An adeno-associated virus vector overexpressing pinch-2 (AAV-PINCH-2), a preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology and provides an adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, its preparation method, and its applications. Lims2 The gene fragment was directionally inserted into the linearized vector pHBAAV-CMV-MCS-T2A-ZsGreen, resulting in the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, named rAAV(BLS)-CMV- Lims2 -ZsGreen; m- Lims2 The gene sequence is shown in SEQ ID NO.5. A mouse carrying the gene was successfully constructed. Lims2 (PINCH-2) gene overexpression adeno-associated virus vector (rAAV(BLS)-CMV-) Lims2 The study identified ZsGreen and determined its optimal overexpression time window after multi-point injection into the mouse bladder, laying the foundation for subsequent in vivo functional validation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an adeno-associated virus vector AAV-PINCH-2 that overexpresses PINCH-2, its preparation method, and its application. Background Technology

[0002] Adeno-associated virus (AAV), belonging to the family Parvoviridae and the genus Dependent Virus in biological classification, has a particle diameter of approximately 20–26 nm. This virus requires the assistance of a helper virus (often adenovirus or herpesvirus) to complete its full replication cycle. Due to its good safety profile and ability to mediate long-term stable gene expression, AAV has become a highly promising gene therapy vector.

[0003] The AAV packaging system comprises three plasmids: the pAAV-RC plasmid encoding the rep and cap proteins, the shuttle plasmid carrying foreign genes, and the pHelper plasmid, which can replace the auxiliary function of adenovirus. Simultaneous introduction of these three plasmids into AAV-293T tool cells for co-transfection may package recombinant AAV viral particles carrying the target gene.

[0004] AAV (Anaerobic Av) is a viral vector with key applications in gene therapy and other fields, and its function is based on its unique structure. AAV particles are non-enveloped, mainly composed of a protein capsid and an encapsulated single-stranded DNA genome; the synergistic work of these two components is crucial for its function. The AAV capsid exhibits an icosahedral symmetry, with a diameter of approximately 22 nanometers, and its structure is formed by the assembly of 60 identical capsid protein subunits. These subunits include three proteins: VP1, VP2, and VP3, which together determine the virus's infectivity and provide physical protection for its genome. Specifically, the VP1 protein is essential for viral internalization into cells; its unique N-terminal phospholipase A2 domain activity facilitates the escape of the viral genome from the endosomal tissue and into the cell nucleus, thereby initiating gene expression. The VP2 and VP3 proteins primarily play a role in maintaining the integrity and stability of the capsid structure.

[0005] The protein capsid of AAV is a core component for its function, providing physical protection for the internal viral genome and serving as a crucial interface mediating the interaction between the virus and host cells. Specific receptor-binding domains are distributed on the capsid surface, enabling it to recognize and bind to specific receptor molecules on the host cell membrane. This process directly determines the cell tropism and host range of AAV. Different serotypes of AAV exhibit distinct tropisms for specific tissues and cells due to significant differences in the amino acid sequences of their capsid proteins. For example, AAV2 serotype primarily utilizes heparan sulfate proteoglycans on the cell surface as its initial attachment receptor, while AAV5 tends to bind to N-linked sialic acid on the cell membrane. These serotype differences constitute the molecular basis for the application of AAV in targeted gene therapy.

[0006] The AAV genome is a single-stranded linear DNA molecule, approximately 4.7 kb in length. Its most prominent structural feature is the presence of two inverted terminal repeat (ITR) sequences, each approximately 145 nucleotides long, at each end of the genome. These ITR sequences fold to form a stable T-shaped hairpin secondary structure. This structure is not merely redundant; it acts as a crucial cis-regulatory element, playing an indispensable regulatory role in multiple stages of the viral life cycle—including genome replication, capsid packaging, and site-specific integration. Specifically, during replication, the hairpin structure of the ITR provides an initiation site for DNA polymerase; during viral assembly, the Rep protein guides the newly formed genome to accurately fit into the pre-formed capsid. The middle segment of its genome mainly contains two open reading frames, encoding the Rep and Cap proteins, respectively. The Rep protein, as a multifunctional regulator, participates in viral DNA replication, gene expression regulation, and assists in the site-specific integration of the viral genome into the host chromosome. The Cap protein is a structural unit constituting the viral outer protein capsid, determining the viral serotype and cell orientation.

[0007] With its unique biological characteristics, AAV (anti-inflammatory vasculature) has demonstrated significant value in several cutting-edge biomedical fields. In gene therapy, it is a powerful tool for delivering therapeutic genes. For example, in treating single-gene genetic diseases such as hemophilia, AAV vectors can deliver functionally normalized clotting factor genes (such as FVIII or FIX) into patients, achieving long-term stable expression of these factors and effectively correcting clotting dysfunction. In veterinary medicine, AAV vector vaccines can also be used to control animal diseases such as swine epidemic diarrhea, effectively activating the host's humoral and cellular immune responses. In neurological diseases, due to its low immunogenicity and certain central nervous system targeting capabilities, AAV can deliver neurotrophic factors or regulatory genes to specific brain regions, providing a new potential strategy for treating intractable diseases such as Parkinson's disease and Alzheimer's disease. In vaccine development, AAV vectors can mimic the natural infection process, effectively presenting antigens and stimulating long-lasting immune protection. Novel vaccines developed based on the AAV platform have shown the potential to induce high levels of neutralizing antibodies and specific T-cell immune responses in exploratory studies of novel coronavirus vaccines. In the veterinary field, AAV-vectored vaccines can also be used to control animal diseases such as porcine epidemic diarrhea, effectively activating the host's humoral and cellular immune responses. Furthermore, AAV plays a crucial role in basic research. Researchers can use it to specifically introduce pathogenic genes, reporter genes, or regulatory elements into experimental animals to construct or intervene in disease models, which is of great value in elucidating the mechanisms of disease development, validating drug targets, and evaluating treatment efficacy.

