to which one polynucleotide is complementary to another polynucleotide) can be quantified in terms of 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 a 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 with a target region within a target nucleic acid sequence to which it is targeted. For example, 18 of the 20 nucleobases of an oligomeric compound are complementary to a target region, and an ASO that will thus specifically hybridize will exhibit 90% complementarity.

[0057] In certain embodiments, 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0058] In some embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0059] In other embodiments, the ASO comprises a nucleotide sequence as 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.

[0060] 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.

[0061] 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.

[0062] In certain embodiments, the ASO comprises a nucleotide sequence as 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0063] 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.

[0064] In other embodiments, the ASO comprises a nucleotide sequence as 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.

[0065] Gene editing tools

[0066] 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 having 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 in the mRNA.

[0067] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, RNA editing tools.

[0068] CRISPR / Cas system

[0069] In certain embodiments, the agent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), which is capable of binding to the target region in the SLC20A2 gene or its pre-mRNA.

[0070] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the Cas enzyme and exerts editing activity.

[0071] 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 in the mRNA.

[0072] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0073] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0074] DNA base editors

[0075] 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.

[0076] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0077] 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.

[0078] In certain embodiments, the Cas protein is a catalytically inactive Cas protein or a Cas nickase protein.

[0079] 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.

[0080] RNA editing tools

[0081] 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, editors that are not dependent on the CRISPR / Cas system, such as base editors based on oligonucleotides, and the like.

[0082] In certain embodiments, the RNA base editor based on the CRISPR / Cas system comprises (i) a fusion protein including an RNA base editing domain (e.g., RNA deaminase) and an RNA binding domain (e.g., Cas13) and (ii) a guiding RNA (gRNA), which 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.

[0083] 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 then recruits the binding of the RNA base editing protein and exerts base editing activity.

[0084] 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.

[0085] application

[0086] 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.

[0087] In certain embodiments, the mutation is located in an exonic region and the subject is heterozygous for the mutation in the exonic region.

[0088] 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 in an intron, and the subject may be heterozygous, homozygous, or compound heterozygous for an intronic mutation located in the target region.

[0089] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0090] In certain embodiments, the PiT2 functional deficiency is SLC20A2 haploinsufficiency.

[0091] In certain embodiments, the drug 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.

[0092] 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).

[0093] In certain embodiments, the disease associated with PiT2 functional defect 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.

[0094] 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.

[0095] In certain embodiments, the reagent is administered by suction, intranasal administration, intratracheal administration or oropharyngeal inhalation. The preparation suitable for inhalation administration can be prepared by mixing the required amount of active ingredient in a suitable solvent and then aseptically filtering. Generally, the preparation for inhalation administration is a sterile solution at 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 solubility of the active ingredient 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.

[0096] In certain embodiments, the agent can be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intraocular, intraperitoneal, intraventricular (e.g., by lumbar puncture) or topical administration. 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, antibacterial water, or phosphate buffered saline (PBS) and the like. Pharmaceutical compositions should remain stable under production and storage conditions, and should prevent the contamination of 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 and 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.

[0097] In certain embodiments, the agent is selected from ASO, and the pharmaceutical composition is preferably configured as a solution, an emulsion, a microemulsion, a foam, or a liposome-containing formulation (e.g., a cationic liposome or a non-cationic liposome). In certain embodiments, the pharmaceutical composition is preferably configured for intraventricular 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 for administration.

[0098] In another aspect, the present invention provides a method for regulating the expression of PiT2 protein 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.

[0099] 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.

[0100] II. Agents targeting the SLC20A2 target region and their applications

[0101] In a second aspect, the present invention relates to agents, methods and uses for targeting a target region in SLC20A2 to restore or enhance the function of the SLC20A2 gene product.

[0102] 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.

[0103] In certain embodiments, the cryptic exon is located within intron 2.

[0104] In certain embodiments, the cryptic exon is located at c.289+901 to c.289+1035 of the SLC20A2 gene or its pre-mRNA.

[0105] In certain embodiments, the c.289+1007C>G mutation of the SLC20A2 gene promotes a cryptic exon insertion in said intron 2.

[0106] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0107] 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.

[0108] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:8.

[0109] In certain embodiments, the cryptic exon is located within intron 10.

[0110] In certain embodiments, the cryptic exon is located at c.1795-1703 to c.1795-1627 of the SLC20A2 gene or its pre-mRNA.

[0111] In certain embodiments, the c.1795-1698A>G mutation of the SLC20A2 gene promotes a cryptic exon insertion in said intron 10.

[0112] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0113] 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.

[0114] In certain embodiments, the target region is located within the sequence shown in SEQ ID NO:9.

[0115] In certain embodiments, 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.

[0116] 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.

[0117] In certain embodiments, the target region is selected from a region upstream of the 5' splicing site (splicing acceptor site, SA) of the cryptic exon, a region across the 5' splicing site, a region within the cryptic exon, a region across the 3' splicing site, or a region downstream of the 3' splicing donor site (splicing donor site, SD).

[0118] 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 of 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 of 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.

[0119] 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 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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).

[0124] 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 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 Cas proteins.

[0125] ASO

[0126] 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.

[0127] In certain embodiments, the ASO consists of 10-30 (eg, 15-30, 15-25, 18-30, 18-25, 20-25) consecutive nucleotides; preferably, consists of 18-25 consecutive nucleotides.

[0128] In certain embodiments, the ASO comprises modification of backbone nucleotides and nucleotide linkages (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 not limited to: 2'-O-methoxyethyl (2'-MOE), morpholino, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), constrained ethyl (constrained ethyl, cEt), locked nucleic acid (LNA), peptide nucleic acid.

[0129] 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 required, for example, ASO may comprise a combination of 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)".

[0130] 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 2'MOE and locked nucleic acid (LNA). In certain embodiments, the ASO has a phosphorothioate-linked backbone.

[0131] 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.

[0132] In certain embodiments, the ASO comprises a phosphorodiamidate linkage in combination with a sugar moiety comprising a morpholine ring (morpholino), such as a phosphorodiamidate morpholinooligomer (PMO).

[0133] 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.

[0134] 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.

[0135] In certain embodiments, 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0136] In some embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0137] In other embodiments, the ASO comprises a nucleotide sequence as 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.

[0138] 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.

[0139] 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.

[0140] In certain embodiments, the ASO comprises a nucleotide sequence as 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 thereof. In certain embodiments, the ASO comprises a nucleotide sequence as shown 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.

[0141] 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.

[0142] In other embodiments, the ASO comprises a nucleotide sequence as 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.

[0143] Gene editing tools

[0144] 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 having 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 in the mRNA.

[0145] Exemplary gene editing tools include, but are not limited to, CRISPR / Cas systems, DNA base editors, RNA editing tools.

[0146] CRISPR / Cas system

[0147] In certain embodiments, the agent is a CRISPR / Cas system. The CRISPR / Cas system typically comprises a Cas protein and a guiding RNA (gRNA), which is capable of binding to the target region in the SLC20A2 gene or its pre-mRNA.

[0148] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the Cas enzyme and exerts editing activity.

[0149] 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 in the mRNA.

[0150] In certain embodiments, the target region is located within the region from c.289+801 to c.289+1135 of the SLC20A2 pre-mRNA.

[0151] In certain embodiments, the target region is located within the region from c.1795-1803 to c.1795-1527 of the SLC20A2 pre-mRNA.

[0152] DNA base editors

[0153] 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.

[0154] In certain embodiments, the gRNA binds to the target region and thereby recruits the binding of the fusion protein and exerts base editing activity.

[0155] 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.

[0156] In certain embodiments, the Cas protein is a catalytically inactive Cas protein or a Cas nickase protein.

[0157] 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.

[0158] RNA editing tools

[0159] 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, base editors based on oligonucleotides, and the like.

[0160] In certain embodiments, the RNA base editor based on the CRISPR / Cas system comprises (i) a fusion protein including an RNA base editing domain (e.g., RNA deaminase) and an RNA binding domain (e.g., Cas13) and (ii) a guiding RNA (gRNA), which 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.

[0161] 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 then recruits the binding of the RNA base editing protein and exerts base editing activity.

[0162] In certain embodiments, the RNA editing tool performs base mutations in 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 in the mRNA.

[0163] In another aspect, the present invention provides a pharmaceutical composition comprising the agent or a combination thereof as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0164] 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.

[0165] In certain embodiments, the pharmaceutical composition is administered by suction, intranasal administration, intratracheal administration or oropharyngeal inhalation. The preparation suitable for inhalation administration can be prepared by mixing the required amount of active ingredient in a suitable solvent and then aseptically filtering. 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 solubility of the active ingredient 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.

[0166] In certain embodiments, the pharmaceutical composition can be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intraocular, intraperitoneal, intraventricular (e.g., by lumbar puncture) or topical administration. 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, antibacterial water, or phosphate buffered saline (PBS) and the like. The pharmaceutical composition should remain stable under production and storage conditions, and should prevent the contamination of 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 and 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.

[0167] In certain embodiments, the agent is selected from ASO, and the pharmaceutical composition is preferably configured as a solution, an emulsion, a microemulsion, a foam, or a liposome-containing formulation (e.g., a cationic liposome or a non-cationic liposome). In certain embodiments, the pharmaceutical composition is preferably configured for intraventricular 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 for administration.

