Compositions for the treatment of intellectual impairment, uses thereof and methods for drug screening

Abstract

The application discloses a composition for treating intellectual disability, application thereof and a drug screening method. The application verifies the effectiveness of the composition in improving the expression level of CAPZA2, restoring synaptic function and improving neural behavior by introducing an AAV virus carrying an hCAPZA2 gene into a CAPZA2 hybrid knockout or mutant mouse model. The application not only provides new hope for the treatment of intellectual disability related to the CAPZA2 gene, but also opens up a new direction for the application of gene therapy in neurodevelopmental diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to compositions for treating intellectual disabilities, their applications, and drug screening methods. Background Technology

[0002] Intellectual disability (ID) is a group of developmental disorders characterized by cognitive impairment and adaptive behavioral deficits, severely affecting patients' learning, communication, and social participation abilities. Genetic factors play a significant role in the pathogenesis of ID. ID is a complex neurodevelopmental disorder, with patients typically exhibiting significant cognitive deficits, difficulties in social interaction, anxiety, and abnormal motor behaviors. Although various methods have been attempted to treat these symptoms, such as pharmacological interventions and behavioral therapies, these methods often fail to cure or significantly improve the neurological deficits caused by gene mutations.

[0003] With the development of gene therapy technology, the delivery of functional genes to specific tissues using adeno-associated virus (AAV) vectors has become a cutting-edge strategy for repairing genetic defects. Gene therapy aims to treat hereditary diseases by introducing specific genes into the body to correct or compensate for diseased genes.

[0004] In recent years, various AAV serotypes and variants have been identified, which can recognize different cellular receptors, thus exhibiting different tissue- and cell-specific orientations. Although the VP3 structure of different AAV serotypes is highly homologous, the genetic differences in the variant region (VR) are significant, affecting their binding and interaction with cell surface receptors. However, this variation usually does not affect viral particle assembly; therefore, when designing new AAV capsid vectors, the main focus is on modifying the VR region to maintain the structural integrity of the capsid while enhancing its targeting and transduction efficiency.

[0005] Recombinant adeno-associated virus (rAAV) has proven its effectiveness as a gene delivery vector in various basic scientific studies and clinical trials. Gene therapy using rAAV vectors offers several advantages: (i) long-term stable transgene expression; (ii) independence from any known human diseases; (iii) inducing a weaker immune response; (iv) effective transduction of both dividing and non-dividing cell types; and (v) the rAAV genome can be packaged in different viral capsids with unique transduction properties and tissue specificity.

[0006] Currently, rAAV-based gene therapy has achieved significant 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. Nevertheless, existing AAV vectors still have limitations in some applications, particularly lacking effective transduction capabilities for specific cell types or tissues. In particular, many neurological diseases require highly efficient central nervous system transduction capabilities; therefore, it is necessary to develop new AAV serotypes or variants to improve the efficacy of gene therapy, reduce the required dosage, and minimize 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 an AAV9 vector to deliver the MECP2 gene into the brain of a mouse model of Rett syndrome in an attempt to correct neurological deficits caused by mutations in the MECP2 gene. Although some behavioral improvements and neuroprotective effects have been observed in some models, this method has significant limitations, mainly in that overexpression of the MECP2 gene may lead to neurotoxicity, and challenges exist in long-term efficacy and dosage control.

[0008] For example, US patent application US20160038613A1 discloses a technique for treating spinal muscular atrophy (SMA) by delivering the SMN1 gene via an AAV9 vector. AAV therapy for the SMN1 gene was developed for SMA patients. This technique delivers the SMN1 gene carried by an AAV9 vector to the patient's motor neurons via systemic intravenous injection, compensating for protein loss caused by SMN1 gene mutations and thus improving motor function. This treatment has shown significant effects in prolonging patient lifespan and improving motor function and has been widely used clinically. AAV9 was chosen as the vector due to its superior ability to cross the blood-brain barrier and its effective transduction in the central nervous system. However, although this technique has achieved good results in the treatment of SMA, it primarily targets motor neurons, limiting its applicability to other types of neurological diseases. Furthermore, while the initial treatment effects are significant, the sustainability of the efficacy over time requires further evaluation.

