Composition for treating dysgnosia, application thereof and drug screening method

Abstract

The invention discloses a composition for treating dysgnosia, application thereof and a medicine screening method. According to the invention, the AAV virus carrying the hCAPZA2 gene is introduced into a CAPZA2 heterozygous knockout or mutated mouse model, and the effectiveness of the AAV virus in the aspects of improving the CAPZA2 expression level, recovering the synaptic function and improving the neural behavior is verified. The invention not only provides a new hope for treating the mental disorder related to the CAPZA2 gene, but also opens up a new direction for the application of gene therapy in neurodevelopment diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition for treating intellectual disability, an application thereof and a drug screening method. Background Art

[0002] Intellectual Disability (ID) is a group of developmental disorders characterized by cognitive impairment and adaptive behavior defects, which seriously affect patients' learning, communication and social participation abilities. Genetic factors play an important role in the pathogenesis of intellectual disability. Intellectual disability is a complex neurodevelopmental disease, and patients usually show symptoms such as significant cognitive deficits, difficulty in social interaction, anxiety and abnormal motor behavior. Although there are currently many methods to try to treat these symptoms, such as drug intervention and behavioral therapy, these methods are often difficult to cure or significantly improve the neurological defects caused by gene mutations.

[0003] With the development of gene therapy technology, the use of adeno-associated virus (AAV) vectors to deliver functional genes to specific tissues has become a cutting-edge strategy for repairing genetic defects. Gene therapy is the process of correcting or compensating for diseased genes by introducing specific genes into the body, thereby achieving the purpose of treating genetic diseases.

[0004] In recent years, multiple AAV serotypes and variants have been identified, which can recognize different cell receptors and thus show different tissue and cell tropisms. Although the VP3 structures of various AAV serotypes are highly homologous, the genes in the variable region (VR) are significantly different, which affects their binding and interaction with cell surface receptors. However, this variation usually does not affect the assembly of viral particles. Therefore, when designing new AAV capsid vectors, the VR region is mainly modified to maintain the structural integrity of the capsid while enhancing its targeting and transduction efficiency.

[0005] Recombinant adeno-associated virus (rAAV) has been shown to be effective as a gene delivery vector in a variety of basic science studies and clinical trials. Gene therapy using rAAV vectors has several advantages: (i) long-term stable transgene expression; (ii) not associated with any known human disease; (iii) triggering a weak immune response; (iv) being able to effectively transduce both dividing and non-dividing cell types; and (v) the rAAV genome can be packaged in different viral capsids that have unique transduction properties and tissue tropisms.

[0006] Currently, rAAV-based gene therapy has achieved remarkable success in clinical trials, including for the treatment of diseases such as Leber's congenital amaurosis (LCA), spinal muscular atrophy (SMA), and lipoprotein lipase deficiency. Despite this, existing AAV vectors still have limitations in certain applications, especially the lack of effective transduction ability for specific cell types or tissues. In particular, many nervous system-related diseases require efficient central nervous system transduction ability, so it is necessary to develop new AAV serotypes or variants to improve the effect of gene therapy, reduce the required dose, and reduce potential side effects.

[0007] For example, AAV vector delivery of the MECP2 gene has been used to treat Rett syndrome, a severe neurodevelopmental disorder. Researchers used AAV9 vectors to deliver the MECP2 gene to the brain of a Rett syndrome mouse model in an effort to correct the neurological deficits caused by MECP2 gene mutations. Although certain behavioral improvements and neuroprotective effects have been observed in some models, this approach has significant shortcomings, mainly in that overexpression of the MECP2 gene may lead to neurotoxicity and faces challenges in long-term efficacy and dose control.