[0168] On the other hand, the present invention provides the use of the reagent or pharmaceutical composition as described above in preventing and / or treating diseases associated with PiT2 functional defects (e.g., lack of quantity or activity) in a subject, or in the preparation of a medicament for preventing and / or treating diseases associated with PiT2 functional defects (e.g., lack of quantity or activity) in a subject. The present invention also provides a method for preventing and / or treating a disease associated with PiT2 functional defects in a subject, the method comprising administering an effective amount of the reagent or pharmaceutical composition as described above to a subject in need thereof.

[0169] In certain embodiments, the disease associated with PiT2 functional defect is brain calcification, such as primary familial brain calcification (PFBC). In certain embodiments, 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. 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.

[0170] 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.

[0171] In certain embodiments, the mutation is located in an exonic region and the subject is heterozygous for the mutation in the exonic region.

[0172] 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 in an intron, and the subject may be heterozygous, homozygous, or compound heterozygous for an intronic mutation located in the target region.

[0173] In certain embodiments, the PiT2 functional deficiency is SLC20A2 haploinsufficiency.

[0174] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0175] 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.

[0176] 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 exon region and the cell is heterozygous for the mutation in the exon region. In certain embodiments, the mutation is an intron mutation, such as a mutation within intron 2 and / or intron 10. In certain embodiments, the mutation is located in the target region in an intron and the cell may be heterozygous, homozygous, or compound heterozygous for the intron mutation located in the target region.

[0177] III. Screening Method

[0178] In a third aspect, the present invention relates to a method for screening an agent that regulates PiT2 expression.

[0179] 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, 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).

[0180] In certain embodiments, the methods are performed in vitro.

[0181] 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. When the expression level is increased compared to the control group, it indicates that the agent is an activator. When the expression level is decreased compared to the control group, it indicates that the agent is an inhibitor.

[0182] In certain embodiments, the expression level of PiT2 refers to the expression level of functional PiT2, such as the mRNA level of the correctly spliced ​​product or the level of the protein encoded by it.

[0183] In certain embodiments, the expression level is measured by RT-PCR, qPCR, or by Western blot.

[0184] In certain embodiments, the target region is selected from a region upstream of the 5' splicing site (splicing acceptor site, SA) of the cryptic exon, a region across the 5' splicing site, a region within the cryptic exon, a region across the 3' splicing site, or a region downstream of the 3' splicing donor site (splicing donor site, SD).

[0185] 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. In certain embodiments, the target region is located within the region of 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.

[0186] 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. In certain embodiments, the target region is located within the region of 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.

[0187] In certain embodiments, the agent targets the target region in the SLC20A2 pre-mRNA. In certain embodiments, the agent is antisense oligonucleotides (ASOs). 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.

[0188] 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 a base editor Adenine / Cytosine base editor.

[0189] In a fourth aspect, the present invention relates to a method for screening for agents that modulate SLC20A2 mRNA splicing.

[0190] 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.

[0191] In certain embodiments, the nucleic acid construct comprises a promoter operably linked to the minigene fragment sequence.

[0192] 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.

[0193] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0194] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0195] 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.

[0196] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10.

[0197] 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.

[0198] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0199] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0200] 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.

[0201] In certain embodiments, the minigene fragment sequence comprises a polyA signal sequence downstream.

[0202] In certain embodiments, the nucleic acid construct further comprises a reporter gene, such as a fluorescent protein (eg, GFP, mCherry) or luciferase.

[0203] On the other hand, the present invention provides an expression vector comprising a nucleic acid construct as described above. In this article, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The 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, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phages or M13 phages and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (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.

[0204] On the other hand, the present invention provides a method for screening 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)-(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.

[0205] 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.

[0206] In certain embodiments, a test agent is selected that is capable of inhibiting alternative splicing (eg, reducing the amount of expression product comprising a cryptic exon insertion), thereby increasing the production of a functional PiT2 protein.

[0207] In certain embodiments, a test agent is selected that enhances alternative splicing (eg, increases the amount of expression product comprising a cryptic exon insertion), thereby reducing the production of functional PiT2 protein.

[0208] In certain embodiments, the detecting comprises analyzing the length of the SLC20A2 mRNA expression product.

[0209] 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.

[0210] In certain embodiments, the detection comprises PCR analysis using primers spanning exons exon2 and exon3. In certain embodiments, an amplicon comprising a cryptic exon insertion of about 135 nt in length is generated. In certain embodiments, the primers comprise a forward primer as shown in SEQ ID NO: 123 and a reverse primer as shown in SEQ ID NO: 124.

[0211] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10. 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. In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0212] In some embodiments, the detection comprises PCR analysis using primers spanning exons exon10 and exon11. In some embodiments, an amplicon comprising a cryptic exon insertion of about 76 nt in length is generated. In some embodiments, the primers comprise a forward primer as set forth in SEQ ID NO:127 and a reverse primer as set forth in SEQ ID NO:128.

[0213] In certain embodiments, the method includes contacting the test agent with a cell comprising the nucleic acid construct in vitro and detecting the alternative splicing of the nucleic acid construct. In certain embodiments, the method includes introducing (e.g., transfection) a nucleic acid construct as described above or a vector comprising the nucleic acid construct into a cell to provide a cell comprising the nucleic acid construct. In certain embodiments, the detection includes analyzing the length of the mRNA expression product of the nucleic acid construct as described above. In certain embodiments, the detection includes analyzing the expression of the reporter gene included in the nucleic acid construct. In certain embodiments, the expression of the reporter gene indicates alternative splicing. In certain embodiments, the expression of the reporter gene indicates correct splicing.

[0214] IV. Brain Calcification Model and Its Preparation

[0215] In a fifth aspect, the present invention relates to constructing a brain calcification model based on alternative splicing in introns of the SLC20A2 gene.

[0216] One aspect of the present invention provides a method for preparing a brain calcification model, wherein the method comprises modifying a target genome so that an intron sequence between exon2 and exon3 of its endogenous Slc20a2 gene is replaced by an intronic genomic fragment corresponding to a human SLC20A2 gene, or an intronic genomic fragment between exon10 and exon11 of its 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, thereby providing a brain calcification model. In certain embodiments, the target genome is homozygous or heterozygous for the replacement.

[0217] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a cryptic exon. In certain embodiments, 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.

[0218] 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.

[0219] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0220] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the human SLC20A2 gene.

[0221] 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.

[0222] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10.

[0223] 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.

[0224] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0225] In certain embodiments, the mutation comprises the c.1795-1698A>G mutation of the human SLC20A2 gene.

[0226] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO:11.

[0227] In certain embodiments, the donor sequence for replacement has the following structure: Exon2 (3'-patial)-intron-crpE2 (or crpE10)-intron-exon3 (5'-partial).

[0228] In certain embodiments, the donor sequence for replacement has the following structure: Exon10 (3'-partial)-intron-crpE2 (or crpE10)-intron-exon11 (5'-partial).

[0229] 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.

[0230] The method of replacing the intron sequence of the endogenous Slc20a2 gene with the intron sequence corresponding to the human SLC20A2 gene can be performed by various suitable genome editing techniques known in the art. In certain embodiments, the replacement method includes: gene knockout, gene editing, RNA interference, epigenetic inhibition or any combination thereof.

[0231] In certain embodiments, the gene knockout comprises: stem cell targeting technology based on homologous recombination, gene editing technology, chemical molecule mutagenesis genome mutation, transposon site-directed inactivation or any combination thereof.

[0232] 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.

[0233] In certain embodiments, the replacement method comprises introducing a targeting vector carrying an insert nucleic acid (e.g., an intronic genomic fragment sequence corresponding to the human SLC20A2 gene to be introduced) flanked by 5' and 3' homology arms into a 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 comprise a site-specific recombination site (e.g., loxP, Frt, etc.). In certain embodiments, the insert nucleic acid further comprises a reporter gene.

[0234] In certain embodiments, the model is a non-human animal model, and the target genome is the genome of a non-human animal.

[0235] 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.

[0236] 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; 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.

[0237] In some embodiments, the model is a cell model, and the target genome is the endogenous genome of the target cell. In some embodiments, the replaced intronic genome fragment corresponding to the human SLC20A2 gene (with or without mutation) is introduced exogenously relative to the target cell.

[0238] In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human epidermal fibroblast or a brain glial cell.

[0239] In certain embodiments, the method comprises: modifying the genome of the target cell so that the modified genome comprises the replacement in the endogenous Slc20a2 gene; thereby obtaining a cell model comprising the modified genome.

[0240] In another aspect, the present invention provides a genetically modified non-human animal, whose genome 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. In certain embodiments, the non-human animal is homozygous or heterozygous for the replacement.

[0241] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a cryptic exon. In certain embodiments, 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.

[0242] 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.

[0243] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0244] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0245] 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.

[0246] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10.

[0247] 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.

[0248] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0249] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0250] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO:11.

[0251] In certain embodiments, the non-human animal is a rodent, such as a mouse or a rat.

[0252] In certain embodiments, the non-human animal is a non-human primate.

[0253] In certain embodiments, the genetically modified non-human animal is an animal model for brain calcification.

[0254] In another aspect, the present invention provides a genetically modified cell, the genome of which comprises: the intron sequence between exon2 and exon3 of the endogenous SLC20A2 gene is 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 replaced by the intron genomic fragment corresponding to the 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. In certain embodiments, the cell is homozygous or heterozygous for the replacement.