[0009] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0010] To address at least some of the technical problems in existing technologies, this invention provides an AAV gene therapy regimen for rescuing intellectual disability caused by CAPZA2 mutations. This regimen uses a recombinant AAV2 / 9 vector (pFD-scAAV-ITR-CBh-opt-hCAPZA2-BGHpA) to optimize hCAPZA2 gene expression and correct neurological dysfunction caused by CAPZA2 mutations. The AAV2 / 9 vector developed in this invention has high transduction efficiency in the central nervous system and incorporates self-complementary AAV (scAAV) technology to accelerate gene expression, thereby achieving a higher therapeutic effect. This invention fundamentally improves behavioral abnormalities in patients with intellectual disabilities by modulating synaptic plasticity in the brain. Specifically, this invention includes the following:

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

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

[0013] In some embodiments, the composition for treating intellectual disability according to the present invention is an injectable preparation.

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

[0015] In some embodiments, the method or application for treating intellectual disability in a subject according to the present invention includes intracerebral injection.

[0016] In some embodiments, the method or application for treating intellectual disability in a subject according to the present invention, wherein the subject is under 18 years of age.

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

[0018] A third aspect of the present invention provides a method for testing the efficacy of a drug for treating intellectual disability (a method for screening drugs useful for treating intellectual disability), comprising the following steps:

[0019] (1) The step of providing an animal model and detecting its behavioral indicators to obtain a first parameter, wherein the animal model has a C.776G>T mutation in CAPZA2, and the first parameter includes social ability, motor ability, anxiety index and cognitive ability;

[0020] (2) The step of administering the test drug to the animal model and subsequently detecting behavioral indicators to obtain a second parameter, the second parameter including the following indicators detected after administration of the test drug: social competence, motor competence, anxiety index, and cognitive competence; and

[0021] (3) The step of comparing the first parameter and the second parameter: if an improvement in the behavioral indicators is observed in the second parameter, the drug to be tested is screened as a drug useful for treating intellectual disability; if no improvement in the behavioral indicators is observed in the second parameter, the drug to be tested is screened as a drug useless for treating intellectual disability.

[0022] In some embodiments, according to the method for testing the efficacy of a drug for treating intellectual disability according to the present invention, behavioral indicators are detected 4 months after administration of the drug.

[0023] In some embodiments, the method for testing the efficacy of a drug for treating intellectual disability according to the present invention includes behavioral indicators such as socialability, motor skills (OFT, RRT), anxiety index (EPM), and cognitive abilities (Y-maze, ORT, MWM).

[0024] This invention, by introducing an AAV virus carrying the hCAPZA2 gene into mouse models of CAPZA2 heterozygous knockout or heterozygous mutation, verified its effectiveness in increasing CAPZA2 expression levels, restoring synaptic function, and improving neurobehavioral responses. This technology not only offers new hope for the treatment of CAPZA2 gene-related intellectual disabilities but also opens up new directions for the application of gene therapy in neurodevelopmental disorders.

[0025] Furthermore, the technical effects of the present invention also include:

[0026] (1) Targeted treatment: This invention provides a specific treatment method for neurological diseases caused by CAPZA2 gene defects. Through precise gene supplementation, it is expected to directly treat the cause of the disease.

[0027] (2) Highly efficient gene transduction: By utilizing the highly efficient transduction capability of the AAV9 vector, this invention achieves specific and efficient gene delivery to neurons, improving the therapeutic effect while reducing the potential impact on non-target tissues.

[0028] (3) Optimized gene expression: By controlling the neuron-specific expression of the CBh promoter, this invention optimizes the expression of the CAPZA2 gene in the central nervous system, which helps to achieve more precise therapeutic effects.

[0029] (4) Comprehensive behavioral improvement: This invention significantly improves a variety of behavioral phenotypes in 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.

[0030] (5) Long-term efficacy maintenance: In long-term behavioral assessments, the therapeutic effects demonstrated by this invention can be sustained for a considerable period of time, indicating that AAV-CAPZA2 treatment may have the potential for long-term efficacy maintenance. In contrast, current similar gene therapy studies do not have long-term follow-up, and most studies only conduct a single behavioral test.

[0031] (6) Safety verification: Through body weight monitoring and biochemical index detection, this invention confirms that AAV-CAPZA2 treatment improves behavioral phenotypes without causing significant adverse effects on the growth and development and important physiological indicators of mice.

[0032] (7) Synaptic function recovery: This invention reveals that AAV-CAPZA2 treatment may promote the repair and functional recovery of the nervous system by restoring the expression of PSD95, a molecule closely related to synaptic plasticity.

[0033] (8) Potential clinical application prospects: This 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.

