AAV vector for treating autism spectrum disorder (ASD)

Abstract

Provided is an AAV vector capable of expressing a human Mef2c gene in the brain. Specifically, provided are an adeno-associated virus vector capable of expressing a full-length MEF2C protein, and the use thereof. The adeno-associated virus vector can significantly ameliorate the social deficit symptoms of mice with Mef2c gene deletion.

Description

AAV vectors for the treatment of autism spectrum disorder (ASD)

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 2024119147904, filed on December 23, 2024, and Chinese application No. 2025119495803, filed on December 22, 2025. The entire contents of both are incorporated herein by reference for all purposes. Technical Field

[0003] This invention relates to the field of biomedicine. Specifically, this invention relates to a method for introducing the MEF2C gene into the brain based on adeno-associated virus. Background Technology

[0004] MEF2C is an important member of the Myocyte Enhancer Factor 2 (MEF2) family, belonging to the MADS-BOX (MCM-1-agamous-deficiens-serum reaction factor) subfamily of transcriptional regulators. The MEF2 family includes MEF2A, MEF2B, MEF2C, and MEF2D. MEF2C is the earliest member expressed in the telencephalon of mouse embryos and is highly expressed in the outer layer of the cerebral cortex, the dentate gyrus (DG) of the hippocampus, and the amygdala in adulthood.

[0005] MEF2 is crucial for the development of various tissues and systems, including skeletal muscle, heart, blood vessels, the immune system, and the nervous system. In the nervous system, the transcriptional activity of MEF2C is regulated by extracellular stimuli, and its expression continues to regulate the expression of downstream genes, thereby inhibiting the formation of excitatory dendritic spines. Loss of MEF2C expression leads to increased dendritic spines and behavioral plasticity deficits. Furthermore, MEF2C participates in neuronal differentiation, development, and neural signal transmission, serving as an important regulator for maintaining normal brain function.

[0006] Clinically, patients carrying MEF2C gene deletions or point mutations are typically diagnosed with MEF2C haploinufficiency syndrome (MCHS), primarily characterized by severe speech impairment, epilepsy, social impairment, and repetitive, stereotyped behaviors. These features align with the diagnostic criteria for Autism Spectrum Disorder (ASD), thus MEF2C is considered an important ASD risk gene and is extensively studied. Therefore, designing vectors capable of expressing the MEF2C gene in the brain holds promise as a crucial strategy for treating ASD. Currently, there is no method for treating ASD by introducing the full-length natural MEF2C gene into the brain; therefore, there is an urgent need to develop gene therapy vectors capable of efficiently expressing the MEF2C protein. Summary of the Invention

[0007] To address the technical problem of providing a vector capable of expressing the complete MEF2C protein in the brain for the treatment of ASD, this invention is the first to use an AAV vector expressing the full-length human MEF2C protein. By intravenously injecting AAV virus, the insufficient expression of MEF2C protein in Mef2c+ / - mice was compensated, thereby improving the social deficit phenotype of the affected animals.

[0008] In a first aspect of the present invention, a MEF2C gene expression cassette is provided, characterized in that the expression cassette comprises:

[0009] (i) The nucleotide sequence encoding the MEF2C protein or a variant thereof;

[0010] (ii) A transcriptional regulatory element operatively linked to a nucleotide sequence encoding a MEF2C protein or a variant thereof, wherein the transcriptional regulatory element is selected from CAG, hSyn, CMV, L7, thy-1, recovery protein, calcium-binding protein, GAD-67, chicken β-actin, Grm6, Grm6 enhancer SV40 fusion protein, simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter or variants thereof.

[0011] In another preferred embodiment, the MEF2C protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] In another preferred embodiment, the nucleotides encoding the MEF2C protein or a variant thereof have sequences selected from the group consisting of:

[0013] (a) a nucleotide sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and

[0014] (b) A nucleotide sequence having more than 90% identity with SEQ ID NO: 3 or SEQ ID NO: 4, preferably having more than 95% identity, and more preferably having more than 99% identity.