[0008] For another example, US patent application US20160038613A1 discloses a technology for treating spinal muscular atrophy (SMA) by delivering the SMN1 gene through an AAV9 vector. The AAV treatment of the SMN1 gene was developed for SMA patients. The technology delivers the SMN1 gene carried by the AAV9 vector to the patient's motor neurons through systemic intravenous injection to compensate for the protein deficiency caused by the SMN1 gene mutation, thereby improving the patient's motor function. This treatment has shown significant effects in prolonging the patient's life and improving motor function, and has been widely used clinically. AAV9 was selected as a vector because of its superior ability to cross the blood-brain barrier and effective transduction of the central nervous system. However, although this technology has achieved good results in the treatment of SMA, it mainly targets motor neurons and its adaptability is limited when facing other types of neurological diseases. In addition, although the initial effect of treatment is significant, the sustainability of the efficacy over time still needs to be further evaluated.

[0009] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0010] In order to solve at least some of the technical problems in the prior art, the present invention provides an AAV gene therapy scheme for rescuing intellectual disability caused by CAPZA2 mutation. The scheme uses a recombinant AAV2 / 9 vector (pFD-scAAV-ITR-CBh-opt-hCAPZA2-BGHpA) to correct neurological dysfunction caused by CAPZA2 mutation with optimized hCAPZA2 gene expression. The AAV2 / 9 vector developed by the present invention has a high transduction efficiency in the central nervous system and combines self-complementary AAV (scAAV) technology to accelerate gene expression, thereby achieving a higher therapeutic effect. The present invention fundamentally improves the behavioral abnormalities of patients with intellectual disabilities by regulating brain synaptic plasticity. Specifically, the present invention includes the following contents.

[0011] In a first aspect of the present invention, a composition for treating intellectual disability is provided, which comprises a recombinant adeno-associated virus carrying the nucleic acid shown in SEQ ID NO.1.

[0012] In certain embodiments, according to the composition for treating intellectual disability of the present invention, the gene sequence of the recombinant adeno-associated virus is as shown in SEQ ID NO.2.

[0013] In certain embodiments, the composition for treating intellectual disability according to the present invention is an injection.

[0014] The second aspect of the present invention provides a method for treating intellectual disability in a subject in need thereof (the use of an agent in the preparation of a medicament for treating intellectual disability in a subject in need thereof), wherein the agent comprises the composition described in the first aspect, and the treatment comprises the step of administering a therapeutically effective amount of the composition described in the first aspect to a subject in need thereof.

[0015] In certain embodiments, according to the method or use for treating intellectual disability in a subject according to the present invention, the administration comprises intracerebral injection.

[0016] In certain embodiments, according to the method or use for treating intellectual disability in a subject according to the present invention, the subject is under 18 years old.

[0017] In certain embodiments, according to the method or use of the present invention for treating intellectual disability in a subject, the subject is diagnosed with CAPZA2 c.776G>T Missense mutation.

[0018] The third aspect of the present invention provides a method for testing the effect of a drug to be tested in treating intellectual disability (a method for screening a drug useful for treating intellectual disability), comprising the following steps: (1) providing an animal model and detecting its behavioral indicators to obtain a first parameter, wherein the animal model contains a C.776G>T mutation in CAPZA2, and the first parameter includes social ability, motor ability, anxiety index and cognitive ability; (2) administering the drug to be tested to the animal model, and then detecting behavioral indicators to obtain a second parameter, wherein the second parameter includes the following indicators detected after administration of the drug to be tested: social ability, motor ability, anxiety index and cognitive ability; and (3) A step of comparing the first parameter and the second parameter, wherein when improvement of the behavioral index is observed in the second parameter, the drug to be tested is screened as a drug useful for treating intellectual disability, and when no improvement of the behavioral index is observed in the second parameter, the drug to be tested is screened as a drug not useful for treating intellectual disability.

[0019] In certain embodiments, according to the method for testing the effect of a test drug in treating intellectual disability of the present invention, behavioral indicators are detected 4 months after administration of the test drug.

[0020] In certain embodiments, according to the method for testing the effect of a drug to be tested in treating intellectual disability according to the present invention, the behavioral indicators include social ability (Socialbility), motor ability (OFT, RRT), anxiety index (EPM), and cognitive ability (Y-maze, ORT, MWM).

