Novel gene therapy method for treating cri du chat syndrome

Abstract

A polynucleotide and nucleic acid construct of truncated CTNND2, an AAV particle comprising the nucleic acid construct, a composition comprising the polynucleotide, the nucleic acid construct or the virus particle, and a method for using the polynucleotide, the nucleic acid construct, the virus particle, or the composition, and a use thereof.

Description

A new gene therapy approach to treat Cry-a-cat syndrome

[0001] priority

[0002] This application claims the benefit of and priority to Chinese application No. 2023109758396, filed on August 3, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to a gene therapy vector. Background Art

[0004] Cri Du Chat syndrome (Cri Du Chat syndrome) is a hereditary neurodevelopmental disorder caused by a partial or complete deletion of the short arm of chromosome 5 (5p microdeletion, 5p deletion). Also known as 5p deletion syndrome or pediatric Cri Du Chat syndrome, the size of the deletion on the short arm of chromosome 5 varies among individuals with Cri Du Chat syndrome (referred to as 5p patients), ranging from 0.5 Mb to 45 Mb (the deletion extends from the 5p15 region to the entire short arm). This syndrome is considered the most common autosomal contiguous gene deletion disorder in children. Targeted treatment is lacking, the prognosis is poor, and there is no precise correlation between gene deletions in the 5p region and clinical phenotypes. The 5p region is known to encompass 563 genes (http: / / genome.ucsc.edu).

[0005] Gene therapy involves the introduction of exogenous, normal genes into target cells to correct or compensate for diseases caused by defective or abnormal genes, thereby achieving therapeutic results. Currently, gene therapy has been applied to treat a variety of fields, including rare genetic diseases, malignant tumors, and cardiovascular disease. Gene therapy has potential for treating rare genetic diseases. For example, gene therapy for hereditary β-thalassemia has officially moved from scientific research to the clinical stage.

[0006] In the 1960s, scientists first proposed the concept of using gene therapy to cure genetic diseases. This novel conceptual strategy aims to achieve long-term therapeutic effects by introducing exogenous genetic material into patients. Gene therapy involves the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes. Over the past few decades, the field of gene therapy has made significant progress in treating previously untreatable genetic diseases.

[0007] Gene therapy for genetic diseases aims to achieve sustained gene expression by delivering therapeutic genes to cells, maintaining sufficient expression to improve or cure the disease with minimal safety risks. In the 1960s, American molecular biologist Lederberg J first proposed the concept of gene therapy, laying the foundation for its development. Adeno-associated virus (AAV), a single-stranded, linear DNA-deficient virus, has been used as a safe and effective viral vector in gene therapy clinical trials due to its efficient transduction ability, non-integration properties, and ability to specifically infect various tissues and organs.

[0008] There is currently no treatment for cat cry syndrome. Only treatment is provided for its corresponding symptoms, such as surgery or rehabilitation training.

[0009] Summary of the Invention

[0010] The present invention provides a polynucleotide sequence encoding a CTNND2 truncation, wherein the protein sequence of the CTNND2 truncation is shown in SEQ ID NO: 2.

[0011] In some embodiments, the polynucleotide sequence is as shown in SEQ ID NO: 1, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1.

[0012] In one aspect, the present invention provides a nucleic acid construct, wherein the construct comprises the polynucleotide sequence.

[0013] In some embodiments, the nucleic acid construct includes a transcriptional regulatory element, which includes a promoter and / or an enhancer.

[0014] In one aspect, the present invention provides a vector, wherein the vector contains the polynucleotide sequence or the construct of the present invention.

[0015] In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0016] In some embodiments, the AAV vector is selected from the group consisting of: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof.

[0017] In one aspect, the present invention provides a viral particle comprising the construct of the present invention.

[0018] In one aspect, the present invention provides a composition comprising the polynucleotide sequence of the present invention, the nucleic acid construct, the vector, or the viral particle, and a pharmaceutically acceptable excipient.

[0019] In another aspect, the present invention discloses the use of the polynucleotide sequence, the nucleic acid construct, the vector, the viral particle, or the composition described in the present invention in the preparation of a drug for treating cat cry syndrome in a subject in need thereof.

[0020] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention can be more fully understood with reference to the following drawings.

[0022] FIG1 shows the map of the pAAV-CMV-EGFP-3xFLAG-tWPA (AAV-Vector) empty vector plasmid.

[0023] FIG2 shows the plasmid map after insertion of rCTNND2.

[0024] FIG3 shows protein markers.

[0025] FIG4 shows the results of Western Blot detection.

[0026] FIG5 shows the experimental process of gene therapy in 5p model rats.

[0027] FIG6 shows the effect of administering a gene-targeted nucleic acid drug (AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP) in early development on improving cognitive function in 5p model rats.

[0028] FIG7 shows the effects of early developmental administration of gene-targeted nucleic acid drugs on anxiety-like behaviors in 5p model rats.

[0029] FIG8 shows the effects of gene-targeted nucleic acid drugs administered in late development on the cognitive and social behaviors of 5p model rats.

[0030] FIG. 9 shows the range of knockout in the 5p model rat—rat chromosome 2.

[0031] FIG10 shows a schematic diagram of the preparation of 5p model rats.

[0032] Figure 11 shows a map of the pre-cut pCS vector.

[0033] FIG12 shows the activity detection results of sgRNA.

[0034] FIG13 shows an RNA electrophoresis diagram during RNA preparation of sgRNA.

[0035] FIG. 14 shows the primer design principles for designing identification primers for genotyping of F0 generation rats.

[0036] FIG15 shows the identification of the tail genotype of F0 generation rats, using primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0037] FIG. 16 shows the primer design principles for designing identification primers for genotyping of F1 generation rats.

[0038] FIG17 shows the identification results of the tail genotype of F1 rats using primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-WT-R.

[0039] FIG18 shows the identification results of the tail genotype of F1 rats using primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0040] FIG. 19 shows key homologous genes in the 5p model rat.

[0041] FIG. 20 shows that 5p model rats have growth retardation.

[0042] FIG. 21 shows that the motor coordination ability of 5p model rats is delayed.

[0043] FIG. 22 shows hypotonia in 5p model rats.

[0044] FIG. 23 shows that 5p model rats have obvious social impairment.

[0045] FIG. 24 shows that 5p model rats have repetitive stereotyped behaviors and lack of curiosity.

[0046] FIG. 25 shows that 5p model rats exhibit obvious anxiety-like behaviors.

[0047] FIG. 26 shows that the limb balance ability of 5p model rats is impaired.

[0048] FIG. 27 shows cognitive impairment in 5p model rats.

[0049] FIG. 28 shows that the activity of 5p model rats decreased, accompanied by obvious anxiety-like behaviors.

[0050] FIG29 shows the abnormal development of dendrites and dendritic spines in different brain regions of 5p model rats.

