Application of CSNK2B in prevention and treatment of nervous system diseases

Abstract

The invention provides a method for preventing or treating diseases related to CSNK2B gene deletion. The disclosure also provides a CSNK2B protein or a variant thereof, a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, an expression cassette comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a vector comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a virus, such as an AAV particle, expressing a CSNK2B protein or a variant thereof, and a method for preparing the CSNK2B protein or a variant thereof. According to the present invention, the use of the cells expressing the CSNK2B protein in the preparation of drugs for treating or preventing CSNK2B gene deletion-related diseases in subjects is provided, and the CSNK2B gene deletion-related diseases are selected from the group consisting of Rett syndrome, autism spectrum disorder (ASD), Porier-Bienvenu neurodevelopment syndrome (POBINDS), epilepsy or broad-spectrum development retardation, or the use of the cells expressing the CSNK2B protein in the preparation of the drugs for treating or preventing the CSNK2B gene deletion-related diseases in the subjects,

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedicine, in particular, to an adeno-associated virus (AAV)-based gene delivery vector for brain and its application. CSNK2B The present disclosure provides a vector for gene delivery into brain and its application, which is particularly suitable for treating neurological development disorders associated with mutations in CSNK2B gene, such as autism spectrum disorder (ASD), Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), and epilepsy. CSNK2B The present disclosure provides a vector for gene delivery into brain and its application, which is particularly suitable for treating neurological development disorders associated with mutations in CSNK2B gene, such as autism spectrum disorder (ASD), Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), and epilepsy. BACKGROUND

[0002] CSNK2B (Casein kinase 2 beta) is a regulatory subunit of casein kinase II (CK2), and its expression level in human brain during embryonic period is significantly higher than that after birth, which is a key regulatory factor in brain development. As a serine / threonine protein kinase highly expressed in brain, CK2 is involved in various core physiological processes such as signal transduction, transcription regulation, neuron survival, and synaptic plasticity regulation. For example, in a rat model of intracerebral hemorrhage, CK2 can regulate the expression of NMDA receptor (NR2B) through phosphorylation, thereby reducing neuron apoptosis and neuroinflammatory response.

[0003] In recent years, the development of gene sequencing technology has confirmed that CSNK2B mutations in CSNK2B gene are closely related to various neurological development disorders. Clinical studies have found that CSNK2B mutations in splicing sites (such as c.176-2A>G, c.292-2A>G), nonsense mutations, and missense mutations can cause diseases such as autism spectrum disorder (ASD) and Poirier-Bienvenu neurodevelopmental syndrome (POBINDS). Among them, patients with POBINDS often show early-onset epilepsy (usually onset before 2 years old, including myoclonic seizures, tonic-clonic seizures, and febrile convulsions), developmental delay, facial deformity, and intellectual disability; patients with ASD are characterized by social interaction deficits and repetitive stereotyped behaviors, and about 70% of CSNK2B patients with mutations in CSNK2B gene also have epilepsy symptoms, and the severity of the seizures is positively correlated with the degree of neurological development defects. So far, at least 25 cases of neurological development disorders associated with mutations in CSNK2B gene have been reported in the literature, but the specific mechanisms of neurological dysfunction and behavioral phenotypes caused by mutations in this gene have not been fully elucidated, and there is a lack of targeted treatment methods. CSNK2B

[0004] Animal model studies have shown that CSNK2B CSNK2B plays an irreplaceable role in neurological development: Csnk2b homozygous knockout mice die due to impaired cell proliferation during embryonic period; knockout of CSNK2B in embryonic neural progenitor cells inhibits their proliferation and differentiation into oligodendrocytes; Csnk2b Csnk2b ​​Heterozygous knockout (Csnk2b + / - ) mice exhibit similar phenotypes to human patients, including ASD-like behaviors (social novelty recognition impairment, elevated anxiety levels), spatial learning and memory deficits, spontaneous seizures, and abnormal brain structures (thinned cortex, disorganized hippocampus, enlarged lateral ventricles), reduced specific cortical neurons (2-3 layer and 6 layer excitatory neurons, small, white protein positive PV interneurons) and survival rates. In addition, CSNK2B It has also been identified as a schizophrenia risk gene, and the expression of patients in the brain CSNK2B is down-regulated, while suppressing the expression of mice Csnk2b will destroy the morphology of neurons and synaptic transmission function.

[0005] Although CSNK2B the role in neurodevelopment and disease has been initially confirmed, there is currently no method to treat related neurodevelopmental disorders by introducing the natural CSNK2B full-length gene into the brain. Therefore, the development of a gene therapy vector that can efficiently and specifically express CSNK2B protein in the brain is the key to solving the above clinical needs. SUMMARY

[0006] The present disclosure provides a method for preventing or treating a CSNK2B gene deletion-related disease, comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic agent, the therapeutic agent comprising one or more of: a CSNK2B protein or a variant thereof, a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, an expression cassette comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a vector comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a virus such as an AAV particle expressing a CSNK2B protein or a variant thereof, a cell expressing a CSNK2B protein.

[0007] The present disclosure provides the use of a CSNK2B protein or a variant thereof, a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, an expression cassette comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a vector comprising a nucleic acid molecule encoding a CSNK2B protein or a variant thereof, a virus such as an AAV particle expressing a CSNK2B protein or a variant thereof, or a cell expressing a CSNK2B protein in the preparation of a medicament for preventing or treating a CSNK2B gene deletion-related disease in a subject.

[0008] In some embodiments, the CSNK2B protein or a variant thereof has a biological activity that regulates CK2 kinase activity, maintains neuronal survival or synaptic function.