[0021] The present invention introduces AAV virus carrying the hCAPZA2 gene into a mouse model with heterozygous knockout or heterozygous mutation of CAPZA2, and verifies its effectiveness in increasing CAPZA2 expression levels, restoring synaptic function, and improving neurobehavior. This technology not only provides new hope for the treatment of intellectual disabilities related to the CAPZA2 gene, but also opens up a new direction for the application of gene therapy in neurodevelopmental diseases.

[0022] In addition, the technical effects of the present invention also include: (1) Targeted treatment: The present invention provides a specific treatment method for neurological diseases caused by CAPZA2 gene deficiency. Through precise gene supplementation, it is expected to directly treat the cause of the disease.

[0023] (2) Efficient gene transduction: By utilizing the efficient transduction ability of the AAV9 vector, the present invention achieves specific and efficient gene delivery to neurons, improving the therapeutic effect while reducing the potential impact on non-target tissues.

[0024] (3) Optimized gene expression: Through the neuron-specific expression control of the CBh promoter, the present invention optimizes the expression of the CAPZA2 gene in the central nervous system, which helps to achieve a more precise therapeutic effect.

[0025] (4) Comprehensive behavioral improvement: The present invention significantly improved multiple behavioral phenotypes of CAPZA2-deficient mice through AAV-CAPZA2 treatment, including motor activity, anxiety-like behavior, motor coordination, social interaction and cognitive ability, demonstrating the comprehensiveness and effectiveness of the treatment.

[0026] (5) Long-term maintenance of efficacy: In long-term behavioral evaluations, the therapeutic effects of the present invention can last for a long time, indicating that AAV-CAPZA2 treatment may have the potential to maintain long-term efficacy. However, similar gene therapy studies currently do not have long-term follow-up, and most studies only conduct behavioral testing once.

[0027] (6) Safety verification: Through body weight monitoring and biochemical index testing, the present invention confirmed that AAV-CAPZA2 treatment improved the behavioral phenotype without causing significant adverse effects on the growth and development and important physiological indicators of mice.

[0028] (7) Synaptic function recovery: The present invention restores the expression of PSD95, a molecule closely related to synaptic plasticity, and reveals that AAV-CAPZA2 treatment may promote the repair and functional recovery of the nervous system by improving synaptic function.

[0029] (8) Potential clinical application prospects: The present invention provides a gene therapy strategy that may be applicable to human CAPZA2-related diseases, providing a scientific basis and therapeutic potential for the development of new clinical treatment methods.

[0030] (9) Scientific and social benefits: The present invention not only provides a deeper scientific understanding of CAPZA2-related diseases, but also has the potential to reduce patients' suffering and medical costs and improve their quality of life at the social level. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The structure of the recombinant scAAV vector constructed in the present invention is shown.

[0032] Figure 2 Shown are the total distance traveled in the open field, open arm retention time in the elevated plus maze, time to fall from the rotarod, and new and old object contact time in novel object recognition and cognitive indices in CAPZA2 heterozygous knockout (Het) mice 2 months after gene therapy.

[0033] Figure 3Shown are the interaction time and interaction index of social interaction and social preference in three boxes 2 months after gene therapy in Het mice.

[0034] Figure 4-5 Shown is the water maze behavioral test of Het mice 2 months after gene therapy.

[0035] Figure 6 Shown are the total distance traveled in the open field, spontaneous alternation rate in the Y-maze, open arm retention time in the elevated plus maze, time to fall from the rotarod, novel object recognition, new and old object contact time, and cognitive index of MUT mice 6 weeks after gene therapy.

[0036] Figure 7 Shown are the interaction time and interaction index of three-week box social interaction and social preference in MUT mice 6 weeks after gene therapy.

[0037] Figure 8 Shown are the total movement distance in the open field, the spontaneous alternation rate in the Y-maze, the open arm retention time in the elevated plus maze, the contact time between the new and old objects in the novel object recognition, and the cognitive index of the MUT mice 4 months after gene therapy.

[0038] Fig. 9 Shown are the interaction time and interaction index of social interaction and social preference in three boxes of MUT mice 4 months after gene therapy.