[0051] Figure 30 shows the pathological increase in neuronal density in the mPFC of 5p model rats. (A) Representative anti-NeuN immunostained sections of the mPFC of experimental group rats. Low-power image, scale bar, 1 mm, high-power image, scale bar, 500 μm. (B) Representative anti-NeuN immunostained sections of the CA1 region and DG region of the hippocampus of experimental group rats. Low-power image, scale bar, 1 mm, high-power image, scale bar, 500 μm. (C) 5p model rats and WT rats in the II / III layer every 10 5 μm 3 The number of NeuN-positive cells in the CA1 region of the hippocampus was significantly higher than that in the control group (Mann-Whitney U test, P = 0.0255). Sample size: WT, n = 33 slices from 6 rats; 5p model rats, n = 42 slices from 7 rats. (D) 5 μm 3 The number of neurons stained positive for anti-NeuN was quantified. Sample size: WT, n = 42 sections from 6 rats; 5p model rats, n = 42 sections from 7 rats. All rats were 8 weeks old. All data are presented as mean ± standard error of the mean (SEM). Statistical significance: *p < 0.05.

[0052] FIG31 shows the RNA sequencing results (RNA-seq) of PFC and hippocampus samples from 8-week-old WT and 5p model rats.

[0053] FIG32 shows the results of cognitive function testing, gene expression levels, immunoblotting, and Golgi staining in 5p model rats after tail vein injection of targeted nucleic acid drugs.

[0054] Figure 33 shows the partial recovery of dendritic complexity and dendritic spine density in 5p model rats after a single injection of targeted nucleic acid drugs. (A) Representative micrographs of Golgi-Cox-stained neurons in the mPFC and hippocampal CA1 regions from WT, AAV-Vector+5p model rats (injected with empty pAAV-CMV-EGFP-3xFLAG-tWPA) and nucleic acid drug+5p model rats (injected with nucleic acid drug AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP). The analyzed cells are marked with red boxes. Low-power images (scale bar: 200 μm) and high-power images (scale bar: 100 μm) are shown. (B) Sholl analysis of neuronal complexity in the mPFC region between WT rats, AAV-Vector+5p model rats, and nucleic acid drug+5p model rats (two-way ANOVA, F(2,3630)=1.274, p>0.05 at distances from 20 μm to 250 μm from the neuronal soma). (C) Dendritic number in the CA1 region of the hippocampus between WT rats and AAV-Vector+5p model rats at distances from the neuronal soma of 45-90 μm (two-way ANOVA, F(2,3672)=79.86, p=0.0014 at 45 μm, p<0.001 at 50-80 μm, p=0.0062 at 85 μm, and p=0.005 at 90 μm). Sample size: mPFC, WT (n = 15 cells from 5 rats), AAV-Vector + 5p model rats (n = 19 cells from 7 rats), and nucleic acid drug + 5p model rats (n = 24 cells from 8 rats); hippocampal CA1, WT (n = 21 cells from 7 rats), AAV-Vector + 5p model rats (n = 18 cells from 6 rats), and nucleic acid drug + 5p model rats (n = 18 cells from 6 rats). (D) Representative images of secondary dendrites of neurons in the mPFC and hippocampal CA1 regions from WT, AAV-Vector + 5p model rats, and nucleic acid drug + 5p model rats. Scale bar: 10 μm. (E) Spine density analysis of secondary dendrites of mPFC neurons. Sample size: WT (n = 77 branches from 5 rats), AAV-Vector + 5p model rats (n = 75 branches from 5 rats), and nucleic acid drug + 5p model rats (n = 84 branches from 6 rats). (F) Density of mushroom-shaped dendritic spines in neurons of the CA1 region of the hippocampus in WT rats, AAV-Vector + 5p model rats (2.76 ± 0.13, Mann-Whitney test, p = 0.03), and nucleic acid drug + 5p model rats (Mann-Whitney test, p = 0.0498).Sample size: WT (n = 77 branches from 5 rats), AAV-Vector + 5p model rats (n = 68 branches from 5 rats), nucleic acid drug + 5p model rats (n = 73 branches from 5 rats). All rats were 8 weeks old. All data are expressed as mean ± standard error (SEM). Statistical significance is as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

[0055] Figure 34 shows the neuronal density of the mPFC in each experimental group 4 weeks after the injection of nucleic acid drugs. (A) Representative sections of the mPFC of the experimental group stained with anti-NeuN antibody, showing low-power microscopy images (scale bar: 1 mm) and high-power microscopy images (scale bar: 500 μm). (B) The neuronal density of mPFC layers II / III and V / VI was measured every 10 5 μm 3 Quantification of neurons positively stained with anti-NeuN antibodies was performed. Sample size: WT, n = 29 sections from 5 rats; AAV-vector + 5p model rats, n = 28 sections from 5 rats; nucleic acid drug + 5p model rats, n = 29 sections from 5 rats. All rats were 8 weeks old. All data are presented as mean ± standard error (SEM). DETAILED DESCRIPTION

[0056] Various features and aspects of the present invention are discussed in greater detail below.

[0057] The present invention provides a polynucleotide sequence encoding a CTNND2 truncation, wherein the protein sequence of the CTNND2 truncation is shown in SEQ ID NO: 2. In some embodiments, the polynucleotide sequence is shown in SEQ ID NO: 1, or a polynucleotide sequence having 90.03% identity with SEQ ID NO: 1.

[0058] In one aspect, the present invention provides a nucleic acid construct, wherein the construct comprises the polynucleotide sequence. Preferably, the nucleic acid construct is a recombinant AAV construct comprising one or two ITRs and a poly A nucleotide sequence (polyA sequence).

[0059] In some embodiments, the nucleic acid construct includes a transcriptional regulatory element, which includes a promoter and / or an enhancer.

[0060] In some embodiments, the nucleic acid construct comprises:

[0061] a) 5' AAV inverted terminal repeat (ITR);

[0062] b) a first expression cassette encoding at least a first open reading frame (ORF) of a CTNND2 truncation under the control of regulatory control sequences directing its expression;

[0063] c) a polyA sequence; and

[0064] d) 3' AAV ITR.

[0065] The first expression cassette further includes transcriptional regulatory elements such as a promoter and / or enhancer.

[0066] In one aspect, the present invention provides a vector, wherein the vector contains the polynucleotide sequence or the construct of the present invention.

[0067] In some embodiments, the vector is an adeno-associated virus (AAV) vector. The recombinant AAV vector can comprise a nucleic acid molecule comprising 5'AAV ITR, an expression cassette as described herein, and 3'AAV ITR packaged in an AAV capsid. As used herein, the expression cassette can comprise regulatory elements for one or more open reading frames within each expression cassette and the nucleic acid molecule can optionally comprise additional regulatory elements. The AAV vector can comprise full-length AAV 5' inverted terminal repeats (ITRs) and full-length 3' ITRs.

[0068] In some embodiments, the AAV vector is selected from the group consisting of: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof.

[0069] In one aspect, the present invention provides a viral particle comprising the construct of the present invention.

[0070] In one aspect, the present invention provides a composition comprising the polynucleotide sequence of the present invention, the nucleic acid construct, the vector, or the viral particle, and a pharmaceutically acceptable excipient.

[0071] In another aspect, the present invention discloses the use of the polynucleotide sequence, the nucleic acid construct, the vector, the viral particle, or the composition described in the present invention in the preparation of a drug for treating cat cry syndrome in a subject in need thereof.

[0072] Any suitable method or route can be used to administer the AAV-containing compositions described herein, and optionally to co-administer other active drugs or therapeutic agents in combination with the AAV-mediated antibodies described herein. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0073] In some embodiments, the subject is a mammal, preferably a human, preferably a child aged 2 to 5 years.

[0074] The following examples are illustrative only and are not limiting of the invention described herein.