[0009] In some embodiments, the therapeutic agent can further comprise a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable delivery vehicle.

[0010] In some embodiments, the CSNK2B protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 1.

[0011] In some specific embodiments, the CSNK2B protein is an amino acid sequence as set forth in SEQ ID NO: 1.

[0012] In some embodiments, wherein the variant of CSNK2B protein comprises one or more substitution, deletion, insertion and / or addition of amino acids compared to the CSNK2B protein, and has at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the CSNK2B protein.

[0013] In some embodiments, the variant of CSNK2B protein comprises or consists of an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and has at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the CSNK2B protein.

[0014] In some embodiments, the CSNK2B The genetic deletion-related disease is selected from one or more of Rett syndrome, autism spectrum disorder (ASD), Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), epilepsy, or broad-spectrum developmental delay.

[0015] In some embodiments, the nucleic acid molecule encoding the CSNK2B protein or a variant thereof is a DNA sequence, a cDNA sequence, or a mRNA sequence.

[0016] In some specific embodiments, the nucleotide sequence encoding the CSNK2B protein or a functional variant thereof comprises a single-stranded sequence or a double-stranded sequence.

[0017] In some embodiments, the nucleic acid molecule encoding the CSNK2B protein or a variant thereof comprises: (a) a nucleic acid sequence as set forth in SEQ ID NO: 2; (b) a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 99% identical to SEQ ID NO: 2, preferably a nucleic acid sequence that is at least 95% identical, more preferably a nucleic acid sequence that is at least 99% identical; or (c) a codon-optimized nucleic acid sequence encoding the CSNK2B protein or a variant thereof.

[0018] In some embodiments, the expression cassette of the nucleic acid molecule encoding the CSNK2B protein or variant thereof comprises: (i) a nucleic acid sequence encoding the CSNK2B protein or variant thereof; and (ii) a transcriptional regulatory element operably linked to the nucleic acid sequence encoding the CSNK2B protein or variant thereof.

[0019] In some embodiments, the transcriptional regulatory element comprises a neuron-specific promoter or a broad-spectrum promoter, specifically including but not limited to: human synapsin promoter (hSyn), CAG promoter (chicken beta-actin promoter fused with CMV enhancer), human cytomegalovirus promoter (hCMV), chicken beta-actin promoter (CBA), simian virus 40 early promoter (SV40), mouse mammary tumor virus promoter (MMTV), human immunodeficiency virus long terminal repeat promoter (HIV LTR), MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter (EBV IE), Rous sarcoma virus promoter (RSV), actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter or functional variants thereof, or a combination thereof.

[0020] In some embodiments, the transcriptional regulatory element comprises the hSyn promoter.

[0021] In some specific embodiments, the expression cassette of the nucleic acid molecule encoding the CSNK2B protein or variant thereof further comprises a tag sequence.

[0022] In some specific embodiments, the tag sequence is selected from an HA tag, a Flag tag, a His tag, a GST tag, a Myc tag, or a combination thereof.

[0023] In some specific embodiments, the expression cassette of the nucleic acid molecule encoding the CSNK2B protein or variant thereof further comprises a nucleic acid sequence encoding a self-cleaving polypeptide, preferably the self-cleaving polypeptide is selected from P2A, T2A, F2A or variants thereof.

[0024] In some specific embodiments, the expression cassette of the nucleic acid molecule encoding the CSNK2B protein or variant thereof optionally comprises a transcription termination signal sequence, preferably the transcription termination signal sequence is a polyA, more preferably the polyA is selected from human growth hormone (hGH) polyA or SV40 polyA.

[0025] In some specific embodiments, the expression cassette of the nucleic acid molecule encoding the CSNK2B protein comprises, in order from 5' to 3', the following elements operably linked: AAV inverted terminal repeat (ITR) 1 ; a transcription regulatory element, preferably, the transcription regulatory element is selected from hSyn or CAG promoter; an optional Kozak tag sequence; a nucleotide sequence encoding a native human CSNK2B protein or a variant thereof, preferably, SEQ ID NO: 2; a tag sequence, preferably, an HA tag sequence; a self-cleaving polypeptide sequence, preferably, P2A; an optional enhanced green fluorescent protein (EGFP) coding sequence; a transcription termination signal, preferably, hGH polyA; and AAV inverted terminal repeat (ITR) 2.

[0026] In some embodiments, the nucleic acid sequence of the expression cassette of the nucleic acid molecule encoding a CSNK2B protein or a variant thereof is set forth in SEQ ID NO: 4 (hSyn-CSNK2B-P2A-EGFP-polyA).

[0027] In some embodiments, the expression cassette of the nucleic acid molecule encoding a CSNK2B protein or a variant thereof optionally further comprises an enhancer, an intron, or a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE).

[0028] In some embodiments, the enhancer is selected from a CMV enhancer.

[0029] In some embodiments, the intron is selected from a chicken beta-actin intron.

[0030] In some embodiments, the expression cassette of the nucleic acid molecule encoding a CSNK2B protein or a variant thereof comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

[0031] In some embodiments, the vector comprising the nucleic acid molecule encoding a CSNK2B protein or a variant thereof is selected from a plasmid, a viral vector, or a non-viral vector.

[0032] In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV). Preferably, the vector is an AAV vector. More preferably, the AAV vector is selected from the group consisting of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB, or a variant thereof.

[0033] In some embodiments, the vector is an AAV vector plasmid pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0034] In some embodiments, the vector is used to express a human CSNK2B protein.

[0035] In some embodiments, the vector is capable of expression in a neuronal cell.

