Method for screening ddx4 protein as a target for gene therapy potentiator and application thereof

Abstract

This invention belongs to the field of gene therapy and drug screening technology, specifically relating to a method and application for screening gene therapy potentiators targeting DDX4 protein. The method includes contacting candidate molecules with a system expressing DDX4 protein; detecting the effect of candidate molecules on the expression level or activity of DDX4 protein; verifying the enhancing effect of candidate molecules that can downregulate DDX4 protein expression or inhibit its activity on gene therapy efficacy; and then selecting candidate molecules that significantly downregulate DDX4 protein expression or inhibit its activity as potentiators. This invention reveals for the first time the association between DDX4 protein and gene therapy efficacy, and the potentiators obtained through screening cover small molecule compounds, nucleic acid molecules, proteins, etc. This invention has a clear target and high screening efficiency, overcoming the shortcomings of unclear targets and low efficiency in gene therapy potentiator screening, providing new targets and efficient drug use strategies for gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy and drug screening technology, specifically relating to a method and application for screening gene therapy enhancers using DDX4 protein as a target. Background Technology

[0002] Gene therapy refers to the technology of introducing exogenous normal genes or therapeutic nucleic acids into target cells to correct or compensate for diseases caused by gene defects or abnormalities, thereby achieving therapeutic goals. In recent years, with the rapid development of viral vector technology and gene editing technology (such as CRISPR-Cas9), gene therapy has shown broad application prospects in the treatment of hereditary diseases, malignant tumors, and infectious diseases. However, gene therapy still faces many challenges in clinical application, including low gene delivery efficiency, insufficient targeting, unstable gene expression levels, and the body's immune response. These factors seriously restrict the efficacy and widespread application of gene therapy. Therefore, improving the efficiency and safety of gene therapy has become a key scientific problem that urgently needs to be solved in this field.

[0003] To improve the efficacy of gene therapy, current technologies mainly explore approaches such as vector optimization, improved administration routes, and combination therapies. Combination therapy strategies, in particular, combine gene therapy with chemotherapy, radiotherapy, or immunotherapy to achieve synergistic effects. However, the synergistic molecules used in these strategies are drugs or treatments with known functions and are not specifically designed to enhance gene therapy efficacy, making optimal synergistic matching difficult. Current research on gene therapy synergists is insufficient, lacking highly specific synergistic molecules with clearly defined mechanisms of action, and even more so, systematic methods for screening such synergists. In existing technologies, the screening of gene therapy synergists largely relies on empirical trial and error or indirect intervention in known signaling pathways. These screening strategies are inefficient, have unclear targets, and struggle to yield highly effective synergists with clinical translational potential.

[0004] DDX4 (DEAD-box helicase 4), also known as VASA, belongs to the DEAD-box RNA helicase family and is known to play a crucial role in germ cell development, participating in biological processes such as RNA metabolism, translation regulation, and maintenance of germline stem cells. Traditional research has mainly focused on the function of DDX4 in reproductive system development and germ cell tumors. To date, there have been no reports of a correlation between DDX4 protein and gene therapy efficacy, nor have there been any technical insights into using DDX4 as a target for screening or developing gene therapy synergists.

[0005] In summary, there is an urgent need in this field to develop a gene therapy enhancer screening strategy based on clearly defined molecular targets to overcome the shortcomings of existing technologies, such as low screening efficiency, unclear targets, and poor reliability of results, and to establish optimized combination therapy regimens to provide efficient and safe combination therapy strategies for the clinical translation of gene therapy. Summary of the Invention

[0006] To address the technical problems of insufficient screening targets and low screening efficiency in existing gene therapy enhancers, this invention provides the application of DDX4 protein as a target in screening gene therapy enhancers. This invention is the first to discover a negative correlation between the expression level and activity of DDX4 protein and the expression efficiency of gene therapy vectors in target cells; inhibiting DDX4 can significantly enhance gene therapy efficacy, providing a novel molecular target for the development of gene therapy enhancers.