[0015] In another preferred embodiment, the nucleotide sequence encoding the MEF2C protein or a variant thereof includes a DNA sequence, a cDNA sequence, or an mRNA sequence.

[0016] In another preferred embodiment, the nucleotide sequence encoding the MEF2C protein or a variant thereof includes single-stranded and double-stranded sequences.

[0017] In another preferred embodiment, the nucleotide sequence encoding the MEF2C protein or a variant thereof comprises a nucleotide sequence that is completely complementary to SEQ ID NO: 3 or SEQ ID NO: 4.

[0018] In another preferred embodiment, the MEF2C gene expression cassette also has a tag sequence.

[0019] In another preferred embodiment, the tag sequence is selected from: Kozak tag sequence, HA tag, Flag tag, His tag, GST tag, Myc tag, or a combination thereof.

[0020] In another preferred embodiment, the MEF2C gene expression cassette also has a transcription termination signal sequence, preferably a polyA sequence.

[0021] In another preferred embodiment, the MEF2C gene expression cassette includes, from the 5′ end to the 3′ end, the following elements operatively connected in sequence: S1-S2-S3-S4-S5-S6-S7-S8-S9.

[0022] Wherein, S1 is the AAV inverted terminal repeat (ITR) sequence 1.

[0023] S2 is the promoter.

[0024] S3 is the Kozak tag sequence.

[0025] S4 is the nucleotide sequence encoding the natural human MEF2C protein.

[0026] S5 is the HA tag sequence.

[0027] S6 is the T2A self-cleaving peptide sequence.

[0028] S7 is an EGFP-enhanced green fluorescent protein sequence.

[0029] S8 is the transcription termination signal sequence.

[0030] S9 is AAV ITR2.

[0031] In another preferred embodiment, the nucleotide sequence encoding the MEF2C gene expression cassette is shown in SEQ ID NO: 5.

[0032] In another preferred embodiment, the MEF2C gene expression cassette further comprises: an enhancer, an intron, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0033] In a second aspect of the invention, a vector is provided containing the MEF2C gene expression cassette as described in the first aspect of the invention.

[0034] In another preferred embodiment, the vector is a plasmid or a viral vector.

[0035] In another preferred embodiment, the vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector; preferably, the vector is an AAV vector.

[0036] In another preferred embodiment, the vector is an AAV vector plasmid pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0037] In another preferred embodiment, the vector is used to express the human MEF2C protein.

[0038] In another preferred embodiment, the carrier is capable of being expressed in neuronal cells.

[0039] In another preferred embodiment, the vector is capable of being expressed in animal brain tissue cells.

[0040] In a third aspect of the invention, an adeno-associated virus (AAV) particle is provided, comprising the vector described in the second aspect of the invention, and a capsid protein.

[0041] In another preferred embodiment, the AAV is selected from serotypes 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.

[0042] In a fourth aspect of the invention, a host cell is provided, the host cell comprising the MEF2C gene expression cassette as described in the first aspect of the invention, or the vector as described in the second aspect of the invention, or the AAV particles as described in the third aspect of the invention.

[0043] In another preferred embodiment, the host cell is a mammalian cell, and the mammal includes humans and non-human mammals.

[0044] In another preferred embodiment, the host cell is selected from HEK293 cells, HEK293T cells, HEK293T-17 cells, nervous system cells, or combinations thereof.

[0045] In another preferred embodiment, the host cell is selected from neuronal cells.

[0046] In a fifth aspect of the invention, a pharmaceutical composition is provided comprising the MEF2C gene expression cassette as described in the first aspect of the invention, the carrier as described in the second aspect, or the AAV particles as described in the third aspect, and a pharmaceutically acceptable carrier or excipient.

[0047] In a sixth aspect of the invention, the use of the MEF2C gene expression cassette of the first aspect, the vector of the second aspect, or the AAV particles of the third aspect, or the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating or preventing disease in a subject is provided.

[0048] In another preferred embodiment, the disease is a disease associated with a mutation in the MEF2C gene.