[0036] In some embodiments, the vector is capable of expression in an animal brain tissue cell.

[0037] In some embodiments, the AAV is selected from the group consisting of AAV9 or AAV-PHP.eB serotype, wherein the AAV-PHP.eB serotype has a high efficiency of crossing the blood-brain barrier, and can achieve whole brain transduction by systemic injection (e.g., retro-orbital injection, intravenous injection), and is particularly suitable for intracerebral gene delivery in neonatal or adult animals.

[0038] In some embodiments, the non-viral vector is selected from the group consisting of a nanoparticle, a nanosome, a liposome, a biodegradable polymer complex, or a combination thereof.

[0039] In some embodiments, the nanoparticle comprises a liposome, a lipid nanoparticle, a polymer nanoparticle, a dendrimer, a cyclodextrin, a silica nanoparticle, a polymer complex, a magnetic nanoparticle, a gold nanoparticle, a quantum dot, a carbon nanotube, or a combination thereof.

[0040] In some embodiments, the therapeutic agent is administered to the brain of the subject, preferably to the whole brain.

[0041] In some embodiments, the therapeutic agent is administered by retro-orbital injection, intravenous injection, or intrathecal injection, preferably by intrathecal injection.

[0042] In some embodiments, the subject is a human or a non-human mammal, preferably a human.

[0043] In some specific embodiments, the cells expressing the CSNK2B protein are mammalian cells, including humans and non-human mammals.

[0044] In some specific embodiments, the cells expressing the CSNK2B protein are selected from HEK293 cells, HEK293T cells, HEK293T-17 cells, nervous system cells, or combinations thereof.

[0045] In some specific embodiments, the cells expressing the CSNK2B protein are selected from neuronal cells.

[0046] In some specific embodiments, the therapeutic agent is an injectable formulation, preferably an intravenous or intrathecal injection formulation.

[0047] In some specific embodiments, the dosage form of the therapeutic agent is selected from the group consisting of lyophilized formulations, liquid formulations, or combinations thereof.

[0048] In some specific embodiments, the viral titer in the therapeutic agent is 1 × 10⁻⁶. 11 - 1×10 14 vg / mL, preferably 1×10 12 - 1×10 13 vg / mL.

[0049] In some specific implementations, the therapeutic agent can increase the expression level of CSNK2B protein in the cerebral cortex, hippocampus, etc. of the subject.

[0050] On the other hand, this disclosure provides a polynucleotide comprising or consisting of an sgRNA sequence as shown in SEQ ID NO: 17 or 18.

[0051] On the other hand, this disclosure provides nucleic acid sequences comprising or consisting of the sgRNA sequence shown in SEQ ID NO: 17 or 18 in the construction of CSNK2B Applications of gene knockout in non-human animal models.

[0052] On the other hand, this disclosure provides a CSNK2B Gene knockout non-human animal models, wherein the non-human animal models are CSNK2B The gene contains the deletion of 841 bases, including exon 3, and the resulting sequence is shown in SEQ ID NO: 3.

[0053] In some implementations, the animal model is used to screen treatments. CSNK2B Drugs for diseases related to gene deletion.

[0054] In some implementations, the animal model is constructed using sgRNA as described above.

[0055] In another aspect, the present disclosure provides a method for constructing a non-human animal model, comprising steps of: (S1) performing gene editing on an animal using sgRNA sequence as shown in SEQ ID NO: 17 or 18, thereby obtaining a gene-edited non-human animal; and (S2) screening the obtained gene-edited non-human animal, thereby obtaining the Figure 1a gene deletion animal model, wherein the animal model comprises a nucleic acid sequence as shown in SEQ ID NO: 3.

[0056] In some embodiments, the gene editing step comprises injecting the sgRNA of the third aspect of the present disclosure into a zygote of an animal and transplanting into a surrogate mother, thereby obtaining a gene-edited animal.

[0057] In some embodiments, the animal is a non-human mammal, preferably a rodent, more preferably a mouse, and more preferably a male mouse.

[0058] It should be understood that, within the scope of the present disclosure, each of the technical features of the present disclosure described above and each of the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0059] The present disclosure can be more fully understood with reference to the following drawings.

[0060] FIG. 1 shows the vector map of AAV-hSyn-CSNK2B-3XFlag-GSG-P2A-EGFP Figure 1b ) and AAV-CAG-CSNK2B-3XFlag-GSG-P2A-EGFP Figure 2 ).

[0061] CSNK2B shows the construction and identification results of the gene deletion mouse. Figure 3

[0062] Figure 1a shows the AAV virus injection scheme, and the rescue of social deficiency in mice after AAV injection. DETAILED DESCRIPTION

[0063] ​The following description of the disclosure is merely intended to illustrate various different embodiments of the disclosure. Thus, the particular modifications discussed should not be construed as limiting the scope of the disclosure. Various different equivalents, changes and modifications which are apparent to one skilled in the art are intended to be included within the scope of the disclosure, and it is therefore intended that the disclosure be taken only by way of the illustrative embodiments. All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference in their entirety.

[0064] In order that the disclosure can be more readily understood, certain terms are first defined. As used in the present disclosure, unless specifically defined otherwise herein, each of the following terms shall have the meaning given to it below. Additional definitions are set forth throughout the application.

[0065] CSNK2B protein or a variant thereof CSNK2B protein is the beta subunit of the Casein Kinase 2 (CK2) complex, which is encoded by the CSNK2B gene. This protein plays an important role in processes such as cell signaling, neural development, and synaptic transmission. The CSNK2B protein described in the present disclosure has biological activities of regulating CK2 kinase activity, maintaining neuronal survival or synaptic function.