[0039] Fig.10 Shown are the total movement distance in the open field, spontaneous alternation rate in the Y-maze, open arm retention time in the elevated plus maze, time to fall from the rotarod, contact time between the new and old objects in novel object recognition, and cognitive index of MUT mice 8 months after gene therapy.

[0040] Fig.11 Shown are the interaction time and interaction index of social interaction and social preference in three boxes of MUT mice 8 months after gene therapy.

[0041] Fig.12 Shown is the water maze behavioral test of MUT mice 8 months after gene therapy.

[0042] Fig.13 Shown are the total distance traveled in the open field, spontaneous alternation rate in the Y-maze, open arm retention time in the elevated plus maze, novel object recognition, new and old object contact time, and cognitive index of MUT mice 13 months after gene therapy.

[0043] Fig.14 Shown is the water maze behavioral test of MUT mice 13 months after gene therapy.

[0044] Fig.15 The figure shows the changes in body weight of mice after sampling.

[0045] Fig.16 The protein and PCR detection results of mice are shown. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] Composition for treating intellectual disability One aspect of the present invention provides a composition for treating intellectual disability, comprising a recombinant adeno-associated virus and an optional pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus carries the nucleic acid shown in SEQ ID NO.1.

[0050] Typically, adeno-associated viruses (sometimes referred to as "AAV") contain rep genes and cap genes, as well as two terminal inverted repeats (ITRs) flanking them, wherein the rep gene encodes Rep78, Rep68, Rep52, and Rep40 proteins for the replication of the AAV genome and the assembly of viral particles. The cap gene encodes three major capsid proteins (VP1, VP2, and VP3), which are produced by alternative transcription and splicing regulation of rare start codons (ACG) and together constitute the capsid of viral particles. In addition, the internal open reading frame (ORF) of the cap gene also includes an assembly activation protein (AAP) that encodes the necessary shell assembly.

[0051] In the present invention, the recombinant adeno-associated virus is an AAV recombinant vector obtained by modifying the wild-type adeno-associated virus. The wild-type adeno-associated virus genome is about 4.7 kb and consists of 60 VP subunits, wherein the ratio of VP1, VP2 and VP3 is about 1:1:10. The recombinant adeno-associated virus (sometimes referred to as "rAAV") usually retains about 4% of the viral genome, i.e., the inverted terminal repeat sequence, does not express viral genes, and exists as a non-integrated fragment, so that it has relatively good tolerance for target gene transfer in different target tissues of different species.

[0052] After in-depth research, the inventors found that by designing the coding region based on rAAV and then performing transcription and translation, there is no need to wait for the second-strand DNA synthesis, thus eliminating the rate-limiting step of rAAV gene expression and shortening the time to start expression. The peak expression level is generally reached 3-5 days after infection.

[0053] Although the advantages of various AAV serotypes in delivering target genes have been reported, there are still great challenges in using AAV as a vector in clinical settings due to difficulties in effectively delivering target genes to target tissues, achieving long-term expression of corrective transgenes, and avoiding harmful effects of the host immune system.

[0054] In a preferred embodiment, the recombinant adeno-associated virus vector of the present invention comprises an operably linked enhancer, intron, promoter, human CAPZA2 complementary DNA, terminator sequence, and ITR sequence flanked by the above sequence, and the preferred ITR sequence is ITR from AAV9. In a specific embodiment, the nucleic acid for overexpressing CAPZA2 in the recombinant adeno-associated virus vector has the sequence shown in SEQ ID NO.1.

[0055] In a preferred embodiment, the coding sequence of the recombinant adeno-associated virus vector of the present invention is shown as SEQ ID NO.2.

[0056] In the present invention, a pharmaceutically acceptable carrier is involved in carrying or transporting the recombinant adeno-associated virus from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and does not harm the patient. In the present invention, the pharmaceutically acceptable carrier includes at least one of a diluent, an absorbent, a wetting agent, a sweetener, a preservative, and an antioxidant. Pharmaceutically acceptable carriers are preferably those administered by injection, examples of which include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion vehicles, and the like.

[0057] In a preferred embodiment, the composition for treating intellectual disability is an injection, and is especially administered to a subject in need thereof in the form of intracerebral injection.