[0008] In basic medical and translational research, AAV vectors have become a key tool for constructing disease models. Researchers can use them to efficiently deliver specific target genes (such as pathogenic genes or reporter genes) into model organisms to simulate the pathological processes of human diseases. For example, in oncology research, the targeted delivery of specific oncogenes to specific tissues and organs in mice via AAV vectors can induce local cell malignant transformation and tumor formation, thus simulating the natural history of human tumors from their inception and development to invasion and metastasis. This AAV-based method for constructing disease models provides a powerful experimental platform for elucidating the molecular mechanisms of diseases, screening potential therapeutic targets, and evaluating the efficacy of new drugs or therapies.

[0009] Current research on AAV is quite extensive, covering topics such as AAV serotypes and tissue / organ affinity, as well as more efficient methods of infecting target organs. Regarding the methods of AAV infection of the bladder, different animal models employ different approaches. In rats, bladder instillation is commonly used, while in mice (using C57BL / 6 mice as an example), in situ bladder injection is primarily employed.

[0010] It is noteworthy that the expression of gene delivery vectors in living tissues exhibits significant spatiotemporal dynamics. Particularly in the aging, severely fibrotic bladder microenvironment, factors such as epithelial turnover, alterations in the local immune microenvironment, and epigenetic modifications can easily lead to premature silencing or attenuation of exogenous genes (especially expression sequences driven by exogenous promoters). If intervention time points conventionally set in literature are mechanically applied, subsequent phenotypic rescue experiments may result in false-negative results due to a decline in target protein expression.

[0011] PINCH-2 (also known as Lims2 PINCH-2 is a focal adhesion protein highly homologous to PINCH-1 (formerly known as PINCH). Structurally similar to PINCH-1, PINCH-2 contains five LIM domains, which mediate protein-protein interactions, enabling its involvement in various biological processes such as cytoskeleton construction, extracellular matrix adhesion, cell migration, proliferation, and survival. PINCH-1, along with integrin-linked kinase (ILK) and Parvin, forms a stable ternary complex, the IPP complex, essential for cytoskeleton organization and signal transduction. Although PINCH-1 and PINCH-2 exhibit high similarity under specific conditions, they differ significantly in their involvement in myelination in the mouse central nervous system, their role in carcinogenesis, cell type distribution, and organ specificity. Regarding the cell type specificity of PINCH-2, for example, in the colon, PINCH-2 expression is limited to the smooth muscle layer, while PINCH-1, in addition to the aforementioned, can also be detected in the epithelial layer. Furthermore, PINCH-2 exhibits an organ-specific expression pattern. In terms of tissue distribution, PINCH-2 expression is significantly enriched in the bladder, while its expression levels are relatively limited in tissues such as the kidneys, liver, heart, and diaphragm. This difference in expression may stem from the greater abundance of smooth muscle layer in the bladder compared to other organs. Previous studies have shown that PINCH-1 is involved in maintaining the integrity of the heart muscle. Although PINCH-2 is specifically expressed in the smooth muscle layer of the bladder, its specific physiological functions in smooth muscle (especially the bladder detrusor muscle) remain unclear.

[0012] Given that PINCH-2 is highly expressed in detrusor smooth muscle (DSM), recent studies suggest that the absence of PINCH-2 may impair the contractile ability of the detrusor muscle and lead to myogenic urinary retention. Summary of the Invention

[0013] This invention provides an adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, its preparation method, and its application.

[0014] This invention is achieved by the following technical solution: an adeno-associated virus vector AAV-PINCH-2, m- overexpressing PINCH-2. Lims2 The gene fragment was directionally inserted into the linearized vector pHBAAV-CMV-MCS-T2A-ZsGreen, resulting in the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, named rAAV(BLS)-CMV- Lims2 -ZsGreen;m- Lims2 The gene sequence is shown in SEQ ID NO.5.

[0015] The method for preparing the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 includes the following steps: double digestion of the empty vector plasmid with BamHI and KpnI restriction endonucleases according to the multiple cloning site of the vector pHBAAV-CMV-MCS-T2A-ZsGreen to obtain a linearized vector; and then inserting the target gene m- Lims2 PCR amplification primers were designed based on the sequence, and the m- sequence was cloned using the HB-infusion™ one-step cloning method. Lims2 The gene fragment was directionally inserted into a linearized vector; the recombinant plasmid was obtained and named pHBAAV-CMV-m- Lims2 -T2A-ZsGreen; This plasmid was co-transfected with three plasmids using AAV-293 cells in logarithmic growth phase and Lipofiter™ liposome transfection reagent, followed by viral packaging and purification to obtain recombinant adeno-associated virus, named rAAV2-CMV-m- Lims2 -ZsGreen; the viral titer is 2.2 × 10⁻⁶. 12 vg / mL.