[0066] In one aspect, the CSNK2B protein used in the methods or uses described in the present disclosure includes naturally occurring CSNK2B. In some embodiments, the naturally occurring CSNK2B includes, but is not limited to, CSNK2B having a wild-type amino acid sequence of CSNK2B typically associated with animals or naturally occurring variants. In another aspect, the CSNK2B used in the compositions and methods described in the present disclosure includes a CSNK2B variant comprising one or more substitutions, deletions, insertions and / or additions of amino acids compared to the wild-type CSNK2B protein, and having at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the wild-type CSNK2B protein. In some embodiments, the variant of the CSNK2B protein comprises or consists of an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to the wild-type CSNK2B amino acid sequence, and having at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the wild-type CSNK2B protein. It is understood that, due to the degeneracy of the genetic code, any variation in the coding sequence of the nucleic acids of the present disclosure that expresses the same polypeptide is included within the scope of the present disclosure.

[0067] In some embodiments, the CSNK2B protein comprises an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the variant of the CSNK2B protein comprises one or more amino acid substitutions, deletions, insertions and / or additions compared to the CSNK2B protein and has at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the CSNK2B protein. In some embodiments, the variant of the CSNK2B protein comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 and has at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the biological activity of the CSNK2B protein. In some embodiments, the biological activity of the CSNK2B variant is determined as a percentage of the activity of another CSNK2B taken as a comparison. The standard CSNK2B used for comparison can be a full-length CSNK2B, a truncated CSNK2B, a mutated CSNK2B, a post-translationally modified CSNK2B, or a fragment of CSNK2B that is different from the CSNK2B being compared.

[0068] In one aspect, the CSNK2B or variant thereof used in the methods or uses described in the present disclosure comprises a polypeptide sequence encoding an animal CSNK2B. The animal encoding CSNK2B can include, but is not limited to, Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, Pan troglodytes, Canis lupus familiaris, Sus scrofa, Oryctolagus cuniculus, Macaca mulatta, Nomascus leucogenys, Cricetulus griseus, Gorilla gorilla, Pongo abelii, Callithrix jacchus, and Ailuropoda melanoleuca. Various CSNK2B polypeptide sequences found in these animals are available in public databases, including the National Center for Biotechnology Information (NCBI). In some embodiments, the amino acid sequence of the CSNK2B protein used in the compositions and methods described in the present disclosure is a human CSNK2B full-length protein comprising an amino acid sequence as set forth in SEQ ID NO: 1.

[0069] A nucleic acid molecule encoding a CSNK2B protein or a variant thereof The term "nucleic acid" encompasses, for example: (a) DNA that is part of a naturally occurring genomic DNA molecule, but which does not flank the portion of the molecule on both sides by the two nucleic acid sequences that flank that portion of the molecule in the genome of the organism in which it naturally occurs; (b) nucleic acid incorporated into a vector or the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is identical to any naturally occurring vector or genomic DNA; (c) a molecule that is isolated, such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present disclosure further include molecules produced synthetically, as well as any nucleic acid that has been chemically altered and / or has a modified backbone. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide.

[0070] In some embodiments, the nucleic acid molecule encoding a CSNK2B protein or a variant thereof comprises: (a) a nucleic acid sequence as set forth in SEQ ID NO: 2; (b) a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 99% identical to SEQ ID NO: 2, preferably a nucleic acid sequence that is more than 95% identical, more preferably more than 99% identical; or (c) a codon-optimized nucleic acid sequence encoding the CSNK2B protein or a variant thereof. In some embodiments, the nucleotide sequence encoding a CSNK2B protein or a variant thereof comprises a DNA sequence, a cDNA sequence, or an mRNA sequence. In some embodiments, the nucleotide sequence encoding a CSNK2B protein or a variant thereof comprises a single-stranded sequence or a double-stranded sequence.

[0071] An expression cassette of a nucleic acid molecule encoding a CSNK2B protein or a variant thereof The terms "expression cassette of a nucleic acid molecule encoding a CSNK2B protein or a variant thereof," "CSNK2B gene expression cassette," and "expression cassette of the present disclosure" are used interchangeably and include: (i) a nucleic acid sequence encoding a CSNK2B protein or a variant thereof; and (ii) a transcriptional regulatory element operably linked to the nucleic acid sequence encoding a CSNK2B protein or a variant thereof.

[0072] In some embodiments, the transcriptional regulatory element comprises a human synapsin promoter (hSyn), a CAG promoter, a human cytomegalovirus promoter (hCMV), a chicken beta-actin promoter (CBA), a simian virus 40 early promoter (SV40), a mouse mammary tumor virus promoter (MMTV), a human immunodeficiency virus long terminal repeat promoter (HIV LTR), a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter (EBV IE), a Rous sarcoma virus promoter (RSV), an actin promoter, a myosin promoter, a hemoglobin promoter, a creatine kinase promoter, or a functional variant thereof. In some embodiments, the transcriptional regulatory element comprises a hSyn promoter, a CAG promoter, or a hCMV promoter, more preferably the transcriptional regulatory element is a hSyn promoter.

[0073] The expression cassettes of the present disclosure preferably comprise one or more regulatory sequences to direct expression of the nucleic acid sequence in the nervous system target cell. Regulatory sequences can include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence. The expression cassettes of the present disclosure can also include a selectable tag, for example to determine expression of the protein of interest in a growth system (e.g., a bacterial cell) or in the nervous system target cell.