[0049] In another preferred embodiment, the gene mutation includes the substitution, deletion, or insertion of at least one base in the MEF2C gene sequence.

[0050] In another preferred embodiment, the diseases associated with the MEF2C gene mutation include: Ritter syndrome, autism spectrum disorder, broad-spectrum developmental delay, or a combination thereof.

[0051] In another preferred embodiment, the pharmaceutical composition is an injectable formulation, preferably an intravenous or intrathecal injection formulation.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of lyophilized formulations, liquid formulations, or combinations thereof.

[0053] In another preferred embodiment, the pharmaceutical composition is used for intravenous or intrathecal injection.

[0054] In another preferred embodiment, the carrier content in the pharmaceutical composition is 1 × 10⁻⁶. 11 -1×10 14 virus / mL, preferably 1×10⁻⁶ 12 -1×10 13 One virus per milliliter.

[0055] In another preferred embodiment, the pharmaceutical composition can increase the expression level of MEF2C protein in the cerebral cortex, hippocampus, etc. of the subject.

[0056] In a seventh aspect of the invention, a method for treating MEF2C gene deletion-related diseases is provided, the method comprising administering the MEF2C gene expression cassette of the first aspect, the vector of the second aspect, or the AAV particles of the third aspect, or the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect to a subject in need.

[0057] In another preferred embodiment, the MEF2C gene expression cassette described in the first aspect, the vector described in the second aspect, or the AAV particles described in the third aspect, or the host cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect, is introduced into the brain of the desired subject, preferably into the whole brain, the hippocampus, or the cortical region.

[0058] In another preferred embodiment, the subjects include humans and non-human mammals.

[0059] In another preferred embodiment, the non-human mammal includes rodents.

[0060] In an eighth aspect of the invention, a polynucleotide is provided, wherein the polynucleotide comprises an sgRNA sequence as shown in SEQ ID NOs: 7-8.

[0061] In a ninth aspect of the invention, the use of the polynucleotide of the eighth aspect of the invention in constructing a non-human animal model of Mef2c gene knockout is provided.

[0062] In a tenth aspect of the present invention, a non-human animal model of Mef2c gene knockout is provided, wherein the non-human animal model has a deletion of 1331 bases in the Mef2c gene, including exon 4, preferably a deletion of the nucleic acid sequence shown in SEQ ID NO: 6.

[0063] In another preferred embodiment, the animal model is used to screen for drugs to treat diseases related to the Mef2c gene deletion.

[0064] In another preferred embodiment, the animal model is constructed using sgRNA as described in the eighth aspect of the invention.

[0065] In the eleventh aspect of the present invention, a method for constructing the non-human animal model described in the tenth aspect of the present invention is provided, comprising the steps of:

[0066] (S1) Gene editing was performed on animals using the sgRNA sequence shown in SEQ ID NOs: 7-8, thereby obtaining gene-edited non-human animals; and

[0067] (S2) The obtained gene-edited non-human animals are screened to obtain the Mef2c gene deletion animal model, wherein the animal model lacks the nucleic acid sequence shown in SEQ ID NO: 6.

[0068] In another preferred embodiment, the gene editing includes the step of injecting the sgRNA described in the eighth aspect of the present invention into an animal fertilized egg and transplanting it into a surrogate mother, thereby obtaining a gene-edited animal.

[0069] In another preferred embodiment, the animal is a non-human mammal, preferably a rodent, more preferably a mouse, and even more preferably a male mouse.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Beneficial effects

[0071] This invention provides an adenovirus-associated vector that can express the complete MEF2C protein in animals, significantly improving the social deficit phenotype in mice with the Mef2c gene deletion. Attached Figure Description

[0072] This disclosure can be more fully understood with reference to the following figures.

[0073] Figure 1 shows the spectrum of the AAV-hSyn-MEF2C-T2A-EGFP-polyA vector.

[0074] Figure 2 shows the construction and identification results of the Mef2c gene deletion mouse.