[0016] The present invention also provides the use of the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 in the preparation of formulations for the treatment of myogenic urinary retention.

[0017] The use of the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 in the preparation of formulations for treating or alleviating PINCH-2 deficiency and impaired bladder detrusor muscle contractility.

[0018] The adeno-associated virus vector AAV-PINCH-2, which overexpresses PINCH-2, was injected orally into bladder tissue. The onset of action was 2-3 weeks after injection. The target gene... Lims2 The relative expression level of mRNA increased.

[0019] This invention successfully constructed a mouse-borne strain. Lims2(PINCH-2) gene overexpression adeno-associated virus vector (rAAV(BLS)-CMV-) Lims2 -ZsGreen), and determined its optimal overexpression time window after multi-point injection into the mouse bladder, laying the foundation for subsequent in vivo functional validation. Successfully, Lims2 The gene was inserted into the pHBAAV-CMV-MCS-T2A-ZsGreen vector, and rigorous bidirectional sequencing confirmed the absence of mutations and frameshifts, ensuring the accuracy of the exogenous gene. Subsequently, the virus was packaged using a three-plasmid co-transfection AAV-293 cell system, purified by column chromatography, and further concentrated by ultrafiltration. Finally, qPCR detection confirmed the presence of the target virus (rAAV2-CMV-). Lims2 The titers of -ZsGreen and the empty virus reached 2.2 × 10⁻⁶. 12 vg / mL and 1.7×10 12 vg / mL. This high viral titer not only demonstrates the efficiency of the packaging system but also fully meets the requirements for high-concentration viral vectors for subsequent local injection into live animal tissues.

[0020] qPCR validation experiments provided clear data to support this: 2-3 weeks after viral injection, the target gene... Lims2 The relative expression level of PINCH-2 mRNA increased sharply (more than 7 times that of the control group); however, 6 weeks after injection, its relative expression level decreased significantly (only about 2.4 times that of the control group). This indicates that in this experimental system, the high-level expression of the AAV-mediated PINCH-2 gene in bladder tissue exhibits a dynamic characteristic of "rapidly reaching a peak in the early stage and gradually declining in the later stage". Attached Figure Description

[0021] Figure 1 Flowchart for constructing adeno-associated virus overexpressing PINCH-2; Figure 2 For those containing the cmv promoter Lims2 Overexpression plasmids; Figure 3 The results of RNA electrophoresis are shown from left to right, with the grouping order consistent with that used in the concentration determination. Figure 4 The DNA electrophoresis results are shown from left to right, with the grouping order consistent with that used in the concentration determination. Figure 5 The graphs show the amplification and melting curves of PINCH-2. The left graph shows the amplification curve, and the right graph shows the melting curve. Figure 6 The results and raw data of qPCR detection of PICNH-2 expression level 2-3 weeks after viral injection; Figure 7 Statistical results and raw data table of PICNH-2 expression level qPCR detection 6 weeks after viral injection; Figure 8 Statistical table and analysis of gray value results of WB test on bladder smooth muscle tissue taken 2-3 weeks after viral injection; Figure 9Paraffin sections of fixed bladder smooth muscle tissue taken 2-3 weeks after viral injection and statistical results of IHC staining area of ​​PINCH-2. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, 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, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0024] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0026] I. Experimental Reagents: Trizon Reagen (CW0580S), Ultrapure RNA Extraction Kit (CW0581M), SuperStar Universal SYBR Master Mix (CW3360M), CWBIO; HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (R223-01), Vazyme; 50×TAE Buffer (T1060), Ampicillin (Amp), Solarbio; 6×DNA Loading Buffer (GH101-01), TRANS; 50bp DNA Ladder (MD108), TIANGEN; Gsafe Red plus nucleic acid dye (GK20002), GLPBIO; Agarose Powder (75510-019), Invitrogen; Vector, HB-infusion™, Hanbio Biotechnology; DH5α Competent Cell, TIANGEN (CB101-02); Phanta Max-Super-Fidlity DNA Polymerase, Vazyme (P505-D1); Plasmid DNA purification, MAHEREY-NAGEL; Gel DNA purification, Generay (GK2041); DNA ladder, Generay; Restriction endonuclease, BamHl, Kpnl; Restriction endonuclease, Thermo Scientific; DH5α competent cells, Tiangen Biotech (CB101-02); Phanta Max Super-Fidelity DNA polymerase, Vazyme (P505-D1); HB-infusion homologous recombinase, LipofiterT transfection reagent (HB-TRCF-1000), Hanheng Biotechnology; Plasmid DNA extraction kit, MAHEREY-NAGEL (740412); Gel extraction kit (GK2041), DL2000 / 5000 DNA Marker, Qingke Biotechnology; DMEM high glucose medium (11965118), 0.25% trypsin-EDTA (LP0042), phosphate-buffered saline (PBS) (10010001), Thermo; proteinase K, Fermentas (EO0491); SYBR Green qPCR Master Mix, Toyobo (QPK-20).

[0027] II. Experimental Methods 1. Plasmids, Cells, and Strains: The empty vector plasmid used to construct the overexpression vector was pHBAAV-CMV-MCS-T2A-ZsGreen (provided by Hanheng Biotechnology Co., Ltd.). Helper plasmids used for viral packaging included pAAV-RC (encoding Rep and Cap proteins) and pHelper (providing adenovirus helper functions). All plasmids were amplified using the Stbl3 strain (E. coli Stbl3). The tool cells used for viral packaging were AAV-293 cells (human embryonic kidney 293 cell line), preserved in the laboratory.