[0034] (9) Scientific and social benefits: This invention not only provides a deeper scientific understanding of CAPZA2-related diseases, but also has the potential to reduce patient suffering and medical costs and improve patients’ quality of life at the social level. Attached Figure Description

[0035] Figure 1 The recombinant scAAV vector structure constructed according to the present invention is shown.

[0036] Figure 2 The total distance traveled in an open field, dwell time in an open arm of an elevated cross maze, time to fall from a rotundus, contact time between new and old objects and cognitive index in new object recognition are shown in CAPZA2 heterozygous knockout (Het) mice 2 months after gene therapy.

[0037] Figure 3The interaction time and interaction index of social interaction and social preference in the three boxes are shown two months after gene therapy in Het mice.

[0038] Figure 4-5 The results show the water maze behavior test of Het mice 2 months after gene therapy.

[0039] Figure 6 The study showed the total distance traveled in an open field, the spontaneous alternation rate in a Y-maze, the dwell time in an open arm of an elevated cross maze, the time to fall from a rotundus, the time to recognize new objects and the cognitive index of MUT mice 6 weeks after gene therapy.

[0040] Figure 7 The interaction time and interaction index of social interaction and social preference in MUT mice were shown for 6 weeks after gene therapy.

[0041] Figure 8 The study showed the total distance traveled in an open field, the spontaneous alternation rate in a Y-maze, the dwell time in an open arm of an elevated cross maze, and the contact time between new and old objects and the cognitive index in new object recognition in MUT mice 4 months after gene therapy.

[0042] Figure 9 The interaction time and interaction index of social interaction and social preference in three boxes were shown 4 months after gene therapy in MUT mice.

[0043] Figure 10 The study showed the total distance traveled in an open field, the spontaneous alternation rate in a Y-maze, the dwell time in an open arm of an elevated cross maze, the time to fall from a rotundus, and the contact time between new and old objects and the cognitive index in new object recognition in MUT mice 8 months after gene therapy.

[0044] Figure 11 The interaction time and interaction index of social interaction and social preference in the three boxes are shown 8 months after gene therapy in MUT mice.

[0045] Figure 12 The water maze behavioral test is shown 8 months after gene therapy in MUT mice.

[0046] Figure 13 The study showed the total distance traveled in an open field, the spontaneous alternation rate in a Y-maze, the open arm dwell time in an elevated cross maze, the contact time between new and old objects and the cognitive index of MUT mice 13 months after gene therapy.

[0047] Figure 14 The water maze behavioral test is shown 13 months after gene therapy in MUT mice.

[0048] Figure 15 The changes in body weight of mice after sampling are shown.

[0049] Figure 16 The results of protein and PCR detection in mice are shown. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Composition for treating intellectual disability

[0054] In one aspect, the present invention provides a composition for treating intellectual disability, the composition comprising a recombinant adeno-associated virus and optionally a pharmaceutically acceptable vector, wherein the recombinant adeno-associated virus carries the nucleic acid shown in SEQ ID NO. 1.

[0055] Typically, adeno-associated virus (sometimes simply called "AAV") contains a rep gene and a cap gene, along with two side-attached terminal inverted repeats (ITRs). The rep gene encodes Rep78, Rep68, Rep52, and Rep40 proteins, which are used for AAV genome replication and viral particle assembly. The cap gene encodes three major capsid proteins (VP1, VP2, and VP3), which are produced through selective transcription and splicing regulation of rare start codons (ACGs) and together constitute the viral particle capsid. Furthermore, the cap gene's internal open reading frame (ORF) also includes an assembly activation protein (AAP) essential for capsid assembly.

[0056] In this invention, the recombinant adeno-associated virus (rAAV) is an AAV recombinant vector obtained by modifying wild-type adeno-associated virus. The wild-type adeno-associated virus genome is approximately 4.7 kb, composed of 60 VP subunits, with VP1, VP2, and VP3 in a ratio of approximately 1:1:10. The recombinant adeno-associated virus (sometimes simply referred to as "rAAV") typically retains approximately 4% of the viral genome, i.e., inverted terminal repeat sequences, does not express viral genes, and exists as a non-integrating fragment. This results in relatively good tolerance for target gene transfer in different target tissues of different species.

[0057] Through in-depth research, the inventors discovered that by designing the coding region of rAAV and then performing transcription and translation, the rate-limiting step of rAAV gene expression is eliminated without waiting for second-strand DNA synthesis, thus shortening the time to start expression. The peak expression can generally be reached 3-5 days after infection.