[0058] Methods for treating intellectual disability in a subject in need thereof One aspect of the present invention provides a method for treating intellectual disability or its related disorders (symptoms or conditions) in a subject in need thereof, which may also be referred to as "the use of an agent in the preparation of a medicament for treating intellectual disability or its related disorders (symptoms or conditions) in a subject in need thereof", wherein the agent includes the composition described in the present invention.

[0059] The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, who are to receive a specific treatment. Generally, "subject" and "patient" are used interchangeably in the present invention to refer to a human subject. In a specific embodiment, the subject has been diagnosed with the presence of CAPZA2 c.776G>T Subjects with missense mutations, particularly those due to CAPZA2 c.776G>T In another embodiment, the subject is under 18 years old.

[0060] The term "effective amount" as used herein means the amount of a drug or medicament that induces a biological or pharmaceutical response of a tissue, system, animal or human being, such as that pursued by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that effectively enhances normal physiological function. Typically, the effective amount herein varies according to various factors, such as a given drug or composition, a pharmaceutical preparation, a route of administration, the type of disease or condition, the subject being treated, etc., but can still be routinely determined by those skilled in the art.

[0061] The term "treating" as used herein refers to improving a condition before or after a disease or disorder occurs. This relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique compared to an untreated control group under the same conditions. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation (whether partial or complete).

[0062] The therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the composition of the present invention is well known to those skilled in the art. The dosage can be administered in a single dosage form or in several, for example, two, three or four dosage forms. The dosage administered is within the predictable range of the clinician or laboratory personnel, for example, the dosage can be appropriately adjusted through effectiveness and safety testing to obtain the optimal dosage.

[0063] In the present invention, "treating intellectual disability" means being able to improve or treat the c.776G>T Missense mutations leading to at least one of the following: (1) Improve cognitive impairment and adaptive behavior defects; (2) Improve learning, communication, and social participation skills; (3) restore or increase CAPZA2 expression levels; (4) restore or increase the expression level of PSD95; (5) effectiveness in restoring synaptic function and improving neurobehavior; (6) Promote the repair and functional recovery of the nervous system.

[0064] In a preferred embodiment, the composition of the present invention is preferably administered to a subject in need thereof by intracerebral injection.

[0065] Method for testing the effect of a drug to be tested in treating intellectual disability One aspect of the present invention provides a method for testing the effect of a drug to be tested in treating intellectual disability, comprising: (1) providing an animal model and detecting its behavioral indicators to obtain a first parameter, wherein the animal model contains a c.776G>T mutation in CAPZA2; (2) administering the drug to be tested to the animal model, and then detecting behavioral indicators to obtain a second parameter; and (3) A step of comparing the first parameter and the second parameter.

[0066] In the present invention, the animal model is not particularly limited, for example, it can be any model animal, such as rats, mice, chickens, rabbits, etc. Methods for performing c.776G>T mutation on CAPZA2 of animal models are known in the art, for example, gene editing methods can be used to introduce the above point mutation.

[0067] In the present invention, the detected indicators also include the amount of CAPZA2 and the amount of PSD95 measured before the administration of the drug, and the amount of CAPZA2 and the amount of PSD95 measured after the administration of the drug.

[0068] In the present invention, when the improvement of behavioral indicators (including improvement of cognitive impairment and adaptive behavior defects; improvement of learning, communication and social participation abilities) is observed in the second parameter, and / or the change of related gene expression level is detected (including increase of CAPZA2 amount and PSD95 amount), it indicates that the drug to be tested can effectively treat intellectual disability. When the improvement of behavioral indicators (including improvement of cognitive impairment and adaptive behavior defects; improvement of learning, communication and social participation abilities) is not observed in the second parameter, and / or the change of related gene expression level is detected (including unchanged or decreased CAPZA2 amount and PSD95 amount), it indicates that the drug to be tested cannot effectively treat intellectual disability.