[0028] 2. Construction process of PINCH-2 overexpressing adeno-associated virus as follows: Figure 1 As shown, the specific method is as follows: (1) Vector linearization and target fragment preparation: Based on the multiple cloning site of the vector pHBAAV-CMV-MCS-T2A-ZsGreen, BamHI and KpnI restriction endonucleases were selected to double-digest the empty vector plasmid to obtain a linearized vector. The enzyme digestion reaction system (40 μL) was as follows: plasmid DNA 1 μg, 10× FastDigest Buffer 4 μL, BamHI 1.5 μL, KpnI 1.5 μL, and ddH2O added to 40 μL. The reaction system was incubated in a 37℃ water bath for 1-2 h. After incubation, the vector was separated by electrophoresis with a concentration of 1% agarose gel, and then the linearized vector band of approximately 4.7 kb was excised and recovered.

[0029] Target gene m- Lims2 The coding sequence (CDS) is directly chemically synthesized by the company. Its sequence accuracy has been verified by the company's quality control system, and it can be used directly for subsequent ligation reactions without the need for additional PCR amplification.

[0030] (2) Homologous recombination ligation: The synthesized m- Lims2 Gene fragments are directionally inserted into linearized vectors. A 20 μL homologous recombination reaction mixture is prepared (the entire process must be performed on ice): linearized vector fragment (~50-100 ng), m- Lims2 Gene fragment (~100-150 ng), 10 μL of 2×HB-infusion™ Master Mix, and ddH2O to a final volume of 20 μL. Gently pipette to mix, then transfer the reaction mixture to a PCR instrument and incubate at 50°C for 30 minutes. After the reaction is complete, immediately place it on ice to cool for 5 minutes.

[0031] (3) Transformation and positive clone screening: All ligation products were gently mixed with 50 μL of DH5α competent cells and incubated on ice for about 30 min. Then, the cells were heat-shocked in a 42°C water bath for 90 s and quickly transferred to ice for 2 min. 500 μL of LB liquid medium (without antibiotics) was added and the cells were incubated at 37°C and 230 rpm for 45 min with shaking to allow the bacteria to recover. The bacterial culture was evenly spread on LB solid plates containing 100 μg / mL ampicillin (Amp) and incubated upside down at 37°C for 12–16 h.

[0032] After single colonies appeared on the plates, 8–12 clones were randomly selected and inoculated into LB liquid medium containing Amp, and cultured at 37°C with shaking for 4–6 hours. A small amount of bacterial culture was taken and used as a template for PCR amplification using universal primers (or gene-specific primers). The specific primer sequences are shown in Table 1. After agarose gel electrophoresis analysis of the PCR products, positive clones showing a band at the expected size (approximately 720 bp) were selected.

[0033] Table 1: Primer Information (4) Sequencing verification and plasmid preparation: Clones that were verified as positive by bacterial culture PCR were sent to the sequencing company, and the universal primers on the vector were used to insert the m- Lims2 Genes were bidirectionally sequenced, m- Lims2 The gene is shown in SEQ ID NO.5 of the sequence listing. The sequencing results were compared with the designed gene sequence to confirm that the sequence was completely correct, without mutations, frameshifts, or premature stop codons. A positive clone verified by sequencing was selected, and its bacterial culture was transferred to a large volume of LB liquid medium supplemented with ampicillin (Amp) for amplification. Using an endotoxin-free plasmid extraction kit, following the instructions, a high-purity, endotoxin-free recombinant plasmid was extracted and named pHBAAV-CMV-m- Lims2 -T2A-ZsGreen. The concentration and purity of the plasmid were measured using a micro spectrophotometer to ensure that the A260 / A280 ratio was between 1.8 and 2.0. The plasmid was then stored at -20°C for subsequent experiments.

[0034] 3. Packaging and Harvesting of Recombinant Adeno-Associated Virus (1) Preparation of packaging cells: In a biosafety cabinet, AAV-293 cells (in the logarithmic growth phase) were taken, digested with 0.25% trypsin, and resuspended in culture medium to prepare a single-cell suspension. After counting, the cells were seeded at an appropriate density in 100 mm cell culture dishes and cultured in DMEM complete medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. When the cell confluence reaches 80%–90%, they can be used for subsequent transfection experiments.

[0035] (2) Co-transfection of three plasmids: Co-transfection of three plasmids was performed using Lipofiter™ liposome transfection reagent. Before transfection, Opti-MEM medium and Lipofiter™ transfection reagent were equilibrated to room temperature. For each 100 mm culture dish, the transfection complex was prepared as follows: 10 μg of shuttle plasmid (pHBAAV-CMV-m- Lims2 Gently mix 10 μg of pAAV-RC plasmid, 20 μg of pHelper plasmid, and 1.2 mL of Opti-MEM medium (Solution A). In another sterile tube, gently mix 120 μL of Lipofiter™ transfection reagent with 1.2 mL of Opti-MEM medium (Solution B) and incubate at room temperature for 5 min. Then, quickly mix Solutions A and B, gently invert to mix thoroughly, and incubate at room temperature for 15–20 min to form a transfection complex. Add this complex dropwise evenly to a cell culture dish that has been replaced with fresh serum-free DMEM medium, gently shaking to mix thoroughly. Approximately 6 h after transfection, replace with DMEM complete medium containing 10% FBS.