[0075] Figure 3 shows the AAV virus injection protocol and the rescue effect of AAV injection on social deficits in mice.

[0076] Figure 4 illustrates the AAV virus injection protocol, showing the rescue effect of CAG promoter vector injection on social deficits in mice.

[0077] Figure 5 shows the differences in the rescue of mouse motor behavior by vectors of hSyn and CAG promoters. Detailed Implementation

[0078] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0079] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0080] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0081] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0082] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or a protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0083] As used herein, the terms “subject” and “required subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, and goats.

[0084] MEF2C Expression Box

[0085] The terms "MEF2C gene expression cassette" and "expression cassette of the present invention" are used interchangeably and both refer to the expression cassette provided in the first aspect of the present invention, wherein the expression cassette comprises:

[0086] (i) The nucleotide sequence encoding the MEF2C protein or a variant thereof;

[0087] (ii) a transcriptional regulatory element operatively linked to a nucleotide sequence encoding the MEF2C protein or a variant thereof.

[0088] The transcriptional regulatory elements mentioned therein are selected from CAG, hSyn, CMV, L7, thy-1, recovery protein, calcium-binding protein, GAD-67, chicken β-actin, Grm6, Grm6 enhancer SV40 fusion protein, simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter or variants thereof.

[0089] In another preferred embodiment, the MEF2C protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0090] In another preferred embodiment, the nucleotides encoding the MEF2C protein or a variant thereof have sequences selected from the group consisting of:

[0091] (a) a nucleotide sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and

[0092] (b) A nucleotide sequence having more than 90% identity with SEQ ID NO: 3 or SEQ ID NO: 4, preferably having more than 95% identity, and more preferably having more than 99% identity.

[0093] The expression cassette of the present invention preferably includes one or more regulatory sequences to guide the expression of a 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 expression cassette of the present invention may also include selective tags, for example, to determine the expression of the target protein in a growth system (e.g., bacterial cells) or in target cells of the nervous system.

[0094] "Operationally linked" means that nucleic acid sequences are functionally related to their operationally linked sequences such that they are linked in a way that causes them to affect each other's expression or function. For example, a nucleic acid sequence operationally linked to a promoter will have an expression pattern influenced by the promoter.

[0095] A promoter mediates the expression of a nucleic acid sequence linked to it. Promoters can be constitutive or inducible. Promoters can direct ubiquitous expression in neural cells or neuron-specific expression. In the latter case, a promoter can direct cell-type-specific expression, such as for nerve cells. Suitable promoters will be known to those skilled in the art. For example, suitable promoters can be selected from the following groups: CAG, hSyn, CMV, L7, thy-1, recovery protein, calcium-binding protein GAD-67, chicken β-actin, Grm6, Grm6 enhancer SV40 fusion protein, simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter or variants thereof. Cell-specific promoters can be used for targeting; for example, the neuron-specific promoter hSyn can be used for selective targeting of neurons. Pan-neuronal promoters can be used for universal expression.

[0096] In a preferred embodiment, the promoters that can be used in the present invention include, but are not limited to: hSyn promoter, CAG promoter (SEQ ID NO: 11), and CMV promoter (SEQ ID NO: 12).

[0097] In a preferred embodiment, the tag sequences that can be used in this invention include, but are not limited to: Kozak tag sequences, HA tags, Flag tags, His tags, GST tags, Myc tags, or combinations thereof.

[0098] In a preferred embodiment, the expression box of the present invention, from end 5' to end 3', sequentially includes the following operably connected elements: S1-S2-S3-S4-S5-S6-S7-S8-S9.

[0099] Wherein, S1 is AAV ITR1,

[0100] S2 is the promoter.

[0101] S3 is the Kozak tag sequence.

[0102] S4 is the nucleotide sequence encoding the natural human MEF2C protein.

[0103] S5 is the HA tag sequence.

[0104] S6 is the T2A self-cleaving peptide sequence.

[0105] S7 is an EGFP-enhanced green fluorescent protein sequence.