[0075] Example

[0076] Example 1: Preparation of carrier

[0077] 1. Instrument information

[0078] Table 1. Main instrument information

[0079] 2. Reagent Information

[0080] Reagents: AxyPrep Plasmid DNA Mini Kit; PrimeSTAR HS DNA Polymerase; and Meta Seamless Cloning Kit.

[0081] We designed and produced an AAV virus carrying a truncated rat Ctnnd2 gene. We also designed and produced a rat truncated Ctnnd2 gene homologous to the human CTNND2X8 gene, and completed the production of the Ctnnd2 nucleic acid drug carried by the AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP virus. The specific method is as follows:

[0082] 1. Obtaining target gene fragments:

[0083] The target gene was amplified by PCR. The target gene and upstream and downstream sequences were searched from GenBank, and primers were designed using VectorNTI software.

[0084] PCR amplification of target gene: Use high-fidelity PrimeSTAR enzyme to amplify the target gene. The reaction system and conditions are as follows:

[0085] Table 2. PCR reaction solution composition

[0086] The PCR reaction conditions were set as follows:

[0087] ①3-Step Method

[0088] Table 3. Primer sequence information

[0089] The PCR products were subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut out from the gel after agarose gel electrophoresis and recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0.

[0090] 2. Preparation of linearized expression vector:

[0091] The expression vector was digested with restriction endonucleases using the following digestion system: 2 μg of plasmid, 5 μL of 10x reaction buffer, 1 μL of each restriction endonuclease, and water to 50 μL. Incubate in a 37°C waterbath for at least 2 hours. The digestion products were examined by agarose gel electrophoresis. The target vector band was excised from the gel and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0.

[0092] 3. Target gene is constructed into linearized expression vector:

[0093] Use a seamless cloning kit suitable for a single target gene insert (Heyuan Seamless Cloning Kit, No.: OBCR(A)-50) to add the target gene fragment and linearized vector to a centrifuge tube at a molar ratio of 2:1 for recombination reaction:

[0094] Optimal amount of insert fragment used = [0.04 × number of insert fragment bases (2082 bp)] = 83.28 ng (0.03 pmol)

[0095] The optimal amount of linearized vector used = [0.02 × linearized vector base number (4696 bp)] = 93.92 ng (0.03 pmol)

[0096] x = optimal amount of insert (83.28 ng) / concentration of the obtained fragment (ng / μL)

[0097] y = optimal amount of linearized vector used (93.92 ng) / concentration of empty plasmid obtained in the experiment (ng / μL)

[0098] After mixing, incubate at 37°C for 30 minutes, then transfer to ice for 5 minutes. Transform directly or store at -20°C and thaw when needed.

[0099] 4. Preparation and transformation of competent cells:

[0100] Preparation and transformation of DH5α competent cells (Thermo Fisher Scientific, Cat. No. 18265017).

[0101] 5. Identification of positive transformants by colony PCR:

[0102] Pick the transformants grown on the plate and resuspend them in 10 μL of LB culture medium. Take 1 μL as a template for colony PCR identification. The reaction system and PCR cycling conditions are as follows:

[0103] Table 4. PCR reaction solution composition

[0104] PCR reaction conditions

[0105] 3-Step PCR

[0106] The primer sequences are SEQ ID No: 3 and SEQ ID No: 4.

[0107] 6. Send positive clones for sequencing:

[0108] Positive clones obtained through colony identification were sent to a sequencing company for sequencing verification. Sequencing results were compared and analyzed using SnapGene software.

[0109] 7. Plasmid extraction:

[0110] Positive clones that passed sequencing were prepared for plasmid miniprep using the AxyPrep Plasmid DNA Miniprep Kit (Axygen; Catalog No.: AP-MN-P-50).

[0111] 8. Plasmid expression detection experiment

[0112] 8.1 Experimental Purpose

[0113] The expression of Ctnnd2 protein was detected after the plasmid was transferred into 293T cells.

[0114] Table 5. Main reagents and instrument information

[0115] 8.2 Experimental methods

[0116] The expression of CTNND2 protein was detected after the plasmid was transferred into 293T cells.

[0117] 8.2.1 Total protein extraction

[0118] Remove Western and IP cell lysis buffer and protease inhibitor PMSF from -20°C, add 10 μL PMSF to every 990 μL cell lysis buffer, and place on ice; remove the sterilized EP tube, label it, and place it on ice at 4°C; remove the cells from the incubator, discard the cell culture medium, and wash 1-2 times with PBS; discard the PBS, add an appropriate amount of pre-cooled Western and IP cell lysis buffer (containing PMSF), lyse the cells on ice for 30 minutes, scrape the cells, and transfer the sample to an EP tube; transfer the centrifuge tube containing the lysed cells to a pre-cooled high-speed low-temperature centrifuge and centrifuge at 12,000 rpm at 4°C for 5-10 minutes.

[0119] 8.2.2 Protein concentration measurement

[0120] Cell protein solution: Draw out 3 μL from the original solution and add 27 μL PBS to dilute 10 times.

[0121] Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution with a volume ratio of A to B of 50:1 and mix thoroughly.

[0122] Prepare a standard protein gradient: dilute 2000 μg / mL BSA standard to 0, 25, 125, 250, 500, 750, 1000, 1500, and 2000 μg / mL.

[0123] Protein concentration determination: 25 μL of each standard and sample were added to a 96-well plate. 200 μL of BCA working solution was added to each well and incubated at 37°C for 30 minutes. The absorbance of A562 was measured using a microplate reader. The protein concentration of the sample was calculated based on the standard curve and the sample volume used.

[0124] 8.2.3 Sample preparation

[0125] The calculated data for each well was expanded and diluted according to the actual amount of sample mother solution, and then loading buffer was added at a ratio of 1:4 (loading buffer: cell solution). Mixed, boiled at 95°C for 5 minutes, placed on a dry ice box, and then centrifuged at 4°C, 12000g, for 5 minutes.

[0126] 8.2.4 SDS-PAGE gel preparation

[0127] Align two clean glass plates, place them in a clamp, and secure them to the gel-casting stand. Prepare the separation gel solution (see Table 5 for the preparation system) and gently stir to mix. Immediately add the separation gel solution to the glass interlayer until the gel is approximately 5 cm high. Slowly fill the interlayer with deionized water. Allow the gel to polymerize at room temperature for 30 minutes. After polymerization, a clear refractive index line will be visible at the interface between the top layer of deionized water and the gel. Pour off the top layer of deionized water and blot as much as possible with absorbent paper. Prepare a 5% concentrated gel solution (see Table 6 for the preparation system) and add the solution to the glass interlayer until it reaches the top. Insert a 1.5 mm thick comb into the concentrated gel solution in the interlayer. If necessary, add more concentrated gel solution to fill any remaining space. Allow the concentrated gel to polymerize at room temperature for 30 minutes.

[0128] Table 6. SDS-PAGE separation gel formula

[0129] Table 7. SDS-PAGE stacking gel (5% acrylamide) recipe

[0130] 8.2.5 SDS-PAGE gel electrophoresis

[0131] Fix the gel plate to the inner tank of the electrophoresis device, place the fixed gel plate into the outer tank, add an appropriate amount of 1×SDS electrophoresis buffer to the outer tank, and add electrophoresis buffer to the inner tank until it just covers the gel loading well;

[0132] Carefully remove the comb to avoid tearing the polyacrylamide gel wells. After removing the comb, rinse and fill the wells with 1× SDS electrophoresis buffer.