[0058] Although various serotypes of AAV have been reported to have advantages in delivering target genes, there are still significant challenges in using AAV as a vector in clinical settings. These challenges stem from difficulties in effectively delivering target genes to target tissues, achieving long-term expression of corrective transgenes, and avoiding harmful effects on the host immune system.

[0059] In a preferred embodiment, the recombinant adeno-associated virus vector of the present invention includes an operatively linked enhancer, intron, promoter, human CAPZA2 complementary DNA, terminator sequence, and an ITR sequence flanked by the above sequences, preferably an ITR from AAV9. In a specific embodiment, the nucleic acid in the recombinant adeno-associated virus vector for overexpressing CAPZA2 has the sequence shown in SEQ ID NO. 1.

[0060] In a preferred embodiment, the coding sequence of the recombinant adeno-associated virus vector of the present invention is shown in SEQ ID NO.2.

[0061] In this invention, pharmaceutically acceptable carriers are used to transport or deliver recombinant adeno-associated virus from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation and does not harm the patient. In this invention, the pharmaceutically acceptable carriers include at least one of diluents, absorbents, wetting agents, sweeteners, preservatives, and antioxidants. Pharmaceutically acceptable carriers are preferably those administered via injection, examples of which include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media.

[0062] In a preferred embodiment, the composition for treating intellectual disability is an injectable preparation, particularly administered to the subject in need via intracerebral injection.

[0063] Methods for treating intellectual disabilities in subjects in need

[0064] One aspect of the present invention provides a method for treating intellectual disability or related conditions (symptoms or conditions) in a subject in need, also referred to as "use of a reagent in the preparation of a medicament for treating intellectual disability or related conditions (symptoms or conditions) in a subject in need", wherein the reagent includes the composition described in the present invention.

[0065] The term "subject" as used in this invention refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, and the like, who are about to receive specific treatment. Generally, "subject" and "patient" are used interchangeably in this invention, both referring to the subject of the study. In a specific embodiment, the subject has been diagnosed with CAPZA2. c.776G>T Subjects with missense mutations, especially those with CAPZA2 c.776G>T Subjects with intellectual disabilities caused by missense mutations. In another embodiment, the subjects are under 18 years of age.

[0066] As used herein, the term "effective amount" refers to the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeutic effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function. Generally, the effective amount as used herein varies depending on various factors, such as the given drug or composition, pharmaceutical preparation, route of administration, type of disease or symptom, subject being treated, etc., but can still be routinely determined by those skilled in the art.

[0067] The term "treatment" as used in this invention refers to improvement of a condition before or after the onset of a disease or dysfunction. This degree of relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete).

[0068] The therapeutic dosage of the present invention can vary widely. Generally speaking, the dosage of the composition used in the present invention is well known to those skilled in the art. The dosage can be administered in a single dose or in several doses, such as two, three, or four doses. The administered dosage is within the range that clinicians or laboratory personnel can expect, for example, by appropriately adjusting the dosage through efficacy and safety testing to obtain the optimal dosage.

[0069] In this invention, "treatment of intellectual disability" refers to the ability to improve or treat intellectual disability caused by the presence of CAPZA2. c.776G>T At least one of the following situations resulting from missense mutations:

[0070] (1) Improve cognitive impairment and adaptive behavior deficits;

[0071] (2) Improve learning, communication and social participation skills;

[0072] (3) Restore or increase CAPZA2 expression levels;

[0073] (4) Restore or improve the expression level of PSD95;

[0074] (5) Effectiveness in restoring synaptic function and improving neurobehavior;

[0075] (6) Promotes the repair and functional recovery of the nervous system.

[0076] In a preferred embodiment, the composition of the present invention is preferably administered to the subject in need by intracerebral injection.

[0077] Methods for testing the efficacy of drugs for treating intellectual disability

[0078] One aspect of the present invention provides a method for testing the efficacy of a drug for treating intellectual disability, comprising:

[0079] (1) The step of providing an animal model and detecting its behavioral indicators to obtain a first parameter, wherein the CAPZA2 in the animal model has a c.776G>T mutation;

[0080] (2) The step of administering the test drug to the animal model and subsequently detecting behavioral indicators to obtain the second parameter; and

[0081] (3) The step of comparing the first parameter and the second parameter.