[0106] S8 is a transcription termination signal sequence with or without a transcription signal.

[0107] S9 is AAV ITR2.

[0108] In a preferred embodiment of the present invention, the expression cassette of the present invention has an hSyn-MEF2C-T2A-EGFP-polyA structure, and more preferably, the encoding nucleotide sequence of the hSyn-MEF2C-T2A-EGFP-polyA structure is shown in SEQ ID NO: 5.

[0109] Adeno-associated virus

[0110] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus *Dependent Virus* of the family Parvoviridae. It is currently the simplest single-stranded DNA-deficient virus discovered, requiring a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in two terminal inverted repeat sequences (ITRs). ITRs play a crucial role in viral replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration.

[0111] Recombinant adeno-associated virus (rAAV) vectors, derived from non-pathogenic wild-type adeno-associated virus (AAV), are considered one of the most promising gene transfer vectors due to their good safety profile, broad host cell range (dividing and non-dividing cells), low immunogenicity, and long in vivo expression time of exogenous genes. They are widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant AAV have been thoroughly understood, especially regarding their effectiveness in various cell, tissue, and in vivo experiments, for which a wealth of data has been accumulated. In medical research, rAAV is used for gene therapy research on various diseases (including in vivo and in vitro experiments); simultaneously, as a distinctive gene transfer vector, it is also widely used in gene function research, disease model construction, and the creation of gene-deleted mice.

[0112] In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can stably and site-specifically integrate into the genome of the cells they infect. They can infect a wide range of cells without affecting cell growth, morphology, or differentiation, and they do not appear to be involved in human pathology. The AAV genome has been cloned, sequenced, and characterized. AAVs contain approximately 4700 bases and include approximately 145 bases at each end of an inverted terminal repeat (ITR) region, which serves as the origin of viral replication. The remainder of the genome is divided into two important regions with capsid functions: the left portion of the genome containing the rep gene, which is involved in viral replication and viral gene expression; and the right portion of the genome containing the cap gene, which encodes viral capsid proteins.

[0113] 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 plasmid containing two AAV inverted terminal repeat (ITR) regions flanking the nucleic acid sequence of interest, and a plasmid carrying the AAV capsidation genes (rep and cap genes). The resulting AAV recombinants are then purified using standard techniques.

[0114] 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).

[0115] 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.

[0116] The serotypes of the AAV vector used in this invention 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 plasmid is selected from: pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0117] Expression vectors and host cells

[0118] The present invention also provides an expression vector for the MEF2C protein, which contains the MEF2C expression cassette of the present invention.

[0119] With the provided sequence information, skilled technicians can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

[0120] 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 MEF2C 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 AAV4YF, AAV 7m8, AAV-PHP.B, and AAV-PHP.eB).

[0121] 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.

[0122] 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 viral vectors 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 invention are preferably adapted for in vivo or in vitro use, and are preferably adapted for human use. The plasmids are preferably AAV vector plasmids pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0123] 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.

[0124] In some embodiments, the vector comprises one or more promoters operatively linked to the expression cassette, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectivity marker, nucleic acid restriction site, and / or homologous recombination site of the present invention.

[0125] Many expression vectors can be used to express the MEF2C protein in mammalian cells (preferably human, and more preferably human nerve cells). This invention preferably uses adeno-associated virus (AAV) as the expression vector.

[0126] The present invention also provides a host cell for expressing the MEF2C protein. Preferably, the host cell is a mammalian cell (preferably human, more preferably human nerve cell) to increase the expression level of the MEF2C protein.

[0127] The present invention also provides a treatment method comprising administering the carrier to a subject in need.

[0128] In another preferred embodiment, 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.

[0129] In another preferred embodiment, the carrier is introduced into a vein, ventricle, or myelin sheath of the desired subject.

[0130] In another preferred embodiment, the required subjects include humans and non-human mammals.

[0131] Pharmaceutical Composition

[0132] The present invention provides a pharmaceutical composition comprising (a) a MEF2C-expressing carrier of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

[0133] In another preferred embodiment, the pharmaceutical composition is an injectable formulation, preferably an intravenous or intrathecal injection formulation.