[0036] (3) Virus harvesting and preliminary processing: 72 h after transfection, a clear green fluorescence signal was observed under an inverted fluorescence microscope, indicating that the reporter gene ZsGreen had been effectively expressed. After slowly and gently scraping the cells with a cell scraper, the collected cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 150 g for 3 min, and the supernatant was discarded. Then, pre-cooled PBS was added to resuspend the cell pellet, and centrifuged and washed again. Finally, the cell pellet was fully resuspended with 300 μL PBS. The obtained cell suspension was repeatedly frozen and thawed three times in liquid nitrogen and a 37 °C water bath to fully lyse the cells and release the virus particles. The lysis buffer was centrifuged at 2000 g for 5 min at 4 °C, and the supernatant was collected, which was the crude extract containing recombinant adeno-associated virus (rAAV), and was immediately purified or temporarily stored at -80 °C.

[0037] (4) Purification and concentration of recombinant adeno-associated virus: Virus purification was achieved using column chromatography. First, Benzonase nuclease (final concentration approximately 50 U / mL) was added to the crude virus extract, and the mixture was incubated at 37°C for 1 hour to degrade residual cellular genomic DNA and packaging plasmid DNA. After the treatment was completed, the mixture was centrifuged at 4°C and 600g for 10 minutes, and the supernatant was collected. Following the instructions of the Biomiga adeno-associated virus purification kit (V1469-01), the supernatant was purified using column chromatography, completing the sample binding, washing, and elution steps sequentially. The eluted virus solution (approximately 4 mL) was collected. To further concentrate the virus, the eluent was added to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuged at 4°C and 1400g for 30 minutes until the virus solution was concentrated to approximately 1 mL. The high-purity rAAV virus stock solution obtained after purification and concentration was collected, aliquoted, and stored at -80°C for long-term storage. This virus was named rAAV2-CMV-m- Lims2 -ZsGreen.

[0038] 4. Aseptic Techniques and Mycoplasma Detection: Take 10 μL of concentrated virus stock solution and add it to HeLa cells cultured in a 96-well plate. Incubate in a cell culture incubator for 24-48 hours and observe under an inverted microscope. The quality control standard is: the culture medium should be clear and transparent, and no obvious bacterial or fungal contamination should be found in the intercellular spaces. Mycoplasma detection is performed using PCR: Take 10 μL of virus solution, heat at 95℃ for 15 min, and use it as a template for amplification. Perform PCR amplification using mycoplasma-specific primers, and then analyze the obtained products by agarose gel electrophoresis. If no specific bands appear in the electrophoresis results, the mycoplasma detection result is considered negative.

[0039] 5. Viral genome titer determination (qPCR): The viral genome copy number (vg) was determined using real-time quantitative PCR (qPCR). Viral samples were treated with DNase I and proteinase K to eliminate unpackaged cell-free DNA and release the viral genome. qPCR was performed using primers designed to target the conserved WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) sequence on the vector. The primer sequences were: upstream 5'-ACGCTATGTGGATACGCTGC-3', downstream 5'-CGGGCCACAACTCCTCATAA-3'. Plasmid standards with known copy numbers (10...) were used... 5 -10 10A standard curve was plotted using copies / μL. The treated viral samples were appropriately diluted and then subjected to qPCR amplification together with the standards. The reaction system (10 μL) was as follows: 4.8 μL of 2×SYBR Green mix, 0.4 μL each of forward and reverse primers, 1 μL of template DNA, and the remaining amount was brought to 10 μL with ddH2O. Reaction conditions: pre-denaturation at 95℃ for 3 min; then denaturation at 94℃ for 30 s, annealing at 62℃ for 30 s, and extension at 72℃ for 30 s, for a total of 40 cycles; finally, a melting curve analysis was performed. Based on the Ct value of the sample, the genome copy number was calculated according to the standard curve fitting equation, and then multiplied by the corresponding dilution factor to obtain the viral titer in vg / mL.

[0040] 6. Virus verification: tissue processing, RNA extraction, nucleic acid concentration determination, reverse transcription, and RNA electrophoresis.

[0041] (1) Experimental materials: bladders of 2-3 week old mice and bladders of 6-week old mice; empty vector virus group (VEC); overexpression (OE).

[0042] (2) Experimental method: Tissue processing: According to the experimental groups, prepare 1.5 mL RNase-free centrifuge tubes in advance and label them with sample group information; add two 5.2 mm and two 3.2 mm grinding beads that have been pre-treated for RNase removal and sterilization to each centrifuge tube, and add 1 mL of Trizon lysis buffer to each tube; use forceps to remove the tissue sample, cut about 50 mg and place it in the centrifuge tube; briefly cool the grinding equipment with liquid nitrogen beforehand; place the centrifuge tube in the grinder, set the grinding parameters to 65 Hz and 60 s, and if the grinding is insufficient, increase the number of grinding cycles until the tissue is completely and evenly ground; after grinding, store in a -20°C freezer to prevent degradation. The remaining samples are returned to a -80°C freezer for long-term storage.