[0255] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene at least comprises a cryptic exon. In certain embodiments, 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.

[0256] In certain embodiments, the intronic genomic fragment corresponding to the human SLC20A2 gene is introduced exogenously relative to the cell. "Exogenous introduction" refers to nucleic acids derived from outside the target cell. Exogenous nucleic acids can be artificially or recombinantly produced, or can be isolated from heterologous cells and introduced into the target cell.

[0257] 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.

[0258] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:2.

[0259] In certain embodiments, the mutation comprises a c.289+1007C>G mutation of the SLC20A2 gene.

[0260] 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.

[0261] In certain embodiments, the alternative splicing comprises alternative splicing in intron 10.

[0262] 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.

[0263] In certain embodiments, the cryptic exon has a sequence as shown in SEQ ID NO:3.

[0264] In certain embodiments, the mutation comprises a c.1795-1698A>G mutation of the SLC20A2 gene.

[0265] In certain embodiments, the intronic genomic fragment of the human SLC20A2 gene replaced between exon10 and exon11 comprises the sequence shown in SEQ ID NO:11.

[0266] In certain embodiments, the cell is a mammalian cell.

[0267] In certain embodiments, the cell is a human dermal fibroblast or a brain glial cell.

[0268] In certain embodiments, the genetically modified cell is a cell model for brain calcification.

[0269] V. Application of brain calcification model

[0270] In a sixth aspect, the present invention also relates to the application of the above-mentioned brain calcification model in disease research and drug screening.

[0271] One aspect of the present invention provides the 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.

[0272] On the other hand, 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.

[0273] In certain embodiments, the disease is a brain calcification disease.

[0274] In certain embodiments, the drug is a drug for preventing or treating brain calcification.

[0275] In certain embodiments, the drug is a drug that restores phosphorus homeostasis in the brain.

[0276] 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 regimens for diseases related to phosphorus homeostasis imbalance.

[0277] 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.

[0278] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the expression level of its encoded product PiT2.

[0279] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the function of its encoded product PiT2.

[0280] In certain embodiments, the agent inhibits alternative splicing of SLC20A2 mRNA and thereby increases the production of functional PiT2 protein.

[0281] In certain embodiments, the drug promotes alternative splicing of SLC20A2 mRNA and thereby inhibits the production of functional PiT2 protein.

[0282] On the other hand, 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 a genetically modified cell; 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 (such as brain glial cells, such as astrocytes), and / or (iii) reduce the level of inorganic phosphate in cerebrospinal fluid.

[0283] In certain embodiments, the disease is a brain calcification disease.

[0284] In certain embodiments, the drug is a drug for preventing or treating brain calcification.

[0285] In certain embodiments, the drug is a drug that restores phosphorus homeostasis in the brain.

[0286] In certain embodiments, the candidate drug or treatment regimen can increase the expression of the full-length mRNA transcript of SLC20A2 and / or its encoded protein product PiT2, for example, up-regulating 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 of the correctly spliced ​​mRNA transcript of SLC20A2 and / or its encoded functional PiT2 protein product, for example, up-regulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0287] 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.

[0288] 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 or 25%, for example, to a level close to a normal reference value (e.g., a healthy control).

[0289] On the other hand, the present invention provides a method for screening candidate drugs or treatment regimens for treating diseases related to 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 a genetically modified cell; 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 (such as brain glial cells, such as astrocytes), and / or (iii) reduce the level of inorganic phosphate in cerebrospinal fluid.

[0290] 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.

[0291] In certain embodiments, the candidate drug or treatment regimen can increase the expression of the full-length mRNA transcript of SLC20A2 and / or its encoded protein product PiT2, for example, up-regulating 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 of the correctly spliced ​​mRNA transcript of SLC20A2 and / or its encoded functional PiT2 protein product, for example, up-regulating by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, etc.

[0292] 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.

[0293] 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 or 25%, for example, to a level close to a normal reference value (e.g., a healthy control).

[0294] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the expression level of its encoded product PiT2.

[0295] In certain embodiments, the drug is a drug that regulates SLC20A2 mRNA splicing and thereby regulates the function of its encoded product PiT2.

[0296] In certain embodiments, the agent inhibits alternative splicing of SLC20A2 mRNA and thereby increases the production of functional PiT2 protein.

[0297] In certain embodiments, the drug promotes alternative splicing of SLC20A2 mRNA and thereby inhibits the production of functional PiT2 protein.

[0298] definition

[0299] 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. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0300] 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), which encodes a 652 amino acid phosphate transporter, called 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. In this article, when it comes to describing the position of the mRNA sequence of the SLC20A2 gene, for example, to describe the position of the cryptic exon, the sequence shown in NCBI: NM_006749 is used as a reference.

[0301] As used herein, the expression "modulation of splicing" refers to changes that affect the level of any RNA or mRNA variant produced by the SLC20A2 natural precursor mRNA. For example, modulation can mean, for example, causing an increase or decrease in the level of abnormal SLC20A2 mRNA containing a cryptic exon insertion, causing an increase or decrease in the level of normal full-length SLC20A2 mRNA, and / or causing an increase or decrease in the level of abnormal SLC20A2 RNA or mRNA containing a premature stop codon (nonsense codon). It is readily understood that any change in the ratio between SLC20A2 splicing variants is also considered to be 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 reducing the level of abnormal (e.g., containing a cryptic exon insertion) SLC20A2 mRNA.

[0302] 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 usually formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can be base-paired 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 RNA is used instead of DNA, the base that is considered to be complementary to adenosine is uracil instead of thymine. However, when U is represented in the context of the present invention, unless otherwise stated, the ability to replace T is implied. "At least partially complementary" means 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 hybridized nucleotides. 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 test system, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, can also be applied.

[0303] 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 the 5' end, and the left direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right end or right direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3' end or 3' direction. Generally speaking, the region or sequence at the 5' of a reference point in a nucleic acid is referred to as "upstream", and the region or sequence at the 3' of a reference point in a nucleic acid is referred to as "downstream". Generally speaking, the 5' direction or end of an mRNA is the position where the 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 the reference point in a nucleic acid can be represented by a negative number, while the nucleotides downstream of the 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 a "zero" position, and the nucleotide immediately adjacent to and upstream of the reference point is represented as "minus one," e.g., "-1," while the nucleotide immediately adjacent to and downstream of the reference point is represented as "plus one," e.g., "+1."

[0304] 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 a subject and an active ingredient, which is well known in the art. "Pharmaceutically acceptable carrier and / or excipient" does not play or is not intended to play a therapeutic effect at the expected dose. Such ingredients can play the following roles: a) assisting the processing of the drug delivery system during manufacturing, b) protecting, supporting or enhancing the stability, bioavailability or patient acceptability of the active ingredient, c) assisting product identification, and / or d) enhancing the overall safety, effectiveness, delivery, etc. of the active ingredient during storage and use. The role of any other attributes. For example, the siRNA of the present invention can be wrapped by a delivery carrier. "Pharmaceutically acceptable carrier and / or excipient" includes, but is not limited to, a pH regulator, a surfactant, an adjuvant, an ionic strength enhancer, a diluent, an agent that maintains osmotic pressure, an agent that delays absorption, and a preservative. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. 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 (such as buffered saline), alcohols, and polyols (such as glycerol), and the like.

[0305] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is 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" may also refer to extending survival compared to the expected survival (if not receiving treatment).

[0306] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient who has already suffered from the disease, such as an amount necessary to improve at least one clinical parameter of the disease suffered by the patient or reduce the severity of at least one clinical parameter of the disease suffered by the patient. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount 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 simultaneously, etc.

[0307] Beneficial effects and summary of the invention

[0308] The present invention first discovered and identified a previously unreported cryptic exon insertion in the abnormal splicing of SLC20A2 mRNA, and thus invented a reagent and method for regulating the abnormal splicing. In human cells of brain calcification and SLC20A2-KI mouse models, regulating the abnormal splicing can upregulate the expression of the SLC20A2 gene product PiT2 protein, reverse the increase in CSF Pi levels, and thus inhibit the progression of brain calcification.

[0309] In the embodiments provided by the present invention, the method for regulating the abnormal splicing of SLC20A2 and thus upregulating the expression of the SLC20A2 gene product PiT2 protein can be mainly achieved through the action of antisense oligonucleotides (ASOs). However, based on the method logic and implementation path described in the present invention, those skilled in the art will understand that the present invention can also be achieved by permanently changing the mRNA splicing mode of SLC20A2 at the genome level through DNA base editing, or by changing the mRNA splicing mode of SLC20A2 at the RNA level through RNA base editing.

[0310] 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.

[0311] 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 the 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 that account for a larger proportion of the population.

[0312] Based on this, the present invention provides a new strategy for treating SLC20A2 haploinsufficiency based on regulating the mRNA splicing of the SLC20A2 gene, which has important clinical value for the treatment of brain calcification or diseases related to phosphorus homeostasis imbalance.

[0313] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0314] Figure 1 : Intronic variants of SLC20A2 detected by WGS in a PFBC family.