[0133] Load the sample and perform SDS-PAGE electrophoresis. Connect the power supply and run the gel at 80V for 30 minutes until the bromophenol blue dye enters the separating gel from the stacking gel. Then increase the voltage to 120V and continue running the gel for 70 minutes until the bromophenol blue reaches the bottom of the gel.

[0134] 8.2.6 Transfer

[0135] Before the end of the electrophoresis run, prepare a PVDF membrane and six filter papers of the same size as the gel. Activate the PVDF membrane with methanol for 5 minutes, then equilibrate in transfer buffer for 15 minutes. Equilibrate the filter papers in transfer buffer for 15 minutes. Place the transfer clamp, two sponge pads, filter paper, and the soaked membrane in a tray containing transfer buffer. Open the clamp so that the black side is level. Place a sponge pad on top. Place three layers of filter paper on the pad, removing any air bubbles. Remove the glass plate and gently scrape off the stacking gel. Rinse the gel once in ddH2O and once in 1x transfer buffer. Carefully place the separating gel over the filter paper, manually adjusting it to align with the filter paper. Place the membrane over the gel, ensuring it completely covers the gel and removing any air bubbles. Place three sheets of filter paper over the membrane and remove any air bubbles. Finally, place another sponge pad and close the clamp. Place the clamp into the transfer tank, with the black side of the clamp facing the black side of the tank and the white side of the clamp facing the red side. Electrotransfer generates heat. Place an ice cube on one side of the tank to cool the heat. Typically, transfer at 300 mA for 2 hours. Turn off the power supply and remove the connected wires. Discard the running buffer and remove the gel sandwich.

[0136] 8.2.7 Immune Response

[0137] Table 8. Antibody information

[0138] The membrane was transferred to an incubation box containing 5% skim milk powder in TBST and blocked by shaking on a decolorization shaker at room temperature for 1 hour; the primary antibody was diluted to an appropriate concentration with 5% skim milk powder in TBST; the membrane was removed from the blocking solution and placed with the protein side facing up on the antibody liquid surface, and the four corners of the membrane were tilted to remove residual bubbles; the membrane was incubated by shaking on a decolorization shaker at 4°C overnight; the membrane was washed three times with TBST on a decolorization shaker at room temperature for 10 minutes each time; the secondary antibody was diluted as above and placed in contact with the membrane, and after incubation at room temperature for 1-2 hours, the membrane was washed three times with TBST on a decolorization shaker at room temperature for 10 minutes each time, and the chemiluminescence reaction was performed.

[0139] 8.2.8 Chemiluminescence

[0140] Mix equal volumes of reagents A and B; place the membrane with the protein side facing up on a white plate, add drops of luminescent liquid to the membrane to fully cover the membrane surface, and then place it in an imager to take a picture.

[0141] 8.3 Results

[0142] The CTNND2 protein was predicted to be approximately 79 / 106 KDa. The experimental results in Figure 4 showed that protein bands were detected between Marker 70-100 KDa.

[0143] Example 2: In vivo experimental results

[0144] As shown in the flow chart in Figure 5, the constructed targeted nucleic acid drug was injected into 5p model rats via the tail vein. Behavioral and related experiments were conducted 4-12 weeks after administration. The animals were then fed normally for a long period of time, and social behavior, anxiety-like behaviors, and cognitive function were tested at 6, 9, and 12 months after treatment to evaluate the long-term efficacy of the drug. The results showed that after 4-8 weeks of treatment in 3-4 week-old rats, the impaired novel object preference memory, object location recognition memory, and object location exchange recognition cognitive functions of the 5p model rats were improved. However, tail vein injection of the gene-targeted nucleic acid drug did not significantly improve the anxiety-like behaviors of the 5p model rats.

[0145] Figure 6A-B shows that 4-8 weeks after the 5p model rats were injected with gene-targeted nucleic acid drugs via the tail vein, the impaired novel object preference memory function of the 5p model rats was significantly improved, both compared with the 5p model rats injected with empty AAV and compared with their own behavior before administration; Figure 6C-D shows that after the 5p model rats were injected with gene-targeted nucleic acid drugs via the tail vein, the object position recognition memory and object position exchange recognition cognitive functions were significantly improved.

[0146] As shown in Figure 7, 4-8 weeks after the tail vein injection of gene-targeted nucleic acid drugs, the 5p model rats still showed obvious anxiety-like behaviors, just like the 5p model rats injected with empty AAV, indicating that the anxiety-like behaviors of the 5p model rats were not improved after the tail vein injection of gene-targeted nucleic acid drugs.

[0147] As shown in Figure 8A-B, administration of gene-targeted nucleic acid drugs to 8-week-old 5p model rats did not significantly improve the novel object preference memory function and three-box social behavior of the 5p model rats, but did improve anxiety-like behavior to a certain extent, suggesting that tail vein injection of gene-targeted nucleic acid drugs in the late puberty development period is not as effective as administration shortly after birth, and the timing of administration is crucial.

[0148] Example 3: Biological preparation of 5p model rats

[0149] 1. Sequencing confirmation of target sequence

[0150] The target gene sequence may vary between strains. To ensure the efficiency of the designed Cas9 / sgRNA, the target site sequence of the SD rat tail must first be PCR amplified and sequenced to ensure that the sgRNA recognition sequence is completely consistent with the SD rat tail DNA sequence. The PCR primers are as follows:

[0151] Table 9. Primer sequence list

[0152] PCR and sequencing were performed on SD rat tail DNA, and the results showed that the SD rat tail target sequence was completely consistent with the sequences given in Genebank and Ensembl.

[0153] 2. Cas9 / sgRNA Design and Construction

[0154] Cas9 / sgRNA design

[0155] Based on the design principles of sgRNA, 8 sgRNAs were designed in the 5' target site and 14 sgRNAs were designed in the 3' target site region. The corresponding targeting sequences are as follows:

[0156] Table 10. Target sequence information corresponding to sgRNA

[0157] 2.2. Construction of Cas9 / sgRNA Plasmid

[0158] Oligos were synthesized according to the designed sgRNA sequence and ligated into the pCS-4G vector via Gibson ligation. After transformation, the ligation product was sequenced to verify correctness. A map of the pre-cut pCS vector is shown in Figure 11.

[0159] 3. Detection of Cas9 / sgRNA Activity

[0160] sgRNA activity was tested using the UCATM method, a CRISPR / Cas9 activity assay developed independently by Biocytogen. This method offers advantages such as species independence, high throughput, broad adaptability, high sensitivity, and simplicity. EGE-ZLM-048-A-sgRNA3 and EGE-ZLM-048-A-sgRNA20 were selected for the next step of the experiment. The test results are shown in Figure 12.

[0161] 4. sgRNA RNA Preparation

[0162] EGE-ZLM-048-A-sgRNA3 and EGE-ZLM-048-A-sgRNA20 were transcribed in vitro to obtain RNA for microinjection. The RNA electrophoresis pattern is shown in Figure 13.

[0163] 5. Microinjection of Cas9 / sgRNA

[0164] Cas9 / sgRNA was microinjected into rat fertilized eggs, and the birth conditions of F0 rats after injection were as follows.