[0043] RNA Extraction: Remove the processed sample (containing Trizol lysis buffer) centrifuge tube from the freezer and thaw it on ice. Add an appropriate amount of chloroform pre-cooled to 4°C to the centrifuge tube. For every 1 mL of Trizol used, add 0.2 mL of chloroform. Vortex vigorously for 15 seconds, then incubate on ice for 5 minutes, avoiding light. Centrifuge at 4°C and 12,000 rpm for 15 minutes. After centrifugation, the sample will separate into three layers: an upper red organic phase, a middle layer, and a lower colorless aqueous phase. RNA is mainly present in the colorless aqueous phase. Transfer this layer (approximately 400-500 μL) to a new pre-cooled 1.5 mL RNase-Free centrifuge tube. Add an equal volume of 4°C anhydrous ethanol to the resulting aqueous solution and mix thoroughly by inverting. Transfer the mixture to the RM adsorption column pre-installed in the collection tube, centrifuge at 4°C and 12,000 rpm for 30 seconds, and discard the filtrate. Wash the precipitate with 700 μl of pre-chilled RW1 buffer at 4°C. Centrifuge at 12000 rpm for 30 s at 4°C, then discard the eluent. Wash the precipitate with 500 μl of pre-chilled RW2 buffer at 4°C. Centrifuge at 12000 rpm for 30 s at 4°C, then discard the eluent. Repeat the washing and centrifugation steps, discarding the eluent and centrifuging for 2 min empty. Transfer the adsorption column to a new pre-chilled 1.5 mL RNase-Free centrifuge tube, place it on ice in a fume hood to air dry for 5-6 min to remove residual ethanol. Add 38 μL of pre-chilled RNase-Free H2O at 4°C, place it on ice for 3-5 min to allow the RNA to dissolve completely, and centrifuge at 12000 rpm for 1 min at 4°C. Then, aspirate the eluent from the centrifugation, drop it onto the adsorption column membrane, place it on ice again for 3-5 min, and centrifuge at 12000 rpm for 1 min at 4°C. The resulting RNA solution was stored at -80°C to prevent degradation.

[0044] RNA concentration and purity determination: Accurately pipette 1.3 μL from a total RNA solution of 38 μL; measure and record data using a UV spectrophotometer at OD260 and OD280; calculate according to the formula: The results are shown in Table 2. Note: Since the RNA concentrations extracted from each group were not exactly the same, the amount of RNA used in each group needed to be adjusted to be consistent before reverse transcription. The required RNA volume for each group was then calculated. Referring to the operating instructions of the HiScript II QRT SuperMix for qPCR reverse transcription kit, the total volume of RNA and water was 12 μL. The obtained RNA was subjected to agarose gel electrophoresis. The chemiluminescence imaging system was set to nucleic acid development mode, and the FILTER 1 filter was selected. After development, the images were saved, and the development information was clearly marked. The integrity of the RNA and whether degradation had occurred were also recorded. High-quality RNA samples showed no significant diffusion in the electrophoretic bands, and the 5S, 18S, and 28S rRNA bands were clearly distinguishable. The brightness ratio of the 28S to 18S bands was approximately 2:1, while the 5S band was the weakest or invisible.

[0045] Table 2: Results of RNA concentration and purity determination RNA reverse transcription: Genomic DNA removal was performed, and the mixture shown in Table 3 was prepared in an RNase-free PCR tube. The mixture was gently mixed and incubated at 42°C for 2 minutes. 4 μL of 5×HiScript II qRT SuperMixII was added sequentially to the PCR tube, bringing the total volume of the mixture to 20 μL. The PCR tube was then placed in a standard PCR amplification instrument and incubated at 50°C for approximately 15 minutes, followed by a temperature increase to 85°C for 5 seconds (if the template contains complex secondary structures or high GC content regions, the reaction temperature can be increased to 55°C to improve amplification efficiency). The resulting product can be used directly for qPCR reactions or stored at -20°C. Repeated freeze-thaw cycles of cDNA should be avoided during use. Primers used for quantitative real-time PCR are shown in Table 1, the operating system is shown in Table 4, and the specific reaction procedure is shown in Table 5. The qPCR products were subjected to agarose gel electrophoresis using a FILTER 1 filter. After development, confirm the approximate length of the target band, save the image and clearly label the development information, and compare it with the expected product length in the primer file to determine whether the amplified product is the target fragment. During normal specific amplification, the target band has moderate brightness, is correctly positioned, and presents as a single, clear band.

[0046] Table 3: Reverse Transcription Reaction Mixture Table 4: Operating System Table 5: Reaction Procedure 7. Statistical Analysis: SPSS 22.0 statistical software was used for data processing. Quantitative data are expressed as mean ± standard deviation (Mean ± SD). Relevant statistical charts were generated using GraphPad Prism 9 software. Independent samples t-tests were used to compare two groups. A p-value < 0.05 was considered statistically significant.

[0047] III. Experimental Results: 1. Construction and sequencing validation of recombinant PINCH-2 overexpressing adeno-associated virus vector: A mouse vector carrying PINCH-2 was successfully constructed. Lims2 (i.e., the PINCH-2 encoding gene) recombinant adeno-associated virus vector (rAAV2-CMV-m- Lims2 -ZsGreen). Using double enzyme digestion technology, the chemically synthesized target gene fragment is precisely and directionally cloned into the target plasmid containing the CMV promoter.