[0315] a, Kinship of six PFBC families with SLC20A2 intronic mutations. Solid symbols, hollow symbols, gray symbols, or slashed symbols represent individuals affected by brain calcification, individuals without brain calcification, individuals who have not yet developed symptoms, and individuals who have died, respectively. Symbols with question marks (?) represent individuals without brain CT imaging data. "#" and "&" represent individuals selected for whole exome sequencing (WES) or whole genome sequencing (WGS), respectively. b, Representative brain CT images of patients in six PFBC families. c, Distribution of five intronic mutations in the SLC20A2 locus. The light purple box is an exon, and the number below it is the exon number. The line in the middle of the exon marks the intron. 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 work by affecting potential cryptic exons; while the mutations in italic non-bold font may produce mutations in splice acceptors or donors.

[0316] Figure 2 :The cryptic exon of SLC20A2 alters the normal splicing of SLC20A2 mRNA and the expression of PiT2 protein due to intronic mutation.

[0317] ab, Schematic diagram of minigene construction and transcription products of SLC20A2 intron mutations c.289+937G>A and c.289+1007C>G (a). (b) is the PCR detection of minigenes to detect changes in splicing products after intron mutations. RT-PCR analysis of Neuro-2A cell samples transfected with SLC20A2 minigenes carrying c.289+937G>A, c.289+1007C>G, and c.289+1021G>C mutations showed that the lower band represents the correctly spliced ​​exon, while the upper band represents the transcript PCR product with 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 intron 10 of human SLC20A2 (c) and PCR analysis of the transcripts (d). The lower band represents the correctly spliced ​​transcript, while the upper band represents the PCR product of the transcript with a cryptic exon inserted between exons 10 and 11 of SLC20A2. e, Schematic diagram of the SLC20A2 CDS construct with two intronic mutations causing aberrant splicing (each with an N-terminal HA tag). f, After transfection of Neuro-2A cells with the HA-tagged SLC20A2 CDS construct, the effect of endogenous mutations of SLC20A2 on the PiT2 protein product was detected by Western blotting (WB). gh, Immunoblotting results of SLC20A2 gene product PiT2 in fibroblast extracts from PFBC patients carrying c.289+1007C>G mutation (P1), heterozygous deletion of SLC20A2 gene (P2) or healthy controls (C1 and C2) showed that intronic mutations led to decreased expression of PiT2, similar to exonic mutations. (h) is the statistical result.

[0318] Figure 3 : Cryptic exons crpE2 and crpE10 are present in SLC20A2 transcripts produced by cells from human brain.

[0319] a, RT-PCR analysis of the presence of cryptic exon insertion events between adjacent exons of the SLC20A2 gene in HEK293T and A172 cells. CHX, cyclohexylamine (a widely used compound that can inhibit the degradation of abnormal transcripts). In general, the bottom band represents normal transcripts; the upper band represents abnormal transcripts containing certain cryptic exons (marked with asterisks, named crpE2 and crpE10). b, Sanger sequencing of the upper bands of cryptic exons (crpE2 and crpE10) in HEK293T and A172 cells in Figure a showed that their sequences were consistent with Figure 2 The cryptic exons found in the minigene experiment have the same sequence, indicating that these cryptic exons are endogenous, but mutations significantly increase their probability of integration into mRNA. c, RT-PCR products of crpE2 and crpE10 from human fibroblasts of human cell lines including HEK293T, HeLa, HepG2, A172, and healthy controls (C1, C2); a PFBC patient with a heterozygous c.289+1007C>G mutation in SLC20A2 (P1); and a PFBC patient with a heterozygous deletion of SLC20A2 (P2).

[0320] Figure 4 : ASO inhibits the integration of the cryptic exon of SLC20A2 in the minigene

[0321] a, Schematic diagram of the cryptic exon carrying the c.289+1007C>G mutation on the SLC20A2 genomic sequence targeted by PMO-modified ASO (PMO-1, PMO-2, PMO-3) and MOE-modified ASO (MOE-A, MOE-B, MOE-C) minigene. b, c, RT-PCR analysis and statistical results of crpE2 cryptic exon integration in Neuro-2A cells transfected with SLC20A2 c.289+1007C>G minigene, treated with PMO-modified ASO (PMO-1, PMO-2, PMO-3) (b), or MOE-modified ASO (MOE-A, MOE-B, MOE-C) (c) compared with control ASO (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 results of crpE10 cryptic exon integration in Neuro-2A cells transfected with SLC20A2 c.1795-1698A>G mutation, 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). The upper band is the transcript with crpE10 inserted (261 bp), and the lower band is the normal transcript (185 bp). The red line represents MOE-modified ASO; the blue line represents PMO-modified ASO. The quantification value (%) of the splicing events with cryptic exon integration was calculated based on the intensity of the RT-PCR band as follows: (transcripts with cryptic exon integration / (transcripts with cryptic exon integration + normal transcripts)) × 100. The relative PiT2 protein was evaluated using Tubulin as an internal reference (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0322] Figure 5 :ASO inhibits the integration of cryptic exons of SLC20A2 mRNA caused by intronic mutations and upregulates PiT2 protein expression

[0323] a, Schematic diagram of the ability of MOE-modified ASOs (MOE-A, MOE-B, MOE-C) to target cryptic exons on pre-mRNAs transcribed from SLC20A2 genes on chromosomes carrying the c.289+1007C>G intron mutation and normal chromosomes without the mutation after culturing fibroblasts from a PFBC patient (P1) carrying the SLC20A2 c.289+1007C>G intron mutation, thereby inhibiting abnormal splicing of SLC20A2. b, RNA was extracted from fibroblasts from a PFBC patient (P1) carrying the SLC20A2 c.289+1007C>G intron mutation after being stably cultured and treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or control MOE-NC), and RT-PCR detection and statistical analysis of the integration of crpE2 cryptic exons into SLC20A2 mRNA levels were performed. c, Western blot results and statistical analysis of PiT2 protein levels in P1-derived fibroblasts after MOE-modified ASOs (MOE-A, MOE-B, MOE-C) intervention compared with MOE-NC. The red line represents the MOE-modified ASO. The quantification value (%) of splicing events integrating cryptic exons was calculated based on the intensity of RT-PCR bands as follows: (transcripts integrating cryptic exons / (transcripts integrating cryptic exons + normal transcripts)) × 100. The relative PiT2 protein was evaluated using Tubulin as an internal reference (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0324] Figure 6 : ASO can also increase PiT2 expression by regulating the splicing events involving the cryptic exon of SLC20A2 in normal human fibroblasts.

[0325] a, Schematic diagram of the use of MOE-modified ASOs (targeting crpE2: MOE-A, MOE-B, MOE-C; targeting crpE10: MOE-J, MOE-K, MOE-L) to regulate the splicing events of the cryptic exon crpE2 or crpE10 on the pre-mRNA transcribed from the SLC20A2 gene on the normal chromosome after culture of human fibroblasts from healthy controls, thereby increasing the expression of SLC20A2 mRNA and PiT2 protein. b, RT-PCR detection and statistical analysis of the expression level of SLC20A2 mRNA transcripts inserted into the cryptic exon of crpE2 after human fibroblasts from healthy controls (C1) were treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or control group MOE-NC). The upper band is the transcript inserted into crpE2 (699 bp), and the lower band is the normal transcript (564 bp). c, Western blot results 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 with MOE-NC. d, RT-PCR detection and statistical analysis of the expression level of SLC20A2 mRNA transcripts inserted into the crpE2 cryptic exon in human fibroblasts from healthy controls (C2) treated with MOE-modified ASOs (MOE-A, MOE-B, MOE-C, or control group MOE-NC). The upper band is the transcript inserted into crpE2 (699 bp), and the lower band is the normal transcript (564 bp). e, Western blot results 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 with MOE-NC. f, RT-PCR detection and statistical analysis of the expression level of SLC20A2 mRNA transcripts inserted into the crpE10 cryptic exon in human fibroblasts from healthy control (C1) treated with MOE-modified ASOs (MOE-G, MOE-H, MOE-I) compared with MOE-NC. The upper band is the transcript inserted into crpE10 (199 bp), and the lower band is the normal transcript (123 bp). g, PiT2 protein levels in human fibroblasts from healthy control C1 after intervention with MOE-modified ASOs (MOE-G, MOE-H, MOE-I) compared with MOE-NC. The quantification value (%) of splicing events integrating the cryptic exon was calculated based on the intensity of the RT-PCR bands as follows: (transcripts integrating the cryptic exon / (transcripts integrating the cryptic exon + normal transcript)) × 100.The relative PiT2 protein was evaluated using Tubulin as an internal reference (i.e., PiT2 / Tubulin). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t-test.

[0326] Figure 7 : Expanded screening of ASO sequences that modulate SLC20A2 splicing

[0327] a, Minigenes were transfected into N2A cells, and the responses of the minigene-expressed splicing products to ASOs were analyzed by PCR, including new ASO sequences targeting crpE2 splicing regulation (cr5E2-m1,

[0328] cr5E2-5,cr5E2-6,cr5E2-8,5crE2-9,cr5E2-10,cr5E2-12,5crE2-13,

[0329] cr5E2-13L,cr5E2-13S,cr5E2-14,cr5E2-16,cr5E2-17,cr5E2-18,cr5E2-19,

[0330] cr5E2-20,cr5E2-21,cr5E2-22,cr5E2-23,cr5E2-24,cr3E2-5,cr3E2-8,

[0331] cr3E2-9,cr3E2-10,cr3E2-11,cr3E2-12,cr3E2-13,cr3E2-14,cr3E2-15,

[0332] 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, cr3E10-14, cr3E10-15), their sequences and related information are listed in the attached table. b, The ASO sequences screened by minigene were further screened on healthy human fibroblasts. The splicing regulatory effects of ASOs on endogenously expressed SLC20A2 were analyzed and evaluated by repeated PCR and protein WB detection. The experimental results of only some sequences are listed above. c, After comprehensive analysis of two rounds of screening, several ASO sequences with good effects were obtained.