[0165] Table 11. F0 rat births after Cas9 / sgRNA injection into rat fertilized eggs

[0166] 6. Genotype identification of F0 generation rats

[0167] Gene knockout rats were constructed by injecting fertilized eggs with Cas9 / sgRNA. Due to the rapid cleavage rate of early embryos, the resulting F0 rats are chimeric. Therefore, the F0 genotype obtained by tail analysis of the F0 rats is for reference only and does not guarantee a heritable gene mutation. Heritable genotypes must be confirmed by tail analysis of the F1 rats.

[0168] 6.1. Identification Primer Design

[0169] The principles of primer design are shown in Figure 14.

[0170] Primer information is as follows:

[0171] Table 12. Identification primer information

[0172] PCR conditions: 2x Taq Plus Master Mix II (Dye Plus) progress

[0173] Enzyme: 2x Taq Plus Master Mix II (Dye Plus)

[0174] Table 13. PCR reaction conditions and procedures

[0175] 6.2. Identification of F0 Generation Rat Tail Genotype

[0176] The identification results are shown in Figure 15. Primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R. PCR amplification and product sequencing showed that EM48-0002, EM48-0004, EM48-0005, EM48-0007, EM48-0008, EM48-0019, EM48-0028, EM48-0032, EM48-0045, and EM48-0052 were positive F0 rats.

[0177] 7. Genotype identification of F1 generation rats

[0178] Some of the F0 generation rats identified as positive were mated with wild-type rats to obtain F1 generation rats with stable genotypes. The mating results are as follows:

[0179] Table 14. Mating of F0 generation rats and wild type rats

[0180] 7.1. Identification Primer Design

[0181] The principles of primer design are shown in Figure 16.

[0182] Primer information is as follows:

[0183] Table 15. Identification primer sequence information

[0184] Polymerase chain reaction conditions: 2x Taq Plus Master Mix II (Dye Plus) progress

[0185] Enzyme: 2x Taq Plus Master Mix II (Dye Plus)

[0186] Table 16. PCR reaction conditions and procedures

[0187] The genotype of the F1 generation rat tail was identified. The identification results are shown in Figure 17, primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-WT-R; as shown in Figure 18, primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0188] F1 generation sequencing results:

[0189] 1EM48-0001, 1EM48-0004, 1EM48-0005, 1EM48-0007, 1EM48-0009, 1EM48-0010, 1EM48-0011, 1EM48-0012, 1EM48-0013, 1EM48-0015, 1EM48-0016 and 1EM48-0020 are positive F1 rats.

[0190] We used positive rats for subsequent experiments.

[0191] The rat gene homologous to the human 5p gene is located on rat chromosome 2. The 1.68Mb knockout fragment contains multiple genes related to growth and development. Preliminary results show that homozygous 5p model mice die in utero, while heterozygous 5p model mice can survive, have slowed growth and development, and are significantly lower in weight and body length than normal rats. Compared with control rats of the same age, the 5p model mice have low muscle tone. These phenotypes are highly consistent with those of 5p patients.

[0192] Figure 19 shows that qPCR experiments confirmed that the 5p model rats lacked the key pathogenic homologous genes of cat cry syndrome, Ctnnd2, Cct5, Marchf6, and Ankrd33b.

[0193] Figures 20A-C show that the weight, body length and tail length of 5p model rats were significantly smaller than those of control rats. Figures 20D-E show that there were no significant changes in the development of the pinna of 5p model rats and the time of first eye opening of suckling rats compared with control rats.

[0194] We further analyzed the brain weight of 5p model rats at 14 weeks of age and found that their brain weight was significantly reduced compared with that of their WT littermates (Figure 20F, WT rats, 1.63±0.03g; 5p model rats, 1.47±0.05g; P<0.01), which is consistent with the microcephaly phenotype commonly observed in 5p patients. These results strongly suggest that the 5p model rats replicate the growth and developmental impairment observed in children with 5p15.2 deletion.

[0195] Interestingly, compared with control rats, 5p model rats showed a significantly earlier first postnatal reactivity to environmental sounds, indicating hypersensitivity to ambient sounds in 5p model rats, a phenotype highly consistent with 5p patients. Similar to Ctnnd2 knockout mice, we assessed the animals' social behavior. Results from a three-chamber social behavior test revealed severe social impairment in 5p model rats. Similar to patients with Cry-a-Cat syndrome, 5p model rats displayed significantly increased fur-stroking time and repetitive, stereotyped behaviors, accompanied by decreased curiosity about their surroundings, suggesting typical autistic-like behaviors. An open-field test revealed a significant decrease in the range of activity in 5p model rats, with activity concentrated primarily in the periphery of the open field, and significantly less time spent in the central area, indicating significant anxiety-like behaviors. An elevated plus maze test also confirmed significant anxiety-like behaviors in 5p model rats. We also assessed the animals' limb balance and postural gait, revealing a degree of impairment in motor balance and coordination in 5p model rats, suggesting abnormalities in cerebellum and extrapyramidal system function. Further behavioral experiments showed that the 5p model rats had obvious deficits in novel object preference memory, object location recognition memory, and object location exchange recognition cognitive functions, which were highly consistent with the intellectual disability of patients with cat cry syndrome.

[0196] Figures 21A-B show delayed development of the righting reflex in the plane and in the air in 5p model rats. Figure 21C shows a slight delay in the onset of the first rotational movement in 5p model rats, but no significant difference compared to control rats. Figure 13D shows no significant difference in the onset of the first crawling and negative geotaxis in 5p model rats compared to control rats. Figure 13E shows that the first postnatal reactivity to environmental sounds in 5p model rats was significantly earlier than in control rats, demonstrating hypersensitivity to environmental sounds in 5p model rats.

[0197] 22A-B show that compared with the control normal rats, the total time and score of the 5p model rats in grasping the rope were significantly decreased, indicating that the muscle tension of the 5p model rats was reduced.

[0198] FIG23 shows that 5p model rats have obvious social impairment.

[0199] Figure 24A shows that the 5p model rats spent significantly more time stroking their fur, suggesting that the 5p model rats exhibit repetitive stereotyped behaviors. Figure 24B shows that compared with the control rats, the 5p model rats spent significantly less time digging in their cages, suggesting that the 5p model rats lack curiosity about their surroundings.

[0200] Figure 25A, open field test: The 5p model rats spent significantly less time in the central area of ​​the open field, exhibiting obvious anxiety-like behavior, but the animal's movement speed in the open field did not change. Figure 25B shows that the 5p model rats also exhibited obvious anxiety-like behavior in the elevated plus maze test, further supporting the results of the open field test.

[0201] Figure 26A shows abnormalities in the limb balance ability test of the 5p model rats. Figure 26B shows that the 5p model rats' movement posture and gait are basically normal. The results show that the 5p model rats have no obvious loss of movement coordination, suggesting that the cerebellum and extrapyramidal system functions of the 5p model rats are abnormal to a certain extent.

[0202] Figure 27A-C shows that the 5p model rats showed obvious deficits in novel object preference memory, object location recognition memory, and object location exchange recognition cognitive functions.

[0203] In summary, the phenotype of these 5p model rats is highly consistent with that of individuals with Cri du Chat syndrome. Our establishment of this first rodent model not only validates the key gene for Cri du Chat syndrome but also lays a solid foundation for in-depth research into the pathogenesis of rare pediatric neurological diseases and the development of drugs for these diseases.