[0007] To achieve the above and other objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention also provides a method for screening gene therapy potentiators targeting DDX4 protein, the method comprising the following steps:

[0009] (1) Contact the candidate molecule with the system expressing DDX4 protein;

[0010] (2) Detect the expression level or activity of DDX4 protein after contact with candidate molecules;

[0011] (3) Verify the enhancing effect of candidate molecules that can downregulate DDX4 protein expression levels or inhibit DDX4 protein activity on gene therapy efficacy;

[0012] (4) Select candidate molecules that can significantly downregulate the expression level of DDX4 protein or significantly inhibit the activity of DDX4 protein and significantly enhance the gene therapy effect as gene therapy enhancers.

[0013] Furthermore, the system for expressing DDX4 protein includes cell, tissue, or cell-free in vitro expression systems that express DDX4 protein.

[0014] Furthermore, the detection of DDX4 protein expression level is performed by detecting the mRNA level or protein translation level of DDX4; the detection of DDX4 protein activity is performed by detecting its ATPase activity, RNA helicase activity, or substrate specificity.

[0015] Secondly, the present invention provides the application of DDX4 protein as a target in screening gene therapy enhancers. The application uses DDX4 protein or its encoding gene as an intervention target to screen enhancers that can enhance the effect of gene therapy.

[0016] Thirdly, the present invention provides a gene therapy enhancer obtained by screening using the above method. The enhancer is a regulator that targets the DDX4 protein or its encoding gene and can downregulate the expression level of the DDX4 protein or inhibit the activity of the DDX4 protein.

[0017] Furthermore, the synergist includes small molecule compounds, nucleic acid molecules, proteins, peptides, or combinations thereof.

[0018] Furthermore, the nucleic acid molecules include siRNA, shRNA, antisense oligonucleotides targeting the DDX4 encoding gene, as well as sgRNA and the CRISPR-Cas9 gene editing system targeting the DDX4 encoding gene; the proteins and peptides include antibodies or antigen-binding fragments thereof that specifically bind to the DDX4 protein and inhibit its activity.

[0019] Furthermore, the synergist is an inhibitor obtained by molecular docking, virtual screening, and molecular dynamics simulation using the three-dimensional structure of the DDX4 protein, or a DDX4 protein inhibitor molecule designed or optimized using artificial intelligence technologies such as machine learning and deep learning.

[0020] Fourthly, the present invention also provides the use of the gene therapy enhancer described in any of the above claims in the preparation of a medicament for enhancing the effect of gene therapy, wherein the gene therapy enhancer is used in combination with a gene therapy medicament.

[0021] Furthermore, the gene therapy includes viral vector-mediated gene therapy, non-viral vector-mediated gene therapy, or gene editing therapy.

[0022] Furthermore, the viral vector is selected from adeno-associated virus vectors, lentiviral vectors, adenovirus vectors, or retroviral vectors.

[0023] Furthermore, the gene therapy is used to treat hereditary diseases, malignant tumors, or infectious diseases.

[0024] Fifthly, the present invention also provides a pharmaceutical composition comprising the gene therapy enhancer and gene therapy drug described in any one of the preceding claims.

[0025] Furthermore, the pharmaceutical composition further includes at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

[0026] Furthermore, the gene therapy enhancer and gene therapy drug are configured to be administered simultaneously, separately, or sequentially.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) Providing a novel target. This invention reveals for the first time the function of DDX4 protein as a negative regulator of gene therapy efficiency in non-germ cells, breaking the technical bias of existing technologies that only recognize DDX4 protein in the field of reproductive biology and germ cell tumor diagnosis. Using DDX4 protein as a screening target for gene therapy enhancers provides a novel molecular target for the development of gene therapy enhancers and opens up new avenues for solving the bottleneck of gene therapy efficiency.

[0029] (2) The screening method is efficient, highly targeted, and reliable. The screening method provided by this invention uses the expression level or activity of DDX4 protein as a direct detection indicator. Compared with traditional empirical trial-and-error screening or non-specific intervention in pathways, it has the advantages of clear target and high screening efficiency. By combining primary screening and secondary screening, this method can effectively avoid the interference of systemic false positives, compound toxicity, or solubility issues on the screening results, and significantly improve the reliability and success rate of the screening results.