[0333] Figure 8:ASO regulates SLC20A2 mRNA splicing and upregulates PiT2 protein expression in patients with heterozygous SLC20A2 deletion

[0334] a. Schematic diagram of the splicing event of the cryptic exon crpE2 in the pre-mRNA transcribed from the SLC20A2 gene on another normal chromosome can be targeted by MOE-modified ASO (MOE-A, MOE-B, MOE-C) after culturing fibroblasts from a patient (P2) carrying a large heterozygous deletion of the SLC20A2 genome, thereby increasing the expression of SLC20A2 mRNA and PiT2 protein. b. RNA was extracted from fibroblasts from a patient (P2) carrying a large heterozygous deletion of the SLC20A2 genome after stable culture and treated with MOE-modified ASO (MOE-A, MOE-B, MOE-C) or control MOE-NC, and RT-PCR detection and statistical analysis of the integration of the cryptic exon of crpE2 into the SLC20A2 mRNA level were performed. c, Western blot results and statistical analysis of PiT2 protein levels in P2 fibroblasts after intervention with MOE-modified ASOs (MOE-A, MOE-B, MOE-C) compared with MOE-NC. The red line represents the MOE-modified ASO. The quantification value (%) of splicing events integrating cryptic exons was calculated based on the intensity of the RT-PCR bands as follows: (transcripts integrating cryptic exons / (transcripts integrating cryptic exons + normal transcripts)) × 100. The relative PiT2 protein was evaluated using Tubulin as an internal reference (i.e., PiT2 / Tubulin). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test.

[0335] Fig. 9 : Construction of humanized SLC20A2 intron knock-in (SLC20A2-KI) model mice and analysis of SLC20A2 expression.

[0336] a, Schematic diagram of the construction of the SLC20A2-KI mouse model with humanized SLC20A2 gene intron 2. CRISPR / Cas9 was used to create an incision at the genomic SLC20A2 intron 2 position, and then the introduced targeted donor sequence was used to guide homologous recombination-based repair, thereby replacing the mouse SLC20A2 intron 2 and the sequence of the bridging part of exon 2 and exon 3 on both sides with the sequence of human SLC20A2 intron 2 carrying the c.289+1007C>G mutation. The transcripts generated by the final sequence matched the overall reading frame of mouse Slc20a2. b, RT-PCR analysis of the expression level of SLC20A2 transcripts integrated with humanized crpE2 in the brain tissues of heterozygous and homozygous SLC20A2-KI mice and wild-type mice. The upper band represents the abnormal transcript (crpE2) produced by the humanized intron 2 with the 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 of SLC20A2 transcript levels (c) and western blot results of PiT2 levels (d) in the brains of WT, HET, and HOM SLC20A2-KI mice showed that humanized mutant mice had similar defects in Slc20a2 mRNA and PiT2 protein expression as Slc20a2 KO mice. e, Compared with wild-type mice, CSF Pi levels in heterozygous and homozygous SLC20A2-KI mice showed a stepwise increase, similar to Slc20a2 KO mice.

[0337] Fig.10 : Micro-CT and RIS staining analysis of brain calcification in humanized SLC20A2 (SLC20A2-KI) model mice.

[0338] a, Calcification of the brain of homozygous SLC20A2-KI mice at 5, 7, and 9.5 months of age after PFA perfusion fixation and micro-CT scanning (voxel resolution: 9um x 9um x 9um). From left to right, the micro-CT images are 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 sections of homozygous SLC20A2-KI mice aged 5, 7, and 9.5 months were stained with the fluorescent dye RIS-AF647 (an imaging reagent that carries the AlexaFluo-647nm fluorescent group and can bind to bisphosphonate calcium salts), and brain calcification images were collected using a VS120 microscope. The images are representative of the analysis results of three mice in each group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t-test. Scale bar, 1 mm.

[0339] Fig.11 : Histochemical staining analysis of brain calcification in humanized SLC20A2 (SLC20A2-KI) model mice.

[0340] Homozygous SLC20A2-KI mice at 7 months (a), 10 months (b), and 12 months (c) were fixed and frozen in OCT sections. Five different histochemical stains were performed, including alizarin red, Von Kossa, Alcian blue, H&E, and PAS. Alizarin red and Von Kossa mainly show calcification deposits, while Alcian blue and PAS mainly show the mucopolysaccharide components attached to the calcification foci, which is a common feature of calcification foci. Scale bar, 500μm.

[0341] Fig.12 :ASO intraventricular intervention restored the expression of SLC20A2 in the brain of SLC20A2-KI mice and downregulated the phosphate level in cerebrospinal fluid.

[0342] a, Experimental design for intracerebroventricular (ICV) administration of ASOs 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. The dose of each MOEs was 50 μg, 100 μg, 200 μg, or 400 μg. Brain tissues were collected from 7-month-old mice and analyzed for crypticexon inclusion of SLC20A2 transcripts, PiT2 protein levels, cerebrospinal fluid phosphate levels, and brain calcification. b, In situ hybridization analysis of MOE-A distribution throughout the mouse brain (cortex, hippocampus, thalamus, and cerebellum) was performed 2 months after ICV injection in 5-month-old SLC20A2-KI mice. RNA probes were designed to bind specifically to the sequence of MOE-A. MOE-A was stained in red and DAPI was stained in blue. c, Expression of normal SLC20A2 mRNA transcripts in SLC20A2-KI mice 4 months after MOE-NC, MOE-A, or MOE-B administration compared with age-matched WT mice and untreated homozygous SLC20A2-KI mice by quantitative PCR analysis. d, RT-PCR analysis of transcripts of crpE2 with cryptic exon insertion and normal SLC20A2 transcripts 4 months after ICV injection of MOE-NC, MOE-A, and MOE-B. The top band represents the transcript of crpE2 with cryptic exon insertion (crpE2), while the bottom band represents the normal SLC20A2 transcript. e, PiT2 protein levels in the thalamus of SLC20A2-KI mice 4 months after ICV injection of MOE-A, showing a significant restoration of expression compared with control MOE and untreated mice, indicating that the effect of ASO is effective. 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.

[0343] Fig.13 :ASO intraventricular intervention in SLC20A2-KI mice significantly inhibits the occurrence and progression of brain calcification

[0344] 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, 400 μg) of MOE-NC, MOE-A, and MOE-B; white highlights are 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, 400 μg) of MOE-NC, MOE-A, and MOE-B, i.e., at 7 months of age. Images are representative of the analysis 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.

[0345] Fig.14 : ICV injection of ASO drugs can curb the further deterioration of brain calcification in SLC20A2-KI mice that already have brain calcification.

[0346] a, Experimental design of ICV injection of PMO-modified ASO in homozygous SLC20A2-KI mice. Mice were treated with ICV injection of PMO-NC or PMO-1 at 5 months of age, which showed obvious calcification deposits in multiple regions throughout the brain. The dose of each PMO was 25 μg or 75 μg. Brain tissue was collected from 7-month-old mice and analyzed for cryptic exon inclusion and brain calcification. CSF was extracted before and at the end of PMO treatment (i.e., 5 and 7 months) for analysis. b, Changes in CSF Pi levels in homozygous SLC20A2-KI mice before and after PMO-NC and PMO-1 administration. c, Sagittal micro-CT images of brain calcification in 7-month-old homozygous SLC20A2-KI mice treated with control and therapeutic ASOs at 25 μg and 75 μg ICV doses. White highlights are brain calcified nodules. Images are representative of the analysis of two to three mice per group. ****P<0.0001, Student's t-test. Scale bar, 1 mm. Example

[0347] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0348] 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 the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can generally be conventional products obtained commercially.

[0349] Materials and Methods

[0350] 1. Subject recruitment, whole genome sequencing, and genetic analysis

[0351] We recruited 778 patients suspected of PFBC from the Department of Neurology, the First Affiliated Hospital of Fujian Medical University between 2012 and 2023. The diagnostic criteria for PFBC were as follows: a) calcifications detected on CT scan were bilateral and symmetrical in the basal ganglia and / or dentate nucleus; b) the total calcification score (TCS) exceeded the age-specific thresholds previously reported; c) there were no biochemical abnormalities, and serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations were normal; d) there were no infectious, toxic, or traumatic causes. Neurological 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 containing mutations in the entire coding region and exonic sequence segments of known PFBC-associated genes such as SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, JAM2, and CMPK2, and then identified PFBC-associated intronic mutation cases and families 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.

[0352] For whole-genome sequencing, after obtaining genomic DNA from the blood of patients with brain calcification, human DNA samples were used to prepare paired-end DNA libraries (Illumina Truseq library construction) according to the instructions of the relevant sequencing library construction kit manufacturer. The constructed DNA library was subjected to paired-end 150-bp length second-generation sequencing using Illumina NovaSeq 6000. The average sequencing depth ranged from 31.35 to 57.77, and 90.1% to 99.2% of the whole genome was covered with at least 20 sequences. Then, the sequencing results (without barcodes) were aligned to the human reference genome hg19 using SpeedSeq. Next, single nucleotide variants and insertions / deletions (Indels) were called using the GenomeAnalysis Toolkit v4.1. Potential genomic structural variants and copy number variants in the patient's genome were extracted by SpeedSeq; or single nucleotide variants, indels, structural variants, and copy number variants were annotated using ANNOVAR.