[0074] "Operably linked" means that the nucleic acid sequence is functionally connected to the sequence with which it is operably linked such that they are connected in a manner that allows them to affect each other's expression or function. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern influenced by the promoter.

[0075] A promoter mediates expression of a nucleic acid sequence to which it is linked. The promoter can be constitutive or can be inducible. The promoter can direct ubiquitous expression in cells of the nervous system, or neuronal specific expression. In the latter case, the promoter can direct cell type specific expression, for example to neural cells. Suitable promoters will be known to those skilled in the art. For example, a suitable promoter can be selected from the group consisting of CAG, hSyn, L7, thy-1, synapsin, calbindin, human CMV, GAD-67, chicken beta actin, Grm6, Grm6 enhancer SV40 fusion protein. Targeting can be achieved using cell specific promoters, for example the neuronal specific promoter hSyn for selective targeting to neuronal cells. Ubiquitous expression can be achieved using a pan-neuronal promoter. However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters.

[0076] In preferred embodiments, the tag sequences useful in the present disclosure include, but are not limited to, a HA tag, a Flag tag, a His tag, a GST tag, a Myc tag, or a combination thereof.

[0077] In preferred embodiments, the expression cassette of the present disclosure comprises, in order from 5’ end to 3’ end, the following elements operably linked: S1-S2-S3-S4-S5-S6-S7-S8-S9, wherein, S1 is an AAV inverted terminal repeat (ITR) 1, S2 is a promoter, S3 is none or a tag sequence, S4 is the nucleotide sequence encoding the native human CSNK2B protein or a variant thereof, S5 is a tag sequence such as HA, S6 is a self-cleaving polypeptide sequence such as P2A, S7 is an optional fluorescent protein such as EGFP enhanced green fluorescent protein sequence, S8 is none or a transcription termination signal sequence, S9 is an AAV ITR2.

[0078] In preferred embodiments of the present disclosure, the expression cassette of the present disclosure has a hSyn-CSNK2B-P2A-EGFP-polyA structure, more preferably, the coding nucleotide sequence of the hSyn-CSNK2B-P2A-EGFP-polyA structure is set forth in SEQ ID NO: 4.

[0079] Adeno-Associated Virus Adeno-associated virus (AAV), also known as adeno-associated dependovirus, belongs to the family of parvoviridae dependovirus, is a class of simple structure single-stranded DNA defective virus found at present, which needs the help of auxiliary virus (usually adenovirus) to participate in replication. It encodes cap and rep genes in two terminal inverted repeat sequences (ITRs). ITRs play a decisive role in virus replication and packaging. The cap gene encodes viral capsid protein, and the rep gene is involved in viral replication and integration.

[0080] Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus, and is considered as one of the most promising gene transfer vectors due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, long expression of exogenous genes in vivo, etc. It is widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been well understood, especially its application effect in various cells, tissues and in vivo experiments has accumulated a lot of data. In medical research, rAAV is used for gene therapy research of various diseases (including in vivo and in vitro experiments); as a characteristic gene transfer vector, it is also widely used in gene function research, construction of disease models, preparation of gene deletion mice, etc.

[0081] In one embodiment of the disclosure, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can integrate into the genomes of the cells they infect in a stable and site-specific manner. They are able to infect a wide range of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. AAV contains approximately 4700 bases and includes an inverted terminal repeat (ITR) region of about 145 bases at each end, which serves as a replication origin for the virus. The remainder of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

[0082] AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of heterozygous vectors is described, for example, in PCT application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publications Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941; and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with the genes of interest, and the uses of these constructs for transporting the genes of interest in vitro (into cultured cells) or in vivo (directly into the organism). Replication-deficient recombinant AAV can be prepared by co-transfecting a cell line infected with a human helper virus (e.g., adenovirus) with a plasmid containing two AAV inverted terminal repeat (ITR) regions flanking the nucleic acid sequence of interest, and a plasmid carrying the AAV capsid genes (rep and cap genes). The resulting AAV recombinants are then purified using standard techniques.

[0083] In some implementations, the recombinant vector is capsidated into viral particles (e.g., AAV viral particles including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB).

[0084] Therefore, this disclosure includes recombinant viral particles containing any of the vectors described herein (recombinant because they contain recombinant polynucleotides). Methods for producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.

[0085] The serotypes of the AAV vectors used in this disclosure are selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB, or variants thereof. Preferably, the AAV vector plasmids are selected from: pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0086] Expression vectors and host cells This disclosure also provides an expression vector for the CSNK2B protein, which contains the CSNK2B expression cassette of this disclosure.

[0087] Vectors can be viral or non-viral (e.g., plasmids). Viral vectors include those derived from: adenoviruses, including mutant forms of adeno-associated virus (AAV), retroviruses, lentiviruses, herpesviruses, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxviruses, and SV40. Preferably, the viral vector is replication-defective, although it is envisioned to be replication-deficient, capable of replication, or conditionally replicating. Viral vectors generally remain in an extrachromosomal state without integrating into the genome of target cells. Preferred viral vectors for introducing nucleic acid sequences encoding the CSNK2B protein into target cells in the brain are AAV vectors, such as self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotypes 2 to 12, AAV-DJ) or modified versions of any of these serotypes (including AAV 4YF, AAV 7m8, AAV-PHP.B, and AAV-PHP.eB).

[0088] Viral vectors can be modified to delete any non-essential sequences. For example, in AAV, the virus can be modified to delete all or part of the IX gene, Ela, and / or Elb gene. For wild-type AAV, the absence of helper viruses such as adenovirus makes replication very inefficient. For recombinant adeno-associated viruses, preferably, the replication gene and capsid gene are provided in trans form (in the pRep / Cap plasmid), and only the ITR of the AAV genome is preserved and packaged into the virion, while the required adenovirus genes are provided by adenovirus or another plasmid. Similar modifications can be made to lentiviral vectors.