[0204] Table 17. Sequence information

[0205] Example 4: Impaired dendritic complexity, alterations in dendritic spines, and pathological increases in neuronal density

[0206] The ASD-like behaviors, impaired cognition, and hypoactivity we observed in the 5p model rats may reflect changes in synapse formation and number. To explore the link between delayed neurodevelopment, behavioral changes, and cognitive deficits in the 5p model rat brain, we quantitatively analyzed dendritic branching and spine formation in different brain regions at different developmental stages.

[0207] Sholl analysis of Golgi-Cox stained sections revealed that the complexity of dendritic branches in the mPFC and hippocampal CA1 regions of 5p model rats was significantly reduced compared with control rats (Figures 29A-B and 29E-F). These differences were seen between 20 μm and 60 μm from the mPFC to the neuronal cell body (Figure 29B) and between 40 μm and 105 μm from the cell body in the CA1 region of the hippocampus (Figure 29F). Interestingly, no difference in the complexity of dendritic branches was found between the 5p model and WT rats in the dentate gyrus (DG) region of the hippocampus (Figure 29I). Our Golgi staining results also highlighted the relatively disordered arrangement of neurons in the CA1 region of the hippocampus in 5p model rats. In contrast, the control group presented relatively smooth neuronal cell body arrangement lines (Figure 29E, upper and middle layers, marked with red circles).

[0208] Next, we conducted an in-depth analysis of dendritic spine morphology and density in the mPFC and hippocampus. We measured the density of spines in secondary dendritic segments of layer II / III pyramidal neurons in the mPFC (Figure 29C) and in dendrites of neurons in the CA1 (Figure 29G) and DG (Figure 29J) regions of the hippocampus. Based on established classifications, these spines were categorized as either elongated spines (length-to-width ratio >2) or mushroom-shaped spines. Given the lack of measurements of postsynaptic density and their presynaptic counterparts, we termed these elongated spine-like structures “filopodia” and “stubby spines.” Detailed quantitative analysis of dendrites of layer II / III neurons in the mPFC revealed a significant reduction in mushroom-shaped spines (Figure 29D). Furthermore, our observations revealed a consistent decrease in the number of mushroom-shaped spines in the dendrites of CA1 neurons in 5p model rats compared to WT rats, while the density of filopodia and stubby spines was higher in 5p model rats (Figure 29H). Notably, a significant decrease in the density of mushroom-shaped spines was observed in the dendrites of DG neurons in 5p model rats (Figure 29K). In contrast, no differences were detected in the overall density of filopodia and stubby spines in the dendritic spines of mPFC and DG neurons in 5p model and WT rats, respectively (Figures 29D and 29K).

[0209] Taken together, our data suggest that rats with a 2q22 deletion in the 5p model, which results in a ~50% decrease in expression of most genes within the homologous 5p15.2 region, exhibit defects in spine density and maturation in the mPFC and hippocampus. Furthermore, this deletion appears to result in disorganized neuronal organization in the hippocampus.

[0210] In addition, we used immunofluorescence staining to assess the neuronal density of the mPFC and hippocampus of 8-week-old 5p model rats (Figures 30A and 30B). Surprisingly, we observed a significant increase in the neuronal density of layer II / III in 5p model rats compared with WT rats (Figure 30C, left panel), but no change in the neuronal density of layer V / VI in the mPFC (Figure 30C, right panel). However, the neuronal density of the CA1 and DG regions of the hippocampus in 5p model rats remained unchanged compared with WT rats (Figure 30D). This result suggests that in early adulthood, a pathological increase in neuronal density occurs specifically in the superficial regions of the mPFC in 5p model rats.

[0211] Example 5: Gene expression profiles of the PFC and hippocampus in 5p model rats

[0212] To gain deeper insights into the effects of 2q22 deletion (a 1.68Mb gene fragment deletion) on neural development in heterozygous rats, particularly on dendritic damage and behavioral deficits during postnatal development, we performed RNA sequencing (RNA-seq) on PFC and hippocampal samples from 8-week-old WT and 5p model rats. We observed that gene expression patterns in the PFC and hippocampus were consistent between WT and 5p model rats (Figure 31A). This finding provides molecular evidence that the 5p model rat is a reliable and reproducible animal model for 5p model studies.

[0213] Differential expression profiling analysis of the 5p model revealed a large number of differentially expressed genes in the PFC region between WT and 5p model rats, including 1,673 upregulated and 1,750 downregulated genes, totaling 3,423 genes (adjusted p-values, Figure 31B). The most significantly downregulated genes included Ctnnd2 (log2FC = -1.13; p.adj = 9.50e-112), Ankrd33b (log2FC = -1.67; p.adj = 2.94e-25), Marchf6 (log2FC = -1.48; p.adj = 8.35e-64), and Cct5 (log2FC = -1.10; p.adj = 1.40e-44) (Figure 31B).

[0214] Gene set enrichment analysis showed that these differentially expressed genes (DEGs) were mainly in “synapse” (FDR=1.06e-171, GO:0045202), “cell anatomical entity” (FDR=6.50e-105, GO:0110165), “nervous system development” (FDR=8.67e-104, GO:0007399), “neuronal projection” (FDR=8.51e-101, GO:0043005) and “intracellular anatomical structure” (FDR=6.97e-96, GO:0005622) ( Figure 31C ).

[0215] Inflammatory signaling and the innate immune system play a crucial role in the degeneration of brain synapses and dendrites. Abnormal activation of inflammatory signaling disrupts synaptic plasticity and stability, ultimately leading to cognitive impairment, psychiatric disorders, and neurodegenerative diseases. Therefore, to elucidate the underlying mechanisms of the 5p rat model and design innovative therapeutic interventions, a deeper understanding of the interplay between inflammatory signaling and the innate immune response is essential. Therefore, we conducted a comprehensive analysis of inflammatory signaling pathways and innate immune responses across functional forebrain regions. Interestingly, our results revealed 163 inflammation- and 499 innate immune-related differentially expressed genes in the PFC of 5p rats. To further elucidate the associations between these differentially expressed genes and synaptic / dendritic alterations, we performed a gene intersection analysis and displayed the results as a Venn diagram (Figure 31D). This comprehensive analysis highlights the key roles of inflammatory signaling pathways and the innate immune response in the pathogenesis of Cri-du-chat syndrome (CdCS) and establishes a link between their differentially expressed genes and synaptic / dendritic alterations.

[0216] Furthermore, combined differential expression and Gene Ontology (GO) analysis revealed that genes related to synaptic and dendritic development were significantly downregulated, particularly Nlgn1, Nrxn1, Nlgn2, Nlgn3, Npas4, Clstn3, Ephb3, Slitrk3, Ptprd, and Rtn4, as well as genes associated with synaptic transmission and plasticity, such as Snap25, Vamp7, Gabrg2, Gabra1, Grin2a, Gria2, and Chrna7. In contrast, genes associated with brain inflammation and innate immune response, such as Cd2ap, C1qa, Cx3cr1, C4b, F2r, Ptn, Wnt5a, and Itgb1, were significantly upregulated in the PFC of 5p model rats (Figure 31E). These findings indicate that rats carrying the 2q22 deletion-5p model exhibit altered synaptic / dendrite development, accompanied by inflammation and innate immune responses in the brain, ultimately manifesting as defects in dendritic spine density and maturation in the PFC.