[0048] To ensure sequence accuracy, positive recombinant plasmids were screened and Sanger sequencing was performed for verification. Figure 2 Bidirectional sequencing alignment results confirmed that the inserted sequence matched the standard m- sequence in the NCBI database. Lims2 The complete coding sequence (CDS) of the gene was 100% matched, with no base mutations, deletions, or frameshifts. This result confirms that the molecular construction of the target overexpression vector was correct.

[0049] Viral plasmid sequencing showed that the sequencing alignment results were completely consistent with the target sequence, with no differences, indicating that the target plasmid was successfully constructed.

[0050] 2. Packaging, purification, and physical titer determination of adeno-associated virus (AAV): The identified recombinant plasmid and packaging helper plasmid were co-transfected into AAV-293 cells to complete viral particle assembly. The final viral stock solution was then obtained through multi-stage purification and ultrafiltration concentration. Quantitative detection showed that the target overexpressed virus (rAAV2-CMV-m-) was... Lims2 The physical titer of -ZsGreen is as high as 2.2 × 10⁻⁶. 12 The titer of the empty vector control virus (rAAV2-CMV-ZsGreen) was also measured at 1.7 × 10⁻⁶ vg / mL. 12 vg / mL.

[0051] 3. Nucleic Acid Sample Quality Evaluation and Specific Amplification Identification: To ensure the accuracy and scientific validity of subsequent real-time quantitative PCR (RT-qPCR) data, strict quality control was performed on the extracted tissue nucleic acids. RNA agarose gel electrophoresis results are shown below. Figure 3As shown in the results, the total RNA bands extracted from the bladder tissue were clear, the brightness ratio of 28S and 18S rRNA was normal, and no obvious genomic DNA contamination or degradation diffusion was found, proving that the RNA integrity was excellent.

[0052] 4. DNA Electrophoresis: After obtaining cDNA, the target gene was amplified using specific primers. DNA agarose gel electrophoresis results showed that the amplification products containing both the internal control β-actin and the target gene PINCH-2 exhibited extremely clear, single target-sized bands. Figure 4 ).

[0053] 5. qPCR result image: The melting curve of the qPCR reaction shows a sharp and single signal peak. Figure 5 (Right) This completely eliminated interference from primer dimers or non-specific amplification, confirming the high specificity and reliability of the experimental reaction system. Figure 5 The results completely eliminated interference from primer dimers or non-specific amplification, confirming the high specificity and reliability of the experimental reaction system.

[0054] 6. qPCR Validation of Overexpression Levels: Total RNA was extracted from mouse bladder tissue using Trizol reagent. mRNA was extracted using an RNA ultrapure extraction kit. The concentration and purity of mRNA (OD260 / OD280) were measured using a UV-Vis spectrophotometer. cDNA was synthesized using an RNA reverse transcription kit and detected using a real-time PCR instrument. The reaction program was: 95℃ pre-denaturation for 10 min; followed by 40 cycles, including 95℃ denaturation for 10 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s. β-actin or U6 was used as an internal control. The relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0055] like Figure 6 , Figure 7 As shown, the coding gene for PINCH-2 was detected by qPCR at 2-3 weeks and 6 weeks after viral injection. Lims2 Expression level. This can confirm viral load 2-3 weeks after injection. Lims2 Gene expression levels were higher than at 6 weeks, and the timeframe for subsequent bladder function testing, related pathological section staining, and molecular experiments was determined to be 2-3 weeks after viral injection.

[0056] like Figure 8 , Figure 9 As shown, bladder smooth muscle tissue was collected 2-3 weeks after viral injection, and the paraffin section images are as follows. Figure 9 As shown, the statistical results of IHC staining area of ​​PINCH-2 are as follows: Figure 9 As shown, the grayscale values ​​of the WB experiment are as follows: Figure 8As shown, the results indicated that the expression levels of the corresponding proteins increased 2-3 weeks after viral injection, and the expression in bladder smooth muscle tissue was dense and continuous with strong positive results. The expression intensity was significantly higher than that in the VEC group, and the difference was statistically significant.

[0057] This invention successfully constructed a mouse-borne strain. Lims2 (PINCH-2) gene overexpression adeno-associated virus vector (rAAV(BLS)-CMV-) Lims2 The study identified the optimal overexpression time window of ZsGreen after multi-point injection into the mouse bladder, laying the foundation for subsequent in vivo functional validation.

[0058] AAV, with its high safety and persistent expression, has become a highly promising tool in gene therapy and disease model construction. This invention successfully... Lims2 The gene was inserted into the pHBAAV-CMV-MCS-T2A-ZsGreen vector, and rigorous bidirectional sequencing confirmed the absence of mutations and frameshifts, ensuring the accuracy of the exogenous gene. Subsequently, the virus was packaged using a three-plasmid co-transfection AAV-293 cell system, purified by column chromatography, and further concentrated by ultrafiltration. Finally, qPCR detection confirmed the presence of the target virus (rAAV2-CMV-). Lims2 The titers of -ZsGreen and the empty virus reached 2.2 × 10⁻⁶. 12 vg / mL and 1.7×10 12 vg / mL. This high viral titer not only demonstrates the efficiency of the packaging system but also fully meets the requirements for high-concentration viral vectors for subsequent local injection into live animal tissues.