[0353] For parametric linkage analysis, DNA samples were isolated from whole blood of 45 family members of Family 1, and whole-genome linkage analysis was performed using HumanOmniZhongHua-8 BeadChip (Illumina). Genotype calling and quality control were processed using the GenomeStudio Genotyping Module (Illumina). SNPs with Mendelian errors or calling rates less than 100% were 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.

[0354] 2. Construction of Minigene Plasmid

[0355] To establish a rapid analysis of splicing events involving potential cryptic exons by 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 in the human SLC20A2 gene on a minigene backbone plasmid with a CMV promoter. To test the possible changes in the translation products of SLC20A2 mRNA caused by the insertion of cryptic exons associated with mutations, we cloned the corresponding SLC20A2 cDNA 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 the Mut Express II Rapid Mutagenesis Kit (Vazyme). The primers for amplifying these fragments are listed in Table 3.

[0356] 3. Antisense Oligonucleotides (ASO)

[0357] Antisense oligonucleotide technology is a drug development strategy based on chemically modified short oligonucleotides (12-24 nt) that bind to their RNA targets via Watson-Crick base pairing. The sequences of ASOs were designed according to the ASO design guidelines. ASOs with MOE and PS modifications (2′-O-methoxyethyl sugars with a phosphorothioate backbone) were commissioned to Integrated DNA Technologies for synthesis and HPLC purification. ASOs with PMO modifications (phosphorodiamidate morpholine backbone) were synthesized by Gen-Tools, LLC. All ASOs were dissolved in nuclease-free water for in vitro cell experiments and in artificial cerebrospinal fluid (aCSF) for in vivo mouse experiments. The detailed sequences are provided in Table 1.

[0358] 4. Construction of SLC20A2-KI mouse model

[0359] To construct a humanized SLC20A2 mouse model, the genomic fragment from exon 2 to exon 3 of the mouse SLC20A2 gene was replaced with a coding frame-matched genomic sequence of the human SLC20A2 gene carrying the c.289+1007C>G mutation using CRISPR / Cas9 technology. Briefly, the targeting sequence with bilateral 1.4 kb homology arms was co-microinjected into the uterus of C57BL / 6J mice with sgRNAs and Cas9 RNA. To screen founder (F0) mice with the correct knock-in sequence, genomic DNA extracted from the tail of the mice was used for genotyping. F1 KI mice were generated by crossing with C57BL / 6J wild-type mice, and the germline transmission of F0 KI mice was verified. To further verify the target mice with correct homologous recombination, we also performed long-segment sequencing and southern-blot analysis 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.

[0360] 5. Cell Culture and Transfection

[0361] HEK293T, Neuro-2A, A172, HepG2, HeLa cells (Cell Bank, Chinese Academy of Sciences) and primary human fibroblasts were cultured in 6-well or 12-well plates (Corning) using DMEM supplemented with 10% (vol / vol) fetal bovine serum (FBS). Human fibroblasts were derived from PFBC patients with SLC20A2 heterozygous c.289+1007C>G mutation and SLC20A2 heterozygous deletion, as well as voluntary donated healthy human controls.

[0362] 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, 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.

[0363] For MOE-modified ASOs, cells were transfected with Lipofectamine 3000 (Thermo Fisher) at a final concentration of 100 nM for 48 h. For PMO-modified ASOs, cells were transfected with 4 μM PMO for 48 h using 6 μL / mL Endo-Porter (Gene-Tools, LLC). To inhibit NMD-mediated RNA degradation, cyclohexanone (CHX, 50 μg / mL) was incubated with cultured human fibroblasts for 6 h before harvesting.

[0364] 6. RNA extraction and splicing analysis

[0365] 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 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, and the primer sequences are listed in Table 3.

[0366] 7. Protein extraction and Western-blot analysis

[0367] Cells or mouse brains were collected 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, then separated by 9% SDS-PAGE gel and blotted on PVDF membranes. The membranes were blocked with 5% skim milk and then detected with PiT2-7B5 (1:1000, monoclonal antibody obtained by immunizing mice with the loop region of PiT2 protein), HA-tag (1:5000, #3724, Cell Signaling), GAPDH (1:3000, KC-5G5, KANGCHEN), α-Tubulin (1:5000, 66031-1-Ig, Proteintech) and corresponding HRP-labeled secondary antibodies (1:2000, Cell Signaling). Bands were observed with ECL (TIANGEN) and measured with Image J software.

[0368] 8. Intracerebral (ICV) injection of ASO in mice

[0369] All animal surgeries were performed under standard aseptic conditions. Mice were anesthetized with isoflurane (5% for induction and 1.5% for maintenance) and placed on a small animal stereotaxic frame (Model 68528, RWD life science) with a heating pad to maintain body temperature. 2% lidocaine was injected at the incision of SLC20A2-KI mice and hair was shaved using a depilatory cream. After three times disinfection of the scalp at the surgical site with betaine and 75% alcohol, a midline incision was made, the scalp was removed to expose the skull, and the periosteum and other soft tissues connected to the skull were cleaned with cotton swabs and a scalpel. After the skull was dried, the skin around the skull was glued to the exposed skull edge using tissue adhesive (3MVetbond Tissue Adhesive). The skull plane was adjusted to the stereotaxic level and the bregma was determined. A layer of blue light-cured self-etching glue (3M ESPESingle Bond Universal, 3M Deutschland Gmbh) was applied on and around the frontal bone to help fixation. Then, a custom-made head plate was 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 intact. The dura was covered with antibiotic ointment and a fast-curing silicone elastomer to protect it. The mice were moved to a custom-made fixation plate with 1.5% isoflurane, and cerebrospinal fluid was collected from the tympanic cavity. Afterwards, the mice were allowed to recover for at least one week before the injection of ASO.

[0370] For ICV injection, mice were anesthetized with isoflurane (5% for induction and 1% for maintenance) and their heads were fixed on a stereotaxic 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 mice to recover from anesthesia on a heating pad before returning them to their cages.

[0371] 9. CSF collection and Pi measurement

[0372] According to the method flow established in the laboratory, the mouse was fixed using a stereotaxic frame (Model 68528, RWD life science), and another ultra-microscale three-way adjustable bracket was used to clamp the collection needle tube. The drawn glass needle was loaded in the needle tube and punctured into the ventricle from the medullary cisterna posterior to the brain of anesthetized adult mice, and then a slight negative pressure was applied to guide CSF into the glass needle. About 5-10 μL 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 635nm using SpectraMax (Molecular device).

[0373] 10. In situ hybridization (ISH) detection of ASO distribution in mouse brain

[0374] 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. The miRNAscope was used according to the manufacturer's instructions. TM ISH was performed on 18-μm thick brain sections prepared with 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.

[0375] 11. Analysis of brain calcification by micro-CT and histochemical staining

[0376] To evaluate 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 obtained by X-ray micro-CT scanning instrument (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.

[0377] For histochemical staining of brain calcification, mouse brains were dehydrated in a series of graded ethanol before paraffin embedding, and 4-μm-thick sections were prepared. Brain calcification was assessed using conventional methods widely recognized in the field: these staining methods include alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E. Each analysis was repeated three times.

[0378] 13. Statistical analysis

[0379] Data are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed by Student's t-test. Differences were considered statistically significant when <0.05 and are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0380] Example 1: Identification of SLC20A2 intronic mutations in PFBC patients and analysis of their resulting abnormalities in SLC20A2 splicing and translation

[0381] After years of hard work, the inventors have accumulated hundreds of cases of brain calcification and constructed a pedigree map. Through genetic analysis of exon sequencing, pathogenic mutations were found in the exons of known PFBC pathogenic genes (SLC20A2, PDGFB, PDGFRB, XPR1, MYORG and JAM2) in 33 of the 53 PFBC families (62.2%). In addition, CNVs analysis confirmed the deletion of exon segments flanking the SLC20A2 gene in another 3 families. Among the remaining 17 families, a large family (family 1) in which no exon mutations were detected ( Figure 1a) Linkage analysis was performed. The genomic DNA samples of 45 members of the family were analyzed by association mapping using the Affymetrix 250K Nsp SNP chip, and a 32Mb segment on chromosome 8 (hg19 chr8:29042453-61341711) was found to be linked to the calcification phenotype in the family, with a LOD score of 4.5. Further WGS analysis revealed a deep-intronic variant of c.289+1007C>G in SLC20A2, and Sanger sequencing confirmed that the variant was commonly associated with brain calcification in the family. In-silico evaluation of this variant was performed using SpliceAI, with an acceptor gain score of 0.11 and a donor gain score of 0.11, indicating that an unreported cryptic exon may have been introduced from the splicing of intron 2 of SLC20A2 (Table 2).