[0089] Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids can be conjugated with agents to facilitate the uptake of the viral vector by target cells. Such agents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems may be used. The vectors used in this disclosure are preferably suitable for in vivo or in vitro use, and are preferably suitable for human use. The plasmids are preferably AAV vector plasmids pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0090] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / RNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a “collapsed” structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0091] Suitable lipids can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dihexadecanyl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY); and cholesterol (“Choi”) is available from Calbiochem. Behring is available; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of the lipids in chloroform or chloroform / methanol can be stored at approximately [time missing]. 20°C. Chloroform was used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of monolayer and multilayer lipid mediators formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions having structures in solution different from normal vesicular structures are also included. For example, lipids may exhibit a micellar structure or exist simply as heterogeneous aggregates of lipid molecules. Liposomes are also considered. Nucleic acid complex.

[0092] Optionally, the method further includes administering a pharmaceutically acceptable carrier or excipient. The phrase "pharmaceutically acceptable" is recognized in the art and refers to a composition, polymer, and other material and / or dosage form suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and commensurate with a reasonable benefit / risk ratio. For example, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relate to carrying or transporting any supplement or composition or its components from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with the other components of the supplement and harmless to the patient. Optionally, a pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and Soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0093] The vector will preferably contain one or more regulatory sequences to guide the expression of the nucleic acid sequence in target cells of the nervous system. The regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence. The vector may also include selective markers, for example, to determine the expression of the vector in a growth system (e.g., bacterial cells) or in target cells of the nervous system.

[0094] In some embodiments, the vector comprises one or more promoters operatively linked to expression cassettes, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, selectivity markers, nucleic acid restriction sites, and / or homologous recombination sites of the present disclosure.

[0095] Many expression vectors can be used to express the CSNK2B protein in mammalian cells (preferably human, and more preferably human nerve cells). This disclosure preferably uses adeno-associated virus (AAV) as the expression vector.

[0096] This disclosure also provides a host cell for expressing the CSNK2B protein. Preferably, the host cell is a mammalian cell (preferably human, more preferably human nerve cell) to increase the expression level of the CSNK2B protein.

[0097] This disclosure also provides a treatment method comprising administering the carrier to a subject in need.

[0098] In some specific embodiments, the vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof. Preferably, the vector is an AAV vector.

[0099] In some specific implementations, the carrier is introduced into a vein, ventricle, or myelin sheath of the desired subject.

[0100] In some specific implementations, the required subjects include humans and non-human mammals.

[0101] Pharmaceutical Composition This disclosure provides a pharmaceutical composition comprising (a) a CSNK2B-expressing carrier of this disclosure, and (b) a pharmaceutically acceptable carrier or excipient.

[0102] In some specific embodiments, the pharmaceutical composition is an injectable formulation, preferably an intravenous or intrathecal injection formulation. The pharmaceutical composition described in some specific embodiments is intended for intravenous or intrathecal injection.

[0103] In some specific embodiments, the vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof, preferably, the vector is an AAV vector.

[0104] In some specific embodiments, the viral titer in the therapeutic agent is 1 × 10⁻⁶. 11 - 1×10 14 vg / mL, preferably 1×10 12 - 1×10 13 vg / mL.

[0105] The pharmaceutical composition may be a liquid or a solid, such as a powder, gel, or paste. Preferably, the pharmaceutical composition is a liquid, and more preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0106] In this disclosure, the carrier can be administered directly to the brain via intraventricular injection and express the CSNK2B protein within the brain. In either administration mode, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0107] Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0108] The composition may comprise physiologically acceptable sterile aqueous or anhydrous water, dispersion, suspension, or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0109] Table 1. Sequence Information

[0110] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0111] The vectors used in the following examples are AAV-hSyn-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA and AAV-CAG-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA, and their vector maps are shown below. Figure 1b and Csnk2b As shown.

[0112] Example To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0113] Example 1: Csnk2b Construction and Genotyping of Gene-Deleted Mice 1.1 Csnk2b Gene deletion mouse construction In this embodiment, a Csnk2b Gene deletion mouse model. First, using the CRISPR-Cas system, targeting mice... Csnk2b Two sgRNAs were designed near exon 3 of the gene: sg1 (+) and sg2 (-), as shown in Table 2 (Figure 2A).

[0114] Synthesized spCas9 mRNA (company catalog number, such as CST) and two sgRNAs were injected into fertilized eggs of 8-week-old C57BL / 6 mice. The fertilized eggs were then transferred to 8-week-old ICR surrogate mother mice (Cyagen Biosciences) to obtain several gene-edited mice.

[0115] Selecting genes resulted in an 841bp deletion (after deleting the exon sequence). Figure 2 The gene sequence is SEQ ID NO: 3. Csnk2b Established lineages of heterozygous female mice (B), specifically founder mice (Csnk2b). + / - F0). 8-week-old Csnk2b+ / - Female mice were backcrossed with 8-week-old C57BL / 6 male mice. Genotypes of the offspring were identified by PCR amplification.

[0116] 1.2 Figure 2 Detection of Csnk2b expression in the brains of gene-deleted mice Take adult wild animals and Csnk2b + / - RNA and protein were extracted from mouse brain tissue. qPCR and Western blot experiments revealed that Csnk2b plays a crucial role in Csnk2b production. + / - The brains of mice showed a reduction of approximately 50% compared to wild-type mice. Figure 1a C-2E).