[0069] In certain embodiments, behavioral indicators are detected at a time point of 4 months after administration of the test drug, wherein the behavioral indicators include indicators in the open field test (such as total movement distance), the Y-maze (including the cycling index), the three-box social test (including social interaction, social preference ability), and the elevated plus maze (such as the retention time in the open arm), and do not include cognitive indexes in new object recognition and indicators of exploration time for new and old objects.

[0070] In certain embodiments, behavioral indicators are detected at a time point of 8 months after administration of the test drug, wherein the behavioral indicators include open field test, Y-maze, three-box social test, elevated plus maze, cognitive index in novel object recognition, and exploration time for new and old objects, and do not include the time indicator for falling from the rod in the rotating rod test.

[0071] In certain embodiments, behavioral indicators are detected at a time point of 13 months after administration of the test drug, wherein the behavioral indicators include open field test, Y-maze, three-box social test, elevated plus maze, water maze (including learning trajectory during training period, time to find the platform period and number of crossings), cognitive index in new object recognition, exploration time for new and old objects, and time index for falling from the rod in the rotating rod test.

[0072] Example 1. AAV vector construction Design and construct AAV9 vector carrying CAPZA2 gene: AAV-CAPZA2 contains human CAPZA2 complementary DNA and the strong neuron-specific promoter CBh drives CAPZA2 gene expression. The final overexpression AAV virus is: PFD-scAAV-ITR-CBh-opt-hCAPZA2-BGHpA (Fiter: 2.8E+13 vg / ml), and the control virus is: AAV2 / 9: PFD-scAAV-CBh-GFP-BGHpolyA (Fiter: 1.7E+13 vg / ml).

[0073] 2. Preparation of experimental animal models Through homology comparison, it was found that the patient carried CAPZA2 c.776G>T A missense mutation, in which the amino acid arginine R is mutated to leucine L, was constructed to generate the corresponding mutant mouse. Precise introduction of point mutations by homology-directed repair to create CAPZA2 c.G776T CAPZA2 heterozygous mice were created by deleting exons 3 to 6 of the CAPZA2 gene located on chromosome VI using CRISPR / Cas technology. + / - ) mice.

[0074] 3. AAV vector administration In order to explore whether AAV-CAPZA2 can rescue the behavioral phenotype of defective mice, this example performed behavioral testing after overexpressing AAV-CAPZA2 in the bilateral lateral ventricles. The AAV-CAPZA2 vector was injected into the lateral ventricle of the mouse brain (X=±1.0, Y=-0.58, Z=-2.0) using the intracerebral stereotaxic injection method. + / - All mice were 5 months old, half male and half female, with 8 mice in each group. The injection scheme set two doses, the low dose group 5 E+9 vg / each side, the high dose group 1.0 E+10 vg / each side, and the control group used the same number of empty virus particles. CAPZA2 c.G776T All mice were 3 months old, with 6 females and 5 males in each group. A single dose injection (5 E+9 vg / each side) was used, and the control group used the same number of empty virus particles. The mice were anesthetized with 1.5-2% isoflurane and placed on the mouse adapter of the stereotaxic instrument. The hair on the head was removed, the skin was cut to expose the skull, and the stereotaxic instrument was used to level the front and back left and right with Bregma as the zero point to keep the mouse head balanced. The AAV virus was slowly injected (50 nL / min) into the bilateral LV brain area and allowed to stay and diffuse for 10 min.

[0075] Use a suture needle to suture the skin of the head. Determine the number of suture needles according to the size of the opening. After suturing, wipe the scalp again with 75% to avoid infection. After completion, remove the animal from the adapter and place it in a water bath until the animal wakes up. Then put the animal in a cage and give it plenty of water and food.