[0082] In this invention, the animal model is not particularly limited; for example, it can be any model animal, such as rat, mouse, chicken, rabbit, etc. Methods for introducing the c.776G>T mutation into the CAPZA2 of the animal model are known in the art; for example, gene editing techniques can be used to introduce the aforementioned point mutation.

[0083] In this invention, the detected indicators also include the amount of CAPZA2 and PSD95 measured before drug administration, and the amount of CAPZA2 and PSD95 measured after drug administration.

[0084] In this invention, when improvements in behavioral indicators (including improvements in cognitive impairment and adaptive behavior deficits; improvements in learning, communication, and social participation abilities) are observed in the second parameter, and / or changes in the expression levels of relevant genes (including increases in CAPZA2 and PSD95) are detected, it indicates that the test drug can effectively treat intellectual disability. When no improvements in behavioral indicators (including improvements in cognitive impairment and adaptive behavior deficits; improvements in learning, communication, and social participation abilities) are observed in the second parameter, and / or changes in the expression levels of relevant genes (including unchanged or decreased levels of CAPZA2 and PSD95) are detected, it indicates that the test drug cannot effectively treat intellectual disability.

[0085] In some implementations, behavioral indicators are detected at a time point of 4 months after administration of the drug to be tested. These behavioral indicators include indicators in open field tests (e.g., total distance traveled), Y-maze tests (including cycle index), three-box social tests (including social interaction and social preference abilities), and elevated cross maze tests (e.g., dwell time in open arms), but do not include cognitive indices in new object recognition and indicators of exploration time for new and old objects.

[0086] In some implementations, behavioral indicators are detected at a time point of 8 months after administration of the drug to be tested. These behavioral indicators include open field test, Y-maze, three-box social test, elevated cross maze, cognitive index in new object recognition, and indicators of exploration time for new and old objects, but do not include the time indicator of falling from the rod in the rotundus test.

[0087] In some implementations, behavioral indicators are detected at a time point 13 months after administration of the drug to be tested. These behavioral indicators include open field test, Y-maze, three-box social test, elevated cross maze, water maze (including learning trajectory during training, time to find plateau and number of crossings), cognitive index in new object recognition, exploration time for new and old objects, and time to fall from the rod in rotundus test.

[0088] Example

[0089] I. Construction of AAV Vector

[0090] An AAV9 vector carrying the CAPZA2 gene was designed and constructed: AAV-CAPZA2 was developed with CAPZA2 gene expression driven by a potent neuron-specific promoter CBh containing complementary human CAPZA2 DNA. The final overexpressing AAV virus was: PFD-scAAV-ITR-CBh-opt-hCAPZA2-BGHpA (Fiter: 2.8E+13 vg / ml), and the control virus was: AAV2 / 9: PFD-scAAV-CBh-GFP-BGHpolyA (Fiter: 1.7E+13 vg / ml).

[0091] II. Preparation of Experimental Animal Models

[0092] Homology comparison revealed that the patient carried CAPZA2. c.776G>T A missense mutation was used, changing the amino acid from arginine (R) to leucine (L), thus creating the corresponding mutant mouse. CAPZA2 was created by precisely introducing a point mutation via homology-guided repair. c.G776T Mice. CAPZA2 heterozygotes were created by deleting exons 3 through 6 of the CAPZA2 gene located on chromosome 6 using CRISPR / Cas technology. + / - Mice.

[0093] III. AAV Vector Drug Delivery

[0094] To investigate whether AAV-CAPZA2 could rescue the behavioral phenotype of defective mice, this study performed behavioral testing after bilateral lateral ventricle overexpression of AAV-CAPZA2. The AAV-CAPZA2 vector was injected into the lateral ventricle of the mouse brain using a stereotactic injection method (X=±1.0, Y=-0.58, Z=-2.0). + / - All mice were 5 months old, with an equal number of males and females, and 8 mice in each group. Two injection regimens were set up: a low-dose group received 5 E+9 vg per side, and a high-dose group received 1.0 E+10 vg per side. The control group received the same number of empty viral 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 / side) was administered, while the control group received the same number of empty viral particles. Mice were anesthetized with 1.5-2% isoflurane and placed on a stereotaxic adapter. Hair was removed from the head, and the skin was incised to expose the skull. The stereotaxic adapter was used to maintain head balance by aligning the mice anteriorly, posteriorly, laterally, and laterally with Bregma as the zero point. AAV virus was slowly injected (50 nL / min) into the bilateral lower left lateral circulation (LV) regions and allowed to diffuse for 10 min.