[0134] In another preferred embodiment, the pharmaceutical composition is used for intravenous or intrathecal injection.

[0135] In another preferred embodiment, 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.

[0136] In another preferred embodiment, the carrier content in the pharmaceutical preparation is 1 × 10⁻⁶. 11 -1×10 14 virus / mL, preferably 1×10⁻⁶ 12 -1×10 13 One virus per milliliter.

[0137] In this invention, the "active ingredient" in the pharmaceutical composition refers to the vector described herein, such as a viral vector (including adeno-associated virus vectors). The "active ingredient," formulation, and / or composition described herein can be used to treat diseases related to MEF2C gene mutations. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier or excipient" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate with the active ingredient of this invention without significantly reducing the efficacy of the active ingredient.

[0138] The composition can be liquid or solid, such as powder, gel, or paste. Preferably, the composition is liquid, and more preferably, an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0139] In this invention, the carrier can be administered directly to the brain via intraventricular injection and express the MEF2C protein within the brain. In any administration mode, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0140] 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.), and emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0141] 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.

[0142] Table 1. Sequence Information

[0143] The present invention will be 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 invention. 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.

[0144] The vector used in the following examples is the AAV-hSyn-MEF2C-T2A-EGFP-polyA vector (CCDS54878.1), and its vector spectrum is shown in Figure 1.

[0145] Example

[0146] 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.

[0147] Example 1: Construction and Genotyping of Mef2c Gene Deletion Mice

[0148] 1.1 Construction of Mef2c gene deletion mice

[0149] In this embodiment, a mouse model with the Mef2c gene deletion was constructed. First, using the CRISPR-Cas system, two sgRNAs, sg1(+) and sg2(-), were designed near exon 4 of the mouse Mef2c gene, as shown in Table 2 (Figure 2A).

[0150] The synthesized spCas9 mRNA (Invitrogen) TM The gene-edited mice were injected together with two sgRNAs (product code A29378) into fertilized eggs of 8-week-old C57BL / 6 mice. The fertilized eggs were then transferred to 8-week-old ICR surrogate mothers to obtain several gene-edited mice.

[0151] Heterozygous female mice with a 1331bp deletion in the Mef2c gene (Figure 2A) were selected and passaged to establish a lineage. These female mice were designated founder mice (Mef2c+ / -, F0). Eight-week-old Mef2c+ / - female mice were backcrossed with eight-week-old C57BL / 6 male mice for stable passage. Genotypes of the offspring were identified by PCR amplification.

[0152] 1.2 Identification of Mef2c gene deletion mice

[0153] PCR primers MEF2C-Mut-F (SEQ ID NO: 9) and MEF2C-Mut-R (SEQ ID NO: 10) were designed near the Mef2c genome to specifically amplify the Mef2c gene fragment. Primer sequences are shown in Table 2 and Figure 2B.

[0154] The presence of a knockout of the Mef2c gene in mice can be determined by performing DNA gel electrophoresis on the PCR amplified fragment (a 256bp band appears), as shown in Figure 2C.

[0155] Table 2. sgRNA and primers

[0156] Example 2: AAV.PHP.eB-hSyn-MEF2C-T2A-EGFP virus was injected into the brains of Mef2c+ / - mice.

[0157] After stabilizing 4-week-old mice, their tails were placed on a heating pad at 37-40℃ for 3-5 minutes to expose the tail vein. The side with the clearly exposed tail vein was selected, disinfected with an alcohol swab, and a syringe was connected to a 30G needle. AAV solution was drawn up, air bubbles were removed, and the syringe was inserted into the tail vein at a 10-15° angle, slowly advancing and injecting 100 μL of AAV solution. After injection, the injection site was pressed with a cotton swab to stop bleeding, and the mice were returned to their cages for observation. Control group mice were injected with AAV.PHP.eB-hSyn-EGFP virus and labeled: Mef2c+ / -(EGFP); experimental group mice were injected with AAV.PHP.eB-hSyn-MEF2C-T2A-EGFP virus (Paizhen Biotechnology, C-12396) and labeled: Mef2c+ / -(MEF2C); littermate wild-type mice were labeled: Mef2c WT (Figure 3A).