[0217] Furthermore, unlike the PFC, our comprehensive RNA-seq analysis of the hippocampus of WT and 5p model rats revealed only 174 differentially expressed genes, including 108 upregulated and 66 downregulated genes (adjusted p-value, p.adj < 0.1; Figure 31F). The most significantly downregulated genes were Cct5 (log2FC = -1.06; p.adj = 3.45e-49), Ctnnd2 (log2FC = -1.05; p.adj = 9.84e-29), and Marchf6 (log2FC = -1.08; p.adj = 4.38e-21) (Figure 31F). The significant difference in the number of differentially expressed genes between the PFC and hippocampus in the context of 2q22 deletion remains largely unknown. This phenomenon may be due to the different temporal developmental windows and underlying mechanisms of these different brain regions.

[0218] GO analysis in the hippocampus revealed significant enrichment of differentially expressed genes in various neuronal structures, including "neuronal projection" (FDR = 7.66e-4, GO:0043005), "somatodendritic compartment" (FDR = 1.07e-3, GO:0036477), "postsynaptic" (FDR = 1.79e-3, GO:0098794), "dendritic tree" (FDR = 1.97e-3, GO:0097447), "dendrite" (FDR = 2.54e-3, GO:0030425), and "synapse" (FDR = 2.65e-3, GO:0045202) (Figure 31G). Notably, these findings are consistent with those obtained in the prefrontal cortex, further confirming that rats with 2q22 deletion 5p model exhibit altered synaptic and dendritic development in the hippocampus.

[0219] Similar to the analysis of prefrontal regions, we performed an extensive genetic intersection analysis in the hippocampus to illustrate the relationship between these differentially expressed genes and synaptic / dendrite alterations in a Venn diagram (Figure 31H). This exhaustive analysis highlighted the role of synapses (especially GAB A These results suggest that damage to synapses and hippocampal dendrites plays a key role in the pathogenesis of CdCS, thus establishing a link between the corresponding differentially expressed genes in this region and synaptic / dendrite disorders.

[0220] Additional analyses revealed that downregulation of specific differentially expressed genes, namely Gabrb1, Gabrg1, Gabrq, Kcna4, Kcnk9, and Cadm1, was closely associated with reduced GABAergic function, impaired neuronal voltage-gated potassium channel activity, and decreased synaptic adhesion in the hippocampus (Figure 31I). Conversely, significant upregulation of genes such as Scn1b, Scn4b, Cacng3, Adora1, Mbp, and Dagla, which are closely related to neuronal voltage-gated sodium channel function, AMPA receptor trafficking and gating, neuronal metabolism, and myelin development, was observed in the hippocampus (Figure 31I). This upregulation strongly suggests an imbalance in excitation / inhibition dynamics within the hippocampal neural network, particularly increased neuronal excitability. Notably, genes closely associated with the innate immune response, Nfil3 and Fcrl2, were also significantly upregulated (Figure 31I), suggesting the presence of inflammation and enhanced innate immune activity in the hippocampus. Ultimately, this immune response further emphasizes the complex interplay between neuronal dysfunction and immune activation in the setting of CdCS.

[0221] Example 6: A single injection of a gene-targeted nucleic acid drug carrying Ctnnd2 can effectively rescue cognitive function and change gene expression profiles, but is insufficient to address social dysfunction and anxiety-like behaviors

[0222] Given the broad coverage of Ctnnd2 in CdCS-associated deletions and its precise modeling in rats, we hypothesized that AAV-PHP.eB vectors expressing gain-of-function Ctnnd2 variants (Mendell, JR, et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs. Mol Ther 29, 464-488 (2021); Liguore, WA, et al. AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice. Mol Ther 27, 2018-2037 (2019); Deverman, BE, et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209 (2016)) could drive therapeutic CNS expression, thereby enhancing social behavior and cognitive function in 5p model rats while minimizing immune responses. AAV-PHP.eB can cross the BBB and widely transduce central nervous system cells. To this end, we developed a Ctnnd2 nucleic acid drug carried by the AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP virus, which carries a Ctnnd2 variant (a gain-of-function mutation with a core element domain similar to full-length Ctnnd2) under the CMV promoter.

[0223] Subsequently, we performed RT-qPCR analysis to investigate whether intravenous administration of the Ctnnd2 nucleic acid drug carried by the AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP virus would lead to upregulation of Ctnnd2 gene expression. Notably, we observed a significant increase in Ctnnd2 gene expression levels in the hippocampus (Figure 32B). Although there also appeared to be a trend toward increased expression in the functional areas of the forebrain, no statistically significant differences were found in this region (Figure 32B). Notably, compared with the 5p model rats injected with pAAV-CMV-EGFP-3xFLAG-Twpa (AAV-Vector) (AAV-Vector+5p model rats), the expression of the Ctnnd2-encoded protein δ-catenin in the PFC and hippocampus of the 5p model rats injected with the nucleic acid drug (AAV.CAP-B10-CMV-rCTNND2-P2A-EGFP) (nucleic acid drug+5p model rats) was significantly increased, but still lower than that of WT rats (Figure 32C).

[0224] Next, we studied the effects of nucleic acid drug injection on social behavior and cognitive function deficits in 5p model rats at different developmental stages. First, 4-week-old rats were intravenously injected with nucleic acid drugs, and behavioral measurements were performed 4 weeks and 8 weeks after virus injection. 5p model rats injected with nucleic acid drugs (nucleic acid drug + 5p model) showed significant enhancement in the new object preference task at both 4 and 8 weeks (Figure 32D). It is worth noting that the injection of nucleic acid drugs can effectively restore the impaired object location memory of rats at two time points (Figure 32E).

[0225] To determine whether the enhanced cognitive function of early 5p model rats treated with nucleic acid drugs was attributed to improvements in dendritic and synaptic structure, we subsequently quantified dendritic arborization and spine formation. Our Golgi staining analysis showed that the complexity of the dendritic arbors in the DG region of the hippocampus of 5p model rats treated with nucleic acid drugs was significantly increased, in sharp contrast to the 5p model rats treated with AAV-Vector (Figure 32G-H). In addition, compared with the 5p model rats treated with AAV-Vector, we observed a sustained increase in mushroom-shaped spines in the dendrites of DG neurons in 5p model rats treated with nucleic acid drugs (Figure 32I-J).

[0226] Despite treatment with nucleic acid drugs, the density of mushroom-shaped spines in the dendrites of mPFC and hippocampal CA1 neurons in 5p model rats still decreased, indicating that the treatment did not completely restore this aspect of spine morphology. In addition, no significant differences were observed in the overall density of filamentous processes and short thick spines on the dendritic spines of mPFC and DG neurons in 5p model rats treated with nucleic acid drugs and 5p model rats treated with AAV-Vector (Figure 33, Figure 32J). However, an interesting finding was that the density of filopodial spines in the mPFC of 5p model rats was significantly reduced compared with WT rats (Figure 33E). It is worth noting that after treatment with nucleic acid drugs, the density of filopodial spines in 5p model rats was significantly higher than that in WT rats (Figure 33E), indicating that the spine morphology has partially recovered to normal levels, especially the filopodial spines.