[0117] Table 2. sgRNA sequences

[0118] Example 2: AAV-hSyn-CSNK2B-P2A-EGFP and AAV-CAG-CSNK2B-P2A-EGFP viruses were injected into the brains of Csnk2b+ / - mice. The sequences of each element of the vector used in this disclosure are shown in Table 1. AAV-PHP.eb-hSyn-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA (AAV-hSyn-CSNK2B-P2A-EGFP) and AAV-PHP.eb-CAG-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA (AAV-CAG-CSNK2B-P2A-EGFP) viruses (from Paizhen Biotechnology Co., Ltd.) were obtained using conventional methods in the art. Figure 1b and Figure 3 As shown, AAV-PHP.eb is the wild-type sequence. The sequences of each element in this disclosure are merely exemplary and can be replaced with other AAV serotypes, promoters, and self-cleaving peptide sequences. AAV-PHP.eb-hSyn-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA (AAV-hSyn-CSNK2B-P2A-EGFP) and AAV-PHP.eb-CAG-CSNK2B-3XFlag-GSG-P2A-EGFP.WPRE3.SV40LpA (AAV-CAG-CSNK2B-P2A-EGFP) viruses (from Paizhen Biotechnology Co., Ltd.) were injected into Csnk2b, respectively. + / - mouse brain ( Figure 3A) Control group mice were injected with AAV-PHP.eB-hSyn-EGFP.WPRE.SV40LpA (AAV-hSyn-EGFP) virus that does not contain the CSNK2B gene.

[0119] First, thaw and mix the AAV virus to be injected, then place it on ice. Next, remove the newborn mouse (P3) and place it on a clean paper towel, away from its parents, before proceeding. Position the newborn mouse in a left lateral decubitus position with its head facing right. Using a BD 31 insulin syringe, draw up an appropriate amount of AAV, and gently stabilize the mouse's head with your left thumb and forefinger. Locate the mouse's eye area, and insert the syringe at a 30-40 degree angle into the edge of the eye at the 3 o'clock position, to a depth of approximately one-third of the needle's length. Gently inject the AAV into the orbital venous plexus, leave the needle in place for 5 seconds, and then gently remove the needle. Finally, gently return the injected newborn mouse to its cage. Control group mice were injected with AAV-hSyn-EGFP virus and labeled as: Csnk2b+ / -(GFP); experimental groups were injected with AAV-hSyn-CSNK2B-P2A-EGFP and AAV-CAG-CSNK2B-P2A-EGFP viruses, respectively, and labeled as: Csnk2b+ / -(hsyn-Csnk2b) and Csnk2b+ / -(CAG-Csnk2b), respectively; littermate wild-type mice were injected with AAV-hSyn-EGFP virus and labeled as: WT(GFP)( Csnk2b B).

[0120] Example 3: AAV-hSyn-CSNK2B-P2A-EGFP and AAV-CAG-CSNK2B-P2A-EGFP viral injections improve the social behavior deficit phenotype in Csnk2b+ / - mice. In this embodiment, the carrier constructed according to this disclosure is tested for its performance on... Figure 3 Improvement of ASD-related behaviors in gene-deleted mice.

[0121] We used the classic three-box behavioral paradigm to assess the social abilities of WT (GFP), Csnk2b+ / - (GFP), Csnk2b+ / - (hsyn-Csnk2b), and Csnk2b+ / - (CAG-Csnk2b) mice. Behavioral results showed that all four groups of mice preferred to interact with unfamiliar mice during the social interaction test, but there was no significant difference in social interaction preference among the four groups. Figure 3B-3C); however, in the social recognition test, Csnk2b+ / -(GFP) showed no significant tendency to contact new, unfamiliar mice compared to WT(GFP). Importantly, we found that Csnk2b+ / -(hsyn-Csnk2b) and Csnk2b+ / -(CAG-Csnk2b) mice showed a significant tendency to contact new, unfamiliar mice. ​ (D-3E). This indicates that gene complementation mediated by AAV-hSyn-CSNK2B-P2A-EGFP and AAV-CAG-CSNK2B-P2A-EGFP viruses significantly improved the abnormal social recognition or social memory abilities of Csnk2b+ / - mice.

[0122] Example 4: Therapeutic effects of CSNK2B expression by different promoters on gene-mutant mice In this embodiment, the hSyn promoter in hSyn-CSNK2B-P2A-EGFP was replaced with CMV or EF1α. AAV-CMV-CSNK2B-P2A-EGFP and AAV-EF1α-CSNK2B-P2A-EGFP were constructed and prepared using the method described above. These were then injected into the brains of Csnk2b+ / - mice, respectively. A three-box experiment was conducted to test the social abilities of WT(GFP) mice, Csnk2b+ / -(EGFP) mice, Csnk2b+ / -(CMV-CSNK2B) mice, and Csnk2b+ / -(EF1α-CSNK2B) mice. After gene complementation, the abnormal social recognition or social memory abilities of Csnk2b+ / - mice were significantly improved.

[0123] Example 5: Therapeutic effects of different viral capsid vectors on gene-mutated mice In this embodiment, AAV2-hSyn-CSNK2B-P2A-EGFP, AAV5-hSyn-CSNK2B-P2A-EGFP, AAV8-hSyn-CSNK2B-P2A-EGFP, and AAV9-hSyn-CSNK2B-P2A-EGFP were prepared by replacing different AAV serotypes as described above and injected into the brains of Csnk2b+ / - mice, respectively. A three-box experiment was conducted to test the social abilities of WT(GFP) mice, Csnk2b+ / -(EGFP) mice, Csnk2b+ / -(AAV2-CSNK2B) mice, Csnk2b+ / -(AAV5-CSNK2B) mice, Csnk2b+ / -(AAV8-CSNK2B) mice, and Csnk2b+ / -(AAV9-CSNK2B) mice. After gene complementation, the abnormal social recognition or social memory abilities of Csnk2b+ / - mice were significantly improved.