[0076] 4. Behavioral Assessment 1. AAV-CAPZA2 vs CAPZA2 + / - Behavioral rescue assessment in mice CAPZA2 + / - The mice were evaluated behaviorally 2 months after virus injection, and the results showed that gene therapy in both dose groups effectively rescued the Figure 2-5The inventors first tested the motor activity of each group of mice and found that CAPZA2 + / - Control mice (CAPZA2 + / - The total distance of movement of mice injected with empty virus in the open field test was significantly increased, indicating abnormally increased motor activity. After AAV-CAPZA2 was administered, the total distance of movement was significantly reduced, indicating that the motor activity was normalized. The following was a test for anxiety-like behavior. The results showed that compared with the normal group, CAPZA2 + / - The control mice spent significantly less time in the open arms of the elevated plus maze, indicating that CAPZA2 deficiency can cause anxiety in mice, and administration of AAV-CAPZA2 significantly reduced anxiety levels. The motor coordination ability of each group of mice was then tested, and it was found that AAV-CAPZA2 could significantly rescue the motor dysfunction of defective mice and increase the time that mice stayed on the balance rod in the rotating rod test.

[0077] Next, the effect of AAV-CAPZA2 on the social ability of CAPZA2 mice was investigated. The results of three social boxes showed that CAPZA2 deficiency would lead to a lack of social interaction in mice and reduce their social preference ability, while AAV treatment could significantly correct the social abnormalities of mice. Finally, the cognitive ability of each group of mice was tested. In the Y-maze, AAV treatment significantly improved the circulation index of defective mice. In the new object recognition test, AAV treatment significantly improved CAPZA2 + / - The recognition index of mice increased, and the exploration time of mice for new objects was increased; similarly, in the 7-day water maze experiment, a significant rescue effect of AAV treatment was observed. The exogenous injection of AAV-CAPZA2 significantly normalized the learning trajectory of defective mice during the training period, significantly improved their spatial learning ability, and in the test period, it was shown that the time and number of crossings in the CAPZA2-treated group mice to find the platform were significantly increased to normal levels, indicating that their cognitive level was significantly restored. After 1 month of behavioral testing, the mice in each group were in good condition. In addition, it was found that high doses of exogenous AAV-CAPZA2 had a significant effect on CAPZA2 + / - The rescue effect of mice was good.

[0078] 2. AAV-CAPZA2 vs CAPZA2 c.G776T Behavioral rescue assessment in mice CAPZA2 c.G776TThe mice were injected with low doses of virus and behaviorally evaluated at different time points after injection to explore the long-term effectiveness of AAV-CAPZA2. The first test point was 6 weeks after virus injection. It was found that the abnormal indicators of the mice in the treatment group in the open field test, Y-maze, and three-box social test were corrected, while the abnormalities in the elevated plus maze, rotating rod test, and new object recognition were not significantly corrected. Specifically, the exploration time of the open arm of the elevated plus maze did not increase, the time for mice to fall from the rod in the rotating rod test did not increase, and it was difficult to distinguish new objects from old objects in new object recognition (such as Figure 6-7 The second test point was 4 months after virus injection. In addition to the callback index in the 6-week test, the anxiety performance in the elevated plus maze test was normalized, while the abnormal cognitive index in the new object recognition still did not change significantly. There was no significant difference in the exploration time of new and old objects, and there was no significant change in the recognition index compared with MUT-GFP mice (as shown in Figure 2A). Figure 8-9 As shown). The third test point was 8 months after virus injection. It was found that except for the time for mice injected with low doses of virus to fall off the rod in the rotating rod test, there was still no increase, indicating that movement disorders were difficult to improve. The other behavioral rescue effects were good. In addition, this embodiment also conducted a water maze experiment on each group of mice to detect the cognitive and learning abilities of the mice. In the 6-day positioning navigation training, MUT-GFP mice and WT-GFP mice showed similar learning curves, but the time to reach the platform was significantly increased. After overexpressing the AAV virus, the latency of MUT-CAPZA2 mice to reach the platform was significantly reduced. In the spatial search on the seventh day, MUT-CAPZA2 mice had a shorter latency to reach the platform than MUT-GFP mice, the number of times they crossed the platform increased, and the active time in the target quadrant where the platform was located was significantly increased. This shows that the overexpression of CAPZA2 significantly rescued the cognitive impairment of defective mice. CAPZA2 c.G776T The abnormal activity, anxiety-like behavior, social deficiency, cognitive impairment and other behaviors of mice were significantly corrected (such as Figure 10-12 The fourth test point was 13 months after virus injection, and AAV-CAPZA2 treatment still showed a satisfactory rescue effect, showing behavioral performance similar to that of WT-GFP mice in the open field, Y maze, high-price plus maze, novel object recognition and water maze (as shown in Figure 2A). Figure 13-14As shown, the detection result is similar to the detection result at the third time point). In this example, behavioral tests were performed on mice at different time points after overexpression of CAPZA2. When the overexpression time was 6 weeks, behavioral rescue was observed only in some behaviors representing motor activity, cognition and sociality; as the virus expression time increased, the rescue effect on the defective mice became gradually obvious. In the open field test, Y maze, high-price plus maze, novel object recognition, three-box social and water maze, it was detected that the abnormal motor activity of the mice in the MUT-CAPZA2 group was normalized, the anxiety level was reduced, and the cognitive and social abilities were restored.