[0095] Use suture needles to suture the scalp. Determine the number of sutures based on the size of the opening. After suturing, wipe the scalp again with 75% water to prevent infection. Once completed, remove the animal from the adapter and place it in a water bath incubator until it awakens. Afterward, place the animal in its enclosure and provide it with ample food and water.

[0096] IV. Behavioral Assessment

[0097] 1. AAV-CAPZA2 versus CAPZA2 + / - Behavioral rescue assessment of mice

[0098] CAPZA2 + / - Behavioral assessments were performed on mice two months after viral injection, and the results showed that gene therapy in both dosage groups effectively rescued the virus. Figure 2-5 The inventors first tested the motor activity of the mice in each group and found that CAPZA2 + / - Control mice (to CAPZA2) + / - Mice injected with empty viral vectors showed a significant increase in total distance movement in the open field test, indicating abnormally elevated motor activity. Administration of AAV-CAPZA2 significantly reduced this total distance, indicating normalization of motor activity. Anxiety-like behavior was then assessed. Results showed that, compared to the normal group, CAPZA2... + / - The control mice showed a significantly reduced dwell time on the open arm in the elevated cross maze, indicating that CAPZA2 deficiency leads to anxiety behavior in mice, and administration of AAV-CAPZA2 significantly reduced anxiety levels. The motor coordination ability of each group of mice was then examined, revealing that AAV-CAPZA2 could significantly rescue the motor dysfunction in the deficient mice and increase their dwell time on the balance bar in the rotarod test.

[0099] Next, the effects of AAV-CAPZA2 on the social abilities of CAPZA2 mice were investigated. Three-box social assessments showed that CAPZA2 deficiency led to a lack of social interaction and reduced social preference abilities in mice, while AAV treatment significantly corrected these social abnormalities. Finally, the cognitive abilities of each group of mice were examined. In the Y-maze test, AAV treatment significantly improved the circulation index of the deficient mice. In the novel object recognition test, AAV treatment significantly improved CAPZA2 levels. + / -The recognition index in mice was improved, increasing the time mice spent exploring new objects. Similarly, in a 7-day water maze experiment, a significant salvage effect of AAV treatment was observed. Exogenous injection of AAV-CAPZA2 significantly normalized the learning trajectory of defective mice during the training period, significantly improving their spatial learning ability. Furthermore, during the testing period, the time to find the plateau and the number of crossings in the CAPZA2-treated group were significantly improved to normal levels, indicating a significant recovery in their cognitive abilities. After one month of behavioral testing, all groups of mice were in good condition. In addition, high-dose exogenous AAV-CAPZA2 was found to have an effect on CAPZA2... + / - The rescue effect in mice was good.

[0100] 2. AAV-CAPZA2 versus CAPZA2 c.G776T Behavioral rescue assessment of mice

[0101] For CAPZA2 c.G776T Mice were injected with a low dose of the virus, and behavioral assessments were performed at different time points after injection to explore the long-term efficacy of AAV-CAPZA2. The first test point was 6 weeks after viral injection. It was found that abnormal indicators in the open field test, Y-maze, and three-box social test were regressed in the treatment group, while abnormalities in the elevated cross maze, robin test, and new object recognition did not show significant regression. Specifically, the exploration time of the open arm in the elevated cross maze did not increase, the time for mice to fall off the robin in the robin test did not lengthen, and mice had difficulty distinguishing between new and old objects in new object recognition (e.g., ...). Figure 6-7 (As shown). The second test point was 4 months after viral injection. Except for the indicators that reverted in the 6-week test, the anxiety performance in the elevated cross maze test was normalized, while the abnormal cognitive index in new object recognition remained unchanged. There was no significant difference in the exploration time for new and old objects, and the recognition index was not significantly different compared with MUT-GFP mice (e.g.). Figure 8-9 (As shown). The third test point was 8 months after viral injection. It was found that, except for the rotarod test where the time for mice injected with low doses of virus to fall off the rod did not increase, indicating that motor impairment was difficult to improve, other behavioral remediation effects were good. In addition, this embodiment also conducted a water maze test on each group of mice to test their cognitive and learning abilities. During the 6-day orientation navigation training, MUT-GFP mice and WT-GFP mice showed similar learning curves, but the time to reach the platform was significantly increased. After overexpression of 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 showed a shorter latency to reach the platform and more platform crossings compared to MUT-GFP mice, and their active time in the target quadrant where the platform was located was significantly increased. This indicates that CAPZA2 overexpression significantly rescued the cognitive impairment in the defective mice. CAPZA2c.G776T The abnormal hyperactivity, anxiety-like behavior, social deficits, and cognitive impairment in mice were all significantly corrected (e.g., Figure 10-12 (As shown). The fourth test point was 13 months after viral injection. AAV-CAPZA2 treatment still showed a relatively satisfactory rescue effect, exhibiting behavioral performance similar to WT-GFP mice in open field, Y maze, high-valence cross maze, new object recognition, and water maze (e.g.). Figure 13-14 As shown, the results are similar to those at the third time point. In this embodiment, behavioral tests were performed on mice at different time points after CAPZA2 overexpression. At 6 weeks of overexpression, behavioral rescue was observed only in some behavioral areas representing motor activity, cognition, and social interaction. As the viral expression time increased, the rescue effect on defective mice became more apparent. In the open field test, Y maze, high-valence cross maze, new object recognition, three-box social interaction, and water maze, the abnormal motor activity of the MUT-CAPZA2 group mice was normalized, anxiety levels were reduced, and cognitive and social abilities were restored.