[0124] Funding Information This research was funded by the National Major Science and Technology Project "Brain Science and Brain-like Research" (#2025ZD0214700).

[0125] By incorporating via reference The entire contents of each patent and scientific document mentioned herein are incorporated herein by reference for all purposes. All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this disclosure, those skilled in the art may make various alterations or modifications to this disclosure, and equivalent forms thereof also fall within this disclosure.

[0126] Equivalence This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A method of prevention or treatment CSNK2B A method for treating gene deletion-related diseases includes administering an effective therapeutic dose to a subject in need, said therapeutic dose comprising one or more of the following: CSNK2B protein or a variant thereof, a nucleic acid molecule encoding CSNK2B protein or a variant thereof, an expression cassette containing a nucleic acid molecule encoding CSNK2B protein or a variant thereof, a vector containing a nucleic acid molecule encoding CSNK2B protein or a variant thereof, a virus such as AAV particles expressing CSNK2B protein or a variant thereof, or a cell expressing CSNK2B protein; preferably, said... CSNK2B Gene deletion-related diseases are selected from one or more of Ritter syndrome, autism spectrum disorder (ASD), Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), epilepsy, or generalized developmental delay.

2. The method of claim 1, wherein the CSNK2B protein comprises or is composed of: The amino acid sequence is as shown in SEQ ID NO: 1; the CSNK2B protein variant comprises one or more amino acid substitutions, deletions, insertions and / or additions compared to the CSNK2B protein, and has at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the CSNK2B protein biological activity; preferably, the CSNK2B protein variant comprises or consists of the following: an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1, and having at least 70%, 80%, 90%, 95%, 100%, 105%, 110% or 120% of the CSNK2B protein biological activity.

3. The method of claim 1 or 2, wherein the nucleic acid molecule encoding the CSNK2B protein or a variant thereof is a DNA sequence, a cDNA sequence, or an mRNA sequence; preferably, the nucleic acid molecule encoding the CSNK2B protein or a variant thereof comprises: (a) A nucleic acid sequence as shown in SEQ ID NO: 2; (b) A nucleic acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity with SEQ ID NO: 2, preferably having at least 95% identity, and more preferably having at least 99% identity; or (c) A codon-optimized nucleic acid sequence encoding the CSNK2B protein or a variant thereof.

4. The method of any one of claims 1 to 3, wherein the expression cassette encoding the nucleic acid molecule encoding the CSNK2B protein or a variant thereof comprises: (i) The nucleic acid sequence encoding the CSNK2B protein or a variant thereof; and (ii) A transcriptional regulatory element operatively linked to a nucleic acid sequence encoding the CSNK2B protein or a variant thereof; preferably, the transcriptional regulatory element comprises a neuron-specific promoter; preferably, the transcriptional regulatory element is selected from the human synaptophysin promoter (hSyn), CAG promoter, human cytomegalovirus promoter (hCMV), chicken β-actin promoter (CBA), simian virus 40 early promoter (SV40), mouse mammary cancer virus promoter (MMTV), human immunodeficiency virus long terminal repeat promoter (HIV LTR), MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter (EBV IE), Russ's sarcoma virus promoter (RSV), actin promoter, myosin promoter, heme promoter, creatine kinase promoter, or functional variants thereof, or combinations thereof; more preferably, the transcriptional regulatory element comprises the hSyn promoter.

5. The method of any one of claims 1 to 4, wherein the expression cassette encoding the nucleic acid molecule encoding the CSNK2B protein or a variant thereof may optionally further comprise one or more of the following: 1) A tag sequence, preferably, the tag sequence is selected from HA tag, Flag tag, His tag, GST tag, Myc tag, or a combination thereof; 2) A nucleic acid sequence encoding a self-cleaving polypeptide, preferably, the self-cleaving polypeptide is selected from P2A, T2A, F2A or variants thereof; 3) Contains transcription termination signal.

6. The method of any one of claims 1 to 5, wherein the expression cassette encoding the nucleic acid molecule encoding the CSNK2B protein or a variant thereof comprises the nucleic acid sequence shown in SEQ ID NO:

4.

7. The method according to any one of claims 1 to 6, wherein the vector comprising a nucleic acid molecule encoding the CSNK2B protein or a variant thereof is selected from plasmids, viral vectors, or non-viral vectors; the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentiviral vector, preferably an AAV vector, wherein the AAV vector is preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB, or variants thereof; the non-viral vector is selected from nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof, wherein the nanoparticles are preferably selected from liposomes, lipid nanoparticles, polymer nanoparticles, dendritic polymers, cyclodextrins, silica nanoparticles, polymer complexes, magnetic nanoparticles, gold nanoparticles, quantum dots, carbon nanotubes, or combinations thereof.

8. The method of any one of claims 1 to 7, wherein the therapeutic agent is applied to the brain of the subject, preferably to the whole brain.

9. The method according to any one of claims 1 to 8, wherein the therapeutic agent is injected retroorbitally, intravenously, or intrathecally, preferably intrathecally.

10. The method of any one of claims 1 to 9, wherein the subject is a human or non-human mammal, preferably a human.

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