[0079] 5. Body mass monitoring CAPZA2 + / - and CAPZA2 c.G776T The mice were weighed after sampling, including body weight and brain weight, to evaluate the potential effects of AAV-CAPZA2 treatment on the growth and development of mice. The results showed that there were no significant differences among the treatment groups (e.g. Fig.15 shown).

[0080] 6. Biochemical index detection This example shows that CAPZA2 + / - and CAPZA2 c.G776T Overexpression of CAPZA2 was verified in each group of mice, and possible pathological mechanisms associated with CAPZA2 mutation were explored. The exogenous supplementation of AAV-CAPZA2 was verified to be successfully transcribed and translated in the central nervous system of mice by qPCR and WB level detection. Then, the expression level of PSD95, a molecule critical for regulating synaptic plasticity, in the prefrontal cortex of each group of mice was detected. The results showed that CAPZA2 deficiency significantly affected PSD95 expression. After AAV treatment, its expression level was greatly adjusted, and the effect was significant (such as Fig.16 shown).

[0081] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A composition for treating intellectual disability, characterized in that: It comprises a recombinant adeno-associated virus carrying the nucleic acid shown in SEQ ID NO.

1.

2. The composition for treating intellectual disability according to claim 1, characterized in that The gene sequence of the recombinant adeno-associated virus is shown in SEQ ID NO.

2.

3. The composition for treating intellectual disability according to claim 1, characterized in that The composition is an injection.

4. Use of the reagent in the preparation of a drug for treating intellectual disability, characterized in that: The reagent comprises the composition according to any one of claims 1 to 3.

5. The use according to claim 4, characterized in that: The treatment is achieved by administering a therapeutically effective amount of the composition to a subject in need thereof, including by intracerebral injection.

6. The use according to claim 4, characterized in that: The subjects are under 18 years old.

7. The use according to claim 4, characterized in that: The subject is diagnosed with CAPZA2 c.776G>T Missense mutation.

8. A method for screening a drug useful for treating intellectual disability, characterized in that: The following steps are involved: (1) providing an animal model and detecting its behavioral indicators to obtain a first parameter, wherein the animal model contains a c.776G>T mutation in CAPZA2, and the first parameter includes social ability, motor ability, anxiety index and cognitive ability; (2) administering the drug to be tested to the animal model, and then detecting behavioral indicators to obtain a second parameter, wherein the second parameter includes the following indicators detected after administration of the drug to be tested: social ability, motor ability, anxiety index and cognitive ability; (3) A step of comparing the first parameter and the second parameter, wherein when improvement of the behavioral index is observed in the second parameter, the drug to be tested is screened as a drug useful for treating intellectual disability, and when no improvement of the behavioral index is observed in the second parameter, the drug to be tested is screened as a drug not useful for treating intellectual disability.

9. The method for screening a drug useful for treating intellectual disability according to claim 8, characterized in that: Behavioral indices were detected 4 months after administration of the test drug.

10. The method for screening a drug useful for treating intellectual disability according to claim 9, characterized in that: The first parameter also includes the gene expression levels of CAPZA2 and PSD95, and the second parameter also includes the gene expression levels of CAPZA2 and PSD95 detected after administration of the drug to be tested.

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