[0059] When exploring the effects of adeno-associated virus (AAV)-mediated gene intervention, accurately determining the onset time window is a crucial prerequisite for evaluating experimental results. Previous literature and routine animal experimental experience generally indicate that the onset and stable expression of AAV in living tissues typically occur around 6 weeks after viral injection. However, in our preliminary experiments, we observed that in bladder tissue samples taken 6 weeks after in situ injection, the target molecule did not exhibit the expected high abundance of expression, and the overexpression efficiency was unsatisfactory. This phenomenon, contrary to conventional understanding, suggests that the expression dynamics of AAV in the specific organ microenvironment of the bladder may have unique characteristics. Therefore, to avoid false negative results in subsequent functional evaluations due to bias in the observation window, this invention specifically designed a timing exploration experiment for target gene expression, aiming to precisely pinpoint the optimal expression time of the virus in local bladder tissue, thereby laying a reliable foundation for subsequent in vivo phenotypic verification.

[0060] qPCR validation experiments provided clear data support: 2-3 weeks after viral injection, the target gene... Lims2 The relative expression level of PINCH-2 mRNA increased sharply (more than 7 times that of the control group); however, 6 weeks after injection, its relative expression level decreased significantly (only about 2.4 times that of the control group). This indicates that in this experimental system, the high-level expression of the AAV-mediated PINCH-2 gene in bladder tissue exhibits a dynamic characteristic of "rapidly reaching a peak in the early stage and gradually declining in the later stage".

[0061] Although AAV can theoretically mediate persistent expression of exogenous genes, its expression attenuation occurs in the mouse bladder, a specific organ, which may be related to the following physiological and molecular mechanisms: Bladder epithelial cell renewal and shedding: The bladder mucosal epithelium is in a state of dynamic renewal. In situ multi-point injection inevitably causes minor mechanical trauma to the local tissue while delivering the virus. This minimally invasive repair response may accelerate the proliferation and shedding of superficial epithelial cells. Since AAV exists primarily as an episome within the host cell nucleus, its viral genome is lost along with the shed infection.

[0062] Furthermore, CMV promoters may cause transcriptional silencing: the vector used in this experiment employs a cytomegalovirus (CMV) promoter to drive the expression of the target gene. Numerous studies have confirmed that although CMV promoters possess strong early-stage driving capabilities in in vitro cells or some in vivo tissues, they are susceptible to interference from epigenetic modifications such as DNA methylation in specific living tissues over time, leading to transcriptional silencing.

[0063] Immune clearance within the microenvironment: Although the AAV vector removes the original pathogenic genes of the virus and has low immunogenicity, the local accumulation of high-titer viral particles may still act as a foreign antigen, gradually inducing a low-intensity immune response in the local microenvironment over several weeks, leading to the recognition and clearance of some target cells that efficiently express foreign proteins by the immune system.

[0064] This invention successfully obtained a high-titer PINCH-2 overexpressing AAV virus that meets the requirements for in vivo animal experiments. More importantly, it corrects the experimental observation window bias that might have been caused by blindly referring to previous literature (6 weeks). Through rigorous timing verification, the observation was precisely anchored 2-3 weeks after virus injection. This ensures that subsequent experiments are conducted within the window period when the PINCH-2 target protein is in absolute dominant expression, thus providing a solid and reliable prerequisite for accurately assessing whether PINCH-2 overexpression can improve bladder function.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, characterized in that: m- Lims2 The gene fragment was directionally inserted into the linearized vector pHBAAV-CMV-MCS-T2A-ZsGreen, resulting in the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2, named rAAV(BLS)-CMV- Lims2 -ZsGreen;m- Lims2 The gene sequence is shown in SEQ ID NO.

5.

2. The method for preparing the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 as described in claim 1, characterized in that: Includes the following steps: Based on the multiple cloning site of the vector pHBAAV-CMV-MCS-T2A-ZsGreen, the empty vector plasmid was double-digested with BamHI and KpnI restriction endonucleases to obtain a linearized vector, into which the target gene m- Lims2 PCR amplification primers were designed based on the sequence, and the m- sequence was cloned using the HB-infusion™ one-step cloning method. Lims2 The gene fragment was directionally inserted into a linearized vector; the recombinant plasmid was obtained and named pHBAAV-CMV-m- Lims2 -T2A-ZsGreen; This plasmid was co-transfected with three plasmids using AAV-293 cells in logarithmic growth phase and Lipofiter™ liposome transfection reagent, followed by viral packaging and purification to obtain recombinant adeno-associated virus, named rAAV2-CMV-m- Lims2 -ZsGreen; the viral titer is 2.2 × 10⁻⁶. 12 vg / mL.

3. The use of the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 as described in claim 1 in the preparation of a formulation for treating myogenic urinary retention.

4. The application according to claim 3, characterized in that: The use of the adeno-associated virus vector AAV-PINCH-2 overexpressing PINCH-2 in the preparation of formulations for treating or alleviating PINCH-2 deficiency and impaired bladder detrusor muscle contractility.

5. The application according to claim 3 or 4, characterized in that: The adeno-associated virus vector AAV-PINCH-2, which overexpresses PINCH-2, was injected orally into bladder tissue. The onset of action was 2-3 weeks after injection. The target gene... Lims2 The relative expression level of mRNA increased.

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