[0158] Example 3: Injection of AAV.PHP.eB-hSyn-MEF2C-T2A-EGFP virus improves the social behavior deficit phenotype in Mef2c+ / - mice.

[0159] In this embodiment, the improvement of ASD-related behaviors in Mef2c gene-deficient mice by the vector constructed in this invention is examined.

[0160] We used the classic three-box behavioral paradigm (Figure 3B) to assess the social abilities of Mef2c WT, Mef2c+ / - (EGFP), and Mef2c+ / - (MEF2C) mice. Behavioral results showed that Mef2c WT, Mef2c+ / - (EGFP), and Mef2c+ / - (MEF2C) mice were more inclined to interact with unfamiliar mice in the social interaction test, with no significant difference in social interaction preference among the three groups (Figures 3C-D, top). However, in the social recognition test, compared to Mef2c WT, which showed a greater inclination to interact with new unfamiliar mice, Mef2c+ / - (EGFP) did not exhibit a significant tendency to interact with new unfamiliar mice. Importantly, we found that Mef2c+ / - (MEF2C) showed a significant tendency to interact with new unfamiliar mice (Figures 3C-D, bottom). This indicates that gene complementation mediated by AAV.PHP.eB-hSyn-MEF2C-T2A-EGFP virus significantly improved the abnormal social recognition or social memory abilities of Mef2c+ / - mice.

[0161] Example 4: Therapeutic effects of MEF2C expression by different promoters on gene-mutant mice

[0162] In this embodiment, the hSyn promoter in hSyn-MEF2C-T2A-EGFP is replaced with the CAG promoter. The effects of different promoters expressing MEF2C on the therapeutic effect are investigated to find the promoter-MEF2C combination with the best therapeutic effect.

[0163] Using the method described above, AAV.PHP.eB-CAG-MEF2C-T2A-EGFP was constructed and injected into the brains of Mef2c+ / - mice. A three-box test was conducted to assess the social abilities of Mef2c WT mice, Mef2c+ / - (CAG-EGFP) mice, and Mef2c+ / - (CAG-MEF2C) mice. Behavioral results showed:

[0164] Mef2c WT, Mef2c+ / -(CAG-EGFP), and Mef2c+ / -(CAG-MEF2C) mice all showed a greater tendency to interact with unfamiliar mice in the social interaction test, with no significant difference in social interaction preference among the three groups (Figure 4C-D, top). However, in the social recognition test, compared to Mef2c WT, Mef2c+ / -(EGFP) showed a greater tendency to interact with new unfamiliar mice, while Mef2c+ / -(EGFP) did not show a significant tendency to interact with new unfamiliar mice. Furthermore, we found that Mef2c+ / -(MEF2C) showed a significant tendency to interact with new unfamiliar mice (Figure 4C-D, bottom). This indicates that after gene complementation mediated by AAV-CAG-MEF2C-T2A-EGFP virus, the abnormal social recognition or social memory abilities of Mef2c+ / - mice were significantly improved, and there was no significant difference compared to gene complementation treatment mediated by AAV-hSyn-MEF2C-T2A-EGFP.

[0165] However, we also observed a significant increase in movement speed in Mef2c+ / - mice during the open field experiment, indicating hyperactivity. For this behavioral phenomenon, we found that AAV-hSyn-MEF2C-T2A-EGFP-mediated gene complementation therapy significantly improved the hyperactivity in Mef2c+ / - mice (Figures 5A-C), while AAV-CAG-MEF2C-T2A-EGFP-mediated gene complementation therapy exacerbated the hyperactivity in Mef2c+ / - mice (Figures 5D-F). Therefore, combining the results of our two behavioral experiments, we believe the hSyn promoter is the superior promoter.