[0382] Using similar WGS analysis and Sanger sequencing approaches to screen the remaining PFBC pedigrees who lacked a clear genetic diagnosis, we found three additional genetic variants located in deep intronic regions in the SLC20A2 gene in four families ( Figure 1 a): c.289+937G>A in intron 2 (family 2 and 3), c.730+768C>T in intron 6 (family 4), c.1795-1698A>G in intron 10 (family 5). These four intron mutations ( Figure 1 c) None of the mutations were registered in major human population genetics databases and were predicted to affect splicing in SliceAI analysis (Table 2). We also found that these mutations were commonly associated with brain calcification in these five families ( Figure 1 b). The subjects' ages ranged from 30 to 60 years. Four of them (families 1, 3, 4, and 5) reported headaches, and one of them (family 2) suffered from dizziness. CT imaging showed 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 1 b). Serum calcium, phosphorus, and PTH levels were normal in all patients (Table 1). These results support that intronic variants of SLC20A2 may be the genetic cause of PFBC.

[0383] In mammals, it is now clear that the 5'splice donor site and 3'splice acceptor site of introns are conserved. Intron mutations affect mRNA processing by destroying splice sites and / or splice regulatory elements, or by changing the secondary structure of mRNA molecules. 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 splicing 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 splicing acceptors or donors, suggesting that they may be through other mechanisms, such as regulating the probability of splicing inclusion of cryptic exons.

[0384] To validate the genetic prediction, 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 junction sequences of its intron 2, the junction sequences 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 Minigene, and then used site-directed mutagenesis to generate point mutations corresponding to the c.289+937G>A and c.1795-1698A>G intronic mutations of the two PFBC families ( Figure 2 a, c).

[0385] Minigene detection confirmed that for families 1 and 5, although the c.289+1007C>G and c.1795-1698A>G mutations did not result in the generation of new splice sites, RT-PCR and Sanger sequencing showed that there were additional splicing integrations of 135-nt and 76-nt cryptic exons in intron 2 and intron 10, respectively ( Figure 2 b, d). In contrast, cells without the expression of the wild-type SLC20A2 minigene showed no significant level of abnormal transcripts in the mutant minigene assay, suggesting that the two mutations may alter the probability of splicing insertion of cryptic exons by affecting adjacent splicing regulatory elements.

[0386] We further analyzed the effect of the above 135-nt and 76-nt cryptic exons on the protein product by inserting them into the corresponding sequence positions of the CDS of the N-terminal HA-tagged SLC20A2. According to sequence prediction, the insertion of these two cryptic exons introduced a premature termination codon ( Figure 2 g), which makes it impossible to translate and synthesize functional PiT2 protein. In cultured Neuro-2A cells, HA-tagged SLC20A2 minigene constructs containing these two cryptic exons or SLC20A2 minigene constructs containing two other new exons generated by new splicing acceptors / donors were transfected. Western blot results showed a truncated PiT2 protein product consistent with the prediction ( Figure 2 h). According to experience, since these abnormal transcripts have PTC mutations in vivo, the NMD degradation mechanism of abnormal transcripts may not produce protein products with significant expression levels. To this end, we compared fibroblasts from healthy controls and found that the level of full-length PiT2 in fibroblasts from patients carrying the c.289+1007C>G mutation was significantly decreased ( Figure 2 i) Together, these findings support a novel and important molecular genetic pathogenesis, whereby intronic mutations in the SLC20A2 gene lead to increased splicing and integration of endogenous cryptic exons, resulting in SLC20A2 haploinsufficiency and causing brain calcifications in mutation carriers of PFBC families.

[0387] Example 2: Endogenous cryptic exons exist in the human brain and are involved in the formation of alternative splicing isoforms of SLC20A2

[0388] The in vitro evidence in the above examples suggests that the c.289+1007C>G and c.1795-1698A>G mutations have the ability to introduce cryptic exons during mRNA splicing in intron 2 and intron 10, and in silico analysis has shown that they do not predict the generation of splice sites. The important question is whether these cryptic exons exist in human cells themselves, but these abnormal splice isoforms produced at a lower ratio under normal splicing conditions are degraded.

[0389] We explored the presence of potential splicing-regulated cryptic exons in 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 that were treated with CHX to inhibit degradation of aberrantly spliced ​​transcripts showed the presence of a cryptic exon inclusion event between exon 2 and exon 3 in HEK293T and A172 cells (c.289+901 to c.289+1035, named "crpE2"); and a clear cryptic exon inclusion event between exon 10 and exon 11 (c.1795-1703 to c.1795-1628, named "crpE10") was observed in A172 cells ( Figure 3 a, b). It is worth mentioning that the expression levels of these abnormally spliced ​​transcripts containing cryptic exons crpE2 or crpE10 are sensitive to cyclohexylamine (CHX), which is a small molecule inhibitor for evaluating the potential involvement of the NMD pathway, suggesting that transcripts normally integrated with crpE2 or crpE10 may be degraded through the NMD pathway in the brain. Sanger sequencing of crpE2 and crpE10 confirmed that they are the 135-nt and 76-nt splicing products of minigenes carrying the c.289+1007C>G and c.1795-1698A>G mutations ( Figure 3 b) These results suggest 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).

[0390] 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 confirmed that the splicing integration of crpE2 and crpE10 in SLC20A2 mRNA transcripts can occur in different cell types from multiple tissue sources ( Figure 3 c), suggesting that this splicing mechanism could be exploited in scenarios aimed at intervening in diseases by modulating PiT2 expression.

[0391] Example 3: ASO can correct abnormal splicing involving crpE2 or crpE10 and improve the translation efficiency of functional PiT2 protein

[0392] To evaluate whether ASOs can inhibit cryptic exon-dependent alternative splicing, we transfected SLC20A2 minigenes constructs into Neuro-2a cells and co-transfected ASO preparations targeting the splicing sites of crpE2 and / or crpE10 to inhibit the production level 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 ASO drugs, and we also used these two types of modified ASOs for separate analysis. 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. Figure 4 ac). 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 4 df). These results support the potential application value of ASO intervention to regulate PiT2 expression for the treatment of brain calcification diseases.

[0393] 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 group (vehicle) or the control group MOE-ASO (MOE-NC), we found that MOE-A and MOE-B could largely remove crpE2 insertion into SLC20A2 mRNA (MOE-A: 14.35±5.2645%; MOE-B: 15.62±5.206%; MOE-C: 15.48±5.200%) ( Figure 5 a, b), and resulted in an increase in PiT2 protein levels (MOE-A: 21.24±7.787%; MOE-B: 29.53±10.26%; MOE-C: 6.632±12.11%) ( Figure 5 c) These findings support ASO-mediated inhibition of aberrant splicing as a feasible approach to rescue insufficient SLC20A2 expression in PFBC patients with intronic mutations.

[0394] Considering that ASO can regulate the nonproductive alternative splicing of the wild-type allele and increase the level of normal SLC20A2 transcripts. In order to verify this issue from another perspective, we cultured two fibroblasts (C1, C2) from voluntary donors of the healthy control population. The ASO cell intervention experiment showed ( Figure 6 ae), the splicing regulatory effects of MOE-A and MOE-B on SLC20A2 mRNA were also observed in healthy control fibroblasts, and the expression level of PiT2 protein was upregulated (C1, C2). MOEs targeting crpE10 (MOE-J, MOE-K, MOE-L) also reduced the insertion of crpE10 and increased the expression of PiT2 protein in wild-type fibroblasts ( Figure 6 fg). This result indicates that for the target gene of autosomal dominant haploid insufficiency disease, the normal gene without pathogenic mutation on the other chromosome can be regulated by ASO intervention to achieve the purpose of changing the progression of the disease. In view of the expansion of practical application value, we expanded the screening range of ASO sequences on healthy control fibroblasts, including new ASO sequences targeting crpE2 splicing regulation (cr5E2-m1, cr5E2-5,

[0395] cr5E2-6,cr5E2-8,5crE2-9,cr5E2-10,cr5E2-12,5crE2-13,cr5E2-13L,

[0396] cr5E2-13S,cr5E2-14,cr5E2-16,cr5E2-17,cr5E2-18,cr5E2-19,cr5E2-20,cr5E2-21,cr5E2-22,cr5E2-23,cr5E2-24,cr3E2-5,cr3E2-8,cr3E2-9,

[0397] cr3E2-10, cr3E2-11, cr3E2-12, cr3E2-13, cr3E2-14, cr3E2-15, cr3E2-16), and novel ASO sequences targeting crpE10 splicing regulation (cr5E10-9, cr5E10-11,

[0398] cr5E10-12, cr5E10-13, cr5E10-15, cr5E10-16, cr5E10-19, cr3E10-9, 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 splicing products of the minigene expression to ASO was analyzed by PCR, and most of the sequences with poor effects were filtered out ( Figure 7 a, c). Next, to evaluate the splicing regulatory effects of the remaining ASO sequences on endogenously expressed SLC20A2, we transfected them into healthy control human fibroblasts and performed repeated PCR and protein WB detection ( Figure 7 b,c), we finally identified 7 ASO sequences that showed good effects in promoting the expression of SLC20A2 or PiT2, including ASO sequences targeting crpE2 splicing regulation cr5E2-10, MOE-B (cr5E2-11), cr5E2-12, cr5E2-13, cr5E2-14 MOE-A (cr5E2-15), and ASO sequence targeting crpE10 splicing regulation cr5E10-12. Due to the limitations of the detection conditions, this result cannot rule out that ASO sequences targeting the regions described in other patents also have good effects. Here we only show the analysis process of the invention process.