[0227] Together, these findings suggest that a single injection of nucleic acid drugs can effectively increase the complexity of dendritic shafts and the density of mature dendritic spines, implying that nucleic acid drugs can truly improve the structure and function of neurons and synapses.

[0228] In addition, we also evaluated the neuronal density in the mPFC four weeks after the injection of nucleic acid drugs (Figure 34). Unexpectedly, compared with WT rats, the neuronal density in the mPFC layer II / III of the 5p model rats injected with AAV-Vector did not change significantly, while the neuronal density of the 5p model rats and WT rats increased significantly (Figure 30C, left panel). This suggests that neurons in the mPFC layer II / III of the 5p model rats may undergo apoptosis and / or necrosis, which may be triggered by inflammation and innate immune responses, ultimately leading to their death. It is worth noting that after nucleic acid drug treatment, the neuronal density in the mPFC layers II / III and V / VI of the 5p model rats remained unchanged. These findings mean that nucleic acid drug treatment did not change the neuronal density in these areas, highlighting that this treatment method does not have any toxic effects on neuronal loss.

[0229] To gain a deeper molecular understanding of how the nucleic acid drug partially restored impaired recognition memory and altered dendritic patterning, we further performed RNA-seq analysis on samples collected from the PFC and hippocampus of 5p model rats four weeks after intravenous administration of the nucleic acid drug. Notably, our analysis revealed a significantly lower number of DEGs in the forebrain of 5p model rats, with only 87 DEGs (including 48 upregulated and 39 downregulated genes) (Figure 32K). In contrast, compared with intravenous AAV-vector administration, the number of DEGs in the hippocampus increased significantly after nucleic acid drug administration, totaling 2,394 DEGs (1,230 upregulated and 1,164 downregulated genes) (Figure 32N). Although the reason for the significantly higher number of DEGs in the hippocampus compared to the forebrain after nucleic acid drug administration remains unclear, this observation appears consistent with the significantly higher expression levels of Ctnnd2 and δ-catenin in the hippocampus compared to the forebrain (Figures 32B and 32C).

[0230] To investigate the complex gene expression patterns associated with the pathogenesis of the 5p model rats, we performed in-depth RNA sequencing analysis and compared the situation before and after administration of nucleic acid drugs. Our results revealed the presence of 40 DEGs in the PFC, 18 of which were overlapping (Figure 32L). After administration of nucleic acid drugs, a significant reversal was observed in the forebrain functional areas, with all 11 genes that were previously downregulated in the 18 DEGs being upregulated (Figure 32M). This group of genes included two downregulated GABA A Receptor genes (Gabra1 and Gabrg2), a downregulated glycine receptor gene (Glrb), and eight downregulated genes closely related to neuronal transmitter release, synaptic and dendritic development (Snap25,, Stxbp1, Dnm1l, Oxr1, Lgi2, Lrrc4c, Map1b and Pja2) (Figure 32M). This comprehensive upregulation means that GABAergic and glycine receptor function, neuronal transmitter release, synaptic and dendritic development in the PFC were substantially restored after injection of nucleic acid drugs. In contrast, the remaining 7 upregulated genes among the 18 DEGs (known to be involved in innate immune responses and inflammation) were completely downregulated in the PFC (Figure 32M). These genes include C4b, Irgm, Ier3, Lgals9, Apoe, Slfn2 and Parp9 (Figure 32M), indicating that the innate immune response and inflammatory response in the PFC were significantly suppressed after administration of nucleic acid drugs.

[0231] In contrast to the 40 DEGs found in the forebrain functional regions, a significant increase of 408 DEGs was observed in the hippocampus (Figure 32O). However, this major change resulted in only 11 overlapping DEGs (Figure 32O), highlighting the target specificity of the treatment effects. Among these 11 DEGs, notable changes included two GABAA Downregulation of receptor genes (Gabra1 and Gabrg2), upregulation of a calcium voltage-gated channel auxiliary gene (Cacng3), upregulation of a sodium voltage-gated channel gene (Scn1b), and upregulation of synaptic-related genes, as well as genes related to synaptic function and neural signal transduction, such as Slc5a7 (choline uptake transporter), Slc8a2 (cation transporter), Crtc1 (CREB regulatory transcription factor), Cebpb (CCAAT / enhancer binding protein), Aplp2 (downregulated), and Psd and Git1 (both upregulated) (Figure 32P). Unfortunately, none of these 11 overlapping DEGs were rescued in the hippocampus after treatment with nucleic acid drugs (Figure 32P). In addition, our findings revealed the upregulation of 8 DEGs in the 397 non-overlapping sets (Figure 32O and Figure 32Q), namely Pvalb, Ngf, Nrgn, Shank3, Stx1a, Stxb1, and Syn1, which are closely related to dendrite and synaptic development, synaptic transmission and plasticity, especially GABAergic function. Notably, all 8 genes were upregulated after nucleic acid drug treatment, indicating that these key neural processes were enhanced, although the above genes were not rescued.

[0232] In addition, we rigorously monitored the health of the animals and collected blood samples to assess liver and kidney function after vector injection. Notably, we observed no mortality in rats, and renal function remained stable and maintained at normal levels up to 8 months after AAV injection (Table 18). However, it is worth noting that systemic injection of AAV resulted in elevated total bilirubin levels in the serum (Table 18). This observation suggests that the AAV-PHP.eB capsid has viral targeting affinity for hepatocytes, subsequently triggering an immune response and leading to mild hepatotoxicity.

[0233] In summary, a single injection of nucleic acid drugs can effectively alleviate cognitive deficits in early development to a certain extent, but cannot alleviate cognitive deficits in adolescence and adulthood. In addition, injection of nucleic acid drugs cannot restore the impaired social skills or anxiety behaviors of 5p model rats.

[0234] Table 18. Analysis of liver and kidney function before and after injection of nucleic acid drugs

[0235] Incorporated by Reference

[0236] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0237] Equivalence

[0238] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A polynucleotide sequence encoding a CTNND2 truncation, wherein the protein sequence of the CTNND2 truncation is shown in SEQ ID NO:

2.

2. The polynucleotide sequence according to claim 1, wherein The polynucleotide sequence is as shown in SEQ ID NO: 1, or the polynucleotide sequence has at least 90% identity with SEQ ID NO:

1.

3. A nucleic acid construct, wherein: The construct contains the polynucleotide sequence according to claim 1 or 2.

4. The construct of claim 3, further comprising a transcriptional regulatory element comprising a promoter and / or an enhancer.

5. A vector, wherein The vector contains the polynucleotide sequence according to claim 1 or 2 or the construct according to claim 3 or 4.

6. The vector of claim 5, wherein the vector is an adeno-associated virus (AAV) vector.

7. The vector of claim 6, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof.

8. A virus particle, wherein It comprises the construct according to claim 3 or 4.

9. A composition comprising the polynucleotide sequence of claim 1 or 2, the nucleic acid construct of claim 3 or 4, the vector of any one of claims 5 to 7, or the virus particle of claim 8, and a pharmaceutically acceptable excipient.

10. Use of the polynucleotide sequence of claim 1 or 2, the nucleic acid construct of claim 3 or 4, the vector of any one of claims 5 to 7, the viral particle of claim 8, or the composition of claim 9 in the preparation of a medicament for treating cat cry syndrome in a desired subject, preferably, the subject is a human being, more preferably a child. The use according to claim 10 , wherein the drug is a gene therapy drug.

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