[0166] Table 3. Promoter Sequences

[0167] By incorporating via reference

[0168] The entire contents of each patent and scientific document mentioned herein are incorporated herein by reference for all purposes. All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that various alterations or modifications can be made to this invention by those skilled in the art after reading the foregoing teachings, and these equivalent forms also fall within the scope of this application.

[0169] Equivalence

[0170] 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 MEF2C gene expression cassette, characterized in that, The expression box includes: (i) The nucleotide sequence encoding the MEF2C protein or a variant thereof; (ii) a transcriptional regulatory element operatively linked to a nucleotide sequence encoding the MEF2C protein or a variant thereof. The transcriptional regulatory elements are preferably selected from CAG, hSyn, CMV, L7, thy-1, recovery protein, calcium-binding protein, GAD-67, chicken β-actin, Grm6, Grm6 enhancer SV40 fusion protein, simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter or variants thereof.

2. The MEF2C gene expression cassette as described in claim 1, optionally comprising a tag sequence, preferably, the tag sequence being selected from Kozak tag sequences, HA tags, Flag tags, His tags, GST tags, and Myc tags.

3. The MEF2C gene expression cassette as described in claim 1 or 2, further comprising a self-cleaving peptide sequence, preferably, the self-cleaving peptide sequence being selected from T2A.

4. The MEF2C gene expression cassette according to any one of claims 1 to 3, optionally comprising a transcription termination signal.

5. The MEF2C gene expression cassette according to any one of claims 1 to 4, characterized in that, The MEF2C gene expression cassette contains the nucleic acid sequence shown in SEQ ID NO:

5.

6. A vector comprising the MEF2C gene expression cassette as claimed in any one of claims 1 to 5.

7. The carrier as described in claim 6, characterized in that, The vector is a plasmid or a viral vector.

8. The vector of claim 7, wherein the viral vector is an AAV vector, an adenovirus vector, or a lentivirus vector.

9. An adeno-associated virus (AAV) particle comprising the vector and capsid protein as described in any one of claims 6 to 8.

10. The AAV particle of claim 9, wherein the AAV is selected from serotypes 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.

11. A host cell comprising the MEF2C gene expression cassette as claimed in any one of claims 1 to 5, the vector as claimed in any one of claims 6 to 7, or the AAV particle as claimed in claim 9 or 10.

12. A pharmaceutical composition comprising a MEF2C gene expression cassette as claimed in any one of claims 1 to 5, a vector as claimed in any one of claims 6 to 7, or an AAV particle as claimed in claim 9 or 10, and a pharmaceutically acceptable carrier or excipient.

13. Use of the MEF2C gene expression cassette as described in any one of claims 1 to 5, the vector as described in any one of claims 6 to 7, or the AAV particle as described in claim 9 or 10, or the pharmaceutical composition as described in claim 12, in the preparation of a medicament for treating or preventing disease in a subject.

14. The use as claimed in claim 10, wherein the disease is a disease related to MEF2C gene mutation, preferably, the disease is selected from Ritter syndrome, autism spectrum disorder, and generalized developmental delay.

15. A method for treating MEF2C gene deletion-related diseases, the method comprising administering the MEF2C gene expression cassette as described in any one of claims 1 to 5, the vector as described in any one of claims 6 to 7, or the AAV particles as described in claim 9 or 10, or the pharmaceutical composition as described in claim 12, to a subject in need.

16. The use as described in claim 13 or 14, or the method as described in claim 15, wherein the drug is applied to the brain of the subject, preferably to the whole brain, the hippocampus, or a cortical region.

17. A method for constructing a non-human animal model of Mef2c gene knockout, including the following steps: (S1) Gene editing was performed on animals using the sgRNA sequence shown in SEQ ID NOs: 7-8, thereby obtaining gene-edited non-human animals; and (S2) The obtained gene-edited non-human animals are screened to obtain the Mef2c gene deletion animal model, wherein the animal model lacks the nucleic acid sequence shown in SEQ ID NO: 6.

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