[0102] V. Body weight monitoring

[0103] For CAPZA2 + / - and CAPZA2 c.G776T Mice were weighed after tissue sampling, including body weight and brain weight, to assess the potential impact of AAV-CAPZA2 treatment on mouse growth and development. Results showed no significant differences among the treatment groups (e.g., ...). Figure 15 (As shown).

[0104] VI. Biochemical Indicator Testing

[0105] This embodiment refers to CAPZA2 + / - and CAPZA2 c.G776T Overexpression of CAPZA2 was validated in each group of mice, and the possible pathological mechanisms associated with CAPZA2 mutations were explored. qPCR and Western blotting confirmed the successful transcription and translation of exogenous AAV-CAPZA2 in the central nervous system of mice. Next, the expression level of PSD95, a molecule crucial for the regulation of synaptic plasticity, in the prefrontal cortex of each group of mice was examined. The results showed that CAPZA2 deficiency significantly affected PSD95 expression, and after AAV treatment, its expression level was significantly reduced, demonstrating a significant effect (e.g., ...). Figure 16 (As shown).

[0106] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A composition for treating intellectual disability, characterized in that, It is an injectable preparation containing recombinant adeno-associated virus PFD-scAAV-ITR-CBh-opt-hCAPZA2-BGHpA and a pharmaceutically acceptable vector. The recombinant adeno-associated virus includes operatively linked enhancers, introns, promoters, human CAPZA2 complementary DNA, terminator sequences, and a side-connected ITR sequence from AAV9. The gene sequence of the recombinant adeno-associated virus is shown in SEQ ID NO.

2.

2. The composition for treating intellectual disability according to claim 1, characterized in that, Pharmaceutically acceptable carriers include physiological saline or aqueous buffer solutions.

3. The use of the composition according to claim 1 or 2 in the preparation of a medicament for treating intellectual disability.

4. The application according to claim 3, characterized in that, The treatment is achieved by administering a therapeutically effective amount of the composition to a subject in need, including via intracerebral injection.

5. The application according to claim 3, characterized in that, The subject was diagnosed with CAPZA2. c.776G>T Missense mutation.

6. A method for screening drugs useful for treating intellectual disability, characterized in that, Includes the following steps: (1) The step of providing a mouse animal model at least 3 months old and detecting its behavioral indicators to obtain a first parameter, wherein the CAPZA2 in the animal model has a c.776G>T mutation, the first parameter includes social ability, motor ability, anxiety index and cognitive ability, 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; (2) The step of administering the test drug to the animal model and then detecting behavioral indicators to obtain a second parameter, the second parameter including the following indicators detected after administration of the test drug: social competence, motor competence, anxiety index and cognitive competence; (3) The step of comparing the first parameter and the second parameter: if an improvement in the behavioral indicators is observed in the second parameter, the drug to be tested is screened as a drug useful for treating intellectual disability; if no improvement in the behavioral indicators is observed in the second parameter, the drug to be tested is screened as a drug useless for treating intellectual disability.

7. The method for screening drugs useful for treating intellectual disability according to claim 6, characterized in that, Behavioral indicators were tested 4 months after administration of the drug to be tested.

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