[0399] In PFBC disease, heterozygous dominant mutations in the SLC20A2 gene account for up to 60%, highlighting that ASO intervention can be used to repair haploinsufficiency of the SLC20A2 gene that is not limited to mutation forms (including intronic mutations, exonic mutations, transcriptional regulatory region mutations, etc.). 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 epidermal fibroblasts from a patient (P2) with heterozygous loss of a genomic segment covering SLC20A2. Compared with the vehicle group or the control group ASO, MOE-A and MOE-B can 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 7 a, b), and caused 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 8 c) Taken together, these results suggest that ASO-mediated correction of cryptic exon-dependent alternative splicing can rescue SLC20A2 haploinsufficiency caused by intronic mutations in SLC20A2 and even other non-intronic mutations.

[0400] Example 4: Construction of a humanized SLC20A2 intron mouse model (SLC20A2-KI) and analysis of progressive brain calcification

[0401] To evaluate whether ASO treatment can effectively intervene in the occurrence and progression of brain calcification at the animal level, we next developed a humanized mouse model. We knocked in the entire human intron 2 carrying the SLC20A2c.289+1007C>G mutation and the flanking linker sequences ( Fig. 9a), so that the transcription product of the chimeric SLC20A2 gene in the mouse model contains the crpE2 cryptic exon, which can be bound and intervened by ASO preparations suitable for humans. Specifically, using CRISPR / Cas9 technology, combined with the simultaneously introduced targeting DNA fragments, the genomic fragments from exon 2 to exon 3 of the mouse SLC20A2 gene were replaced with the genomic sequence of the human SLC20A2 gene carrying the c.289+1007C>G mutation with a coding frame match. In short, the targeting sequence with bilateral 1.4kb homology arms was co-microinjected with sgRNAs and Cas9 RNA into the uterus of C57BL / 6J mice. In order to screen founder (F0) mice with the correct gene knock-in sequence, genomic DNA extracted from the mouse tail was used for genotyping. F1 KI mice were generated by hybridization with C57BL / 6J wild-type mice, and the germline transmission of F0 KI mice was verified. To further validate the target mice with correct homologous recombination, we also performed long-segment sequencing and southern-blot analysis on F1 mice. Heterozygous and homozygous SLC20A2-KI mice were selected for biochemical and phenotypic analysis.

[0402] In heterozygous and homozygous KI mice, RT-PCR and sequencing analysis observed a significant increase in crpE2 insertion into SLC20A2 mRNA ( Fig. 9 b), which was accompanied by a significant decrease in SLC20A2 mRNA and PiT2 protein levels in these animals ( Fig. 9 c, d). Analysis of inorganic phosphate in cerebrospinal fluid showed that the levels of inorganic phosphate (Pi) in the cerebrospinal fluid (CSF) of heterozygous and homozygous SLC20A2-KI mice were significantly increased ( Fig. 9 e), thereby metabolically reproducing the brain Pi balance disorder of PFBC patients with SLC20A2 mutations. This suggests that SLC20A2 c.289+1007C>G mutations can be successfully used to model SLC20A2 haploinsufficiency.

[0403] We then used micro-CT, fluorescent RIS staining that can identify calcified deposits ( Fig.10 a, b), and histological staining (including alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E) to examine brain calcification in SLC20A2-KI mice. MicroCT scans showed ( Fig.10a), Approximately 71% of SLC20A2-KI mice began to develop obvious symmetrical brain calcification in the bilateral basal forebrain, hypothalamus, and dorsal midbrain regions at 5 months of age, which was 3 months later than that of SLC20A2-KO mice. At 7 months of age, calcification deposits in homozygous SLC20A2-KI mice were widely distributed in many brain regions, such as the basal forebrain, thalamus, hypothalamus, midbrain, and cerebellar dentate nucleus region ( Fig.10 a). At 9.5 months of age, the number and volume of calcified nodules continued to increase, and significant large calcified deposits appeared in the thalamus, which was highly similar to PFBC patients. Using fluorescent RIS-AF647 staining, which can identify calcified deposits, calcified deposits in similar brain regions can also be seen on microCT ( Fig.10 b), but the calcification foci displayed by fluorescence are clearer and more suitable for quantitative statistics. In addition, in summary, histological staining analysis, including alizarin red, Von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E), further showed that the calcification foci in the mouse model are highly consistent with the brain calcification deposition in humans in terms of morphology ( Fig.11 ), indicating that they are driven by the same pathological mechanism. These results indicate that our humanized SLC20A2-KI mouse model can reproduce the brain calcification distribution pattern of PFBC patients with SLC20A2 haploinsufficiency, brain Pi disorder, and SLC20A2 mutations.

[0404] Example 5: ASO intervention effectively inhibits brain calcification in SLC20A2-KI mice

[0405] Given that mutations in the SLC20A2 gene are the most common cause of PFBC, our humanized SLC20A2-KI mouse model therefore represents a valuable model for testing novel ASO-based therapies to treat brain calcification. We term this Calcification Repression by ASO Corrected Expression (CRACE) therapy. We designed four doses of MOE-ASOs (50, 100, 200, 400 μg) to evaluate whether splicing correction mediated by a single dose of ASO intraventricular injection can suppress brain calcification in SLC20A2-KI mice before the onset of brain calcification at 3 months ( Fig.12 a). MOE-A and MOE-B (collectively referred to as therapeutic MOE) were selected for in vivo administration due to their significant effects in restoring PiT2 expression in cell experiments.

[0406] Four months after a single 200 μg ICV injection of MOE-A, in situ hybridization using a probe that specifically recognizes MOE-A revealed widespread ASO signals throughout the brain ( Fig.12 b), indicating that MOEs can effectively spread in the brain and persist for a long time. In terms of target gene expression, compared with SLC20A2-KI mice injected with control MOE, a significant decrease in abnormal SLC20A2 transcripts containing crpE2 insertions was detected in the thalamus of homozygous SLC20A2-KI mice injected with therapeutic MOEs, while normal SLC20A2 transcripts showed a significant increase in expression, and the level of its translation product PiT2 protein was also significantly enhanced ( Fig.12 c, d, e). At the same time, CSF Pi levels in SLC20A2-KI mice were also significantly decreased after 4 months of intervention with 100 μg and 200 μg of therapeutic MOE-ASOs ( Fig.12 f), suggesting that abnormal phosphate homeostasis in the brain was repaired.

[0407] 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 ( Fig.13 a, b). Thus, MOE-modified ASOs-mediated splicing correction can prevent brain calcification in PFBC modeled in humanized SLC20A2-KI mice.

[0408] To further evaluate the intervention effect of ASO on mice with calcification, we injected ASO into the brain of 5-month-old homozygous SLC20A2-KI mice. Fig. 9 As a result, calcification had already appeared in the mouse brain. Previous experiments on cells have shown that both MOE-modified ASO and PMO-modified ASO can repair the expression of SLC20A2. In this animal experiment, we selected 25μg and 75μg PMO-ASOs for 5-month-old homozygous SLC20A2-KI mice ( Fig.14 a). At the end of PMO-1 administration, a significant increase in the normal SLC20A2 transcript expression level was detected in the thalamus of SLC20A2-KI mice ( Fig.14 b), and the Pi concentration in CSF also decreased significantly ( Fig.14c). Similarly, micro-CT showed that the distribution of brain calcification in 7-month-old SLC20A2-KI mice was significantly reduced after a single treatment with 25 μg or 75 μg PMO-1 compared with PMO-NC (Figure 15d). Collectively, these findings indicate that CRACE therapy can effectively increase the expression of PiT2, restore the balance of Pi in the brain, and suppress or reduce brain calcification in SLC20A2-KI mice. Therefore, these data and inventions support that ASO-mediated CRACE therapy can be used to treat brain calcification in PFBC patients.

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Claims

1. 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.

2. The use according to claim 1, 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.

3. The use according to any one of claims 1 to 2, 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.

4. The use according to any one of claims 1 to 2, wherein The alternative splicing includes alternative splicing in intron 10; Preferably, the alternative splicing in intron 2 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.

5. The use according to any one of claims 1 to 4, 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 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; 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).

6. The use according to claim 5, 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.

7. The use according to claim 5, 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.

8. The use according to any one of claims 1 to 7, 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.

9. The use according to claim 8, 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% (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: 8; 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.

10. The use according to claim 8, 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.

11. The use according to any one of claims 1 to 10, 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.

12. The use according to any one of claims 1 to 11, 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); 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.

13. 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 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; Preferably, the target area has the features defined in any one of claims 5-7.

14. The reagent according to claim 13, wherein The reagent has the characteristics defined in any one of claims 8-9.

15. A pharmaceutical composition comprising the agent according to any one of claims 13 to 14 or a combination thereof, and a pharmaceutically acceptable carrier and / or excipient.

16. Use of the agent according to any one of claims 13 to 14 or the pharmaceutical composition according to claim 15 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); 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.

17. The use according to claim 16, 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.

18. 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 1-9.

19. A method for regulating the expression of PiT2 protein in a cell, comprising contacting the agent according to any one of claims 13 to 14 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.

20. 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.

21. 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.

22. A genetically modified cell, the genome of which 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 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 introduced exogenously relative to the cell; 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.

23. 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.

24. 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.

25. A method for screening an agent capable of regulating SLC20A2 mRNA splicing, comprising: Providing (i) a cell comprising the nucleic acid construct of claim 24, (ii) a genetically modified non-human animal or cell as defined in claim 23, or (iii) a brain calcification model obtained by the method of claim 21; 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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