[0030] (3) A wide variety of synergists. This invention covers a broad range of gene therapy synergists, including small molecule compounds, various nucleic acid molecules (siRNA, shRNA, antisense oligonucleotides, sgRNA, and the CRISPR-Cas9 system), antibodies and their antigen-binding fragments, as well as inhibitors obtained through computer-aided design and artificial intelligence technology, providing diverse options for different application scenarios. It also covers bifunctional molecules designed based on PROTAC technology, which can achieve specific downregulation of DDX4 at the protein level by mediating ubiquitination and proteasome-dependent degradation of DDX4 protein.

[0031] (4) Wide range of applications. The synergist of this invention can be used in combination with various gene therapy methods (viral vector-mediated, non-viral vector-mediated, gene editing therapy), and is applicable to the treatment of various diseases such as hereditary diseases, malignant tumors, and infectious diseases, with broad clinical application prospects. The synergist's effect generally increases the gene expression level of the entire cell population, verifying its robustness as a gene therapy synergist.

[0032] (5) Optimization of combination drug use strategy. The pharmaceutical composition provided by the present invention combines the synergist with the gene therapy drug and clarifies the administration methods of simultaneous, separate or sequential administration, providing an optimized strategy for clinical combination drug use, which is beneficial to improving the efficacy and safety of gene therapy. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0034] Figure 1The results show the effects of candidate small molecule compounds on DDX4 protein expression levels and cell viability. In the figure, A represents the results of Western blot detection of DDX4 protein expression levels, and B represents the results of CCK-8 assay detection of cell viability.

[0035] Figure 2 The figure shows the evaluation results of the effect of candidate molecule A1 on AAV-GFP expression efficiency, where A is a scatter plot of flow cytometry, B is the flow cytometry statistical results, and C is the histogram of GFP fluorescence intensity superimposed in each treatment group.

[0036] Figure 3 The graph shows the effect of siRNA on DDX4 expression and ATPase activity. In the graph, A represents the relative expression level of DDX4 mRNA (qPCR), B represents the expression level of DDX4 protein (Western blot), and C represents the results of DDX4 ATPase activity detection.

[0037] Figure 4 Figure 1 shows the CCK-8 cell viability assay results of the killing effect of siDDX4 on the LV-TK / GCV system.

[0038] Figure 5 The graph shows the results of ATPase activity detection of DDX4 in the precipitate;

[0039] Figure 6 The image shows the evaluation of the combined application of anti-DDX4 monoclonal antibody and CRISPR-Cas9 gene editing. In the image, A is the proportion of PD-L1 positive cells detected by flow cytometry, B is the efficiency of targeted PD-L1 gene editing detected by T7E1 enzyme digestion, and C is the result of Sanger sequencing and TIDE / ICE analysis.

[0040] Figure 7 The figure shows the results of verifying the synergistic effect of anti-DDX4 monoclonal antibody on CRISPR-Cas9 gene editing efficiency. In the figure, A is the PD-L1 protein level detected by Western blot, and B is the fluorescence intensity superimposed histogram of PD-L1 direct-label flow cytometry antibody staining.

[0041] Figure 8 This is a schematic diagram of the docking pattern between compound D4-IN-1 and the DDX4 protein molecule;

[0042] Figure 9 The graph shows the inhibitory effect of the candidate compounds on DDX4 ATPase activity.

[0043] Figure 10 This demonstrates the enhancing effect of compound D4-IN-1 on the transduction efficiency of AAV-Luc. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0046] Unless otherwise specified, all materials used in the following implementation are new materials purchased from the market.

[0047] Example 1: Screening of small molecule synergists based on DDX4 protein expression levels

[0048] This embodiment illustrates a method for screening small molecule compounds that can downregulate DDX4 expression as gene therapy enhancers, using DDX4 protein expression level as a detection index:

[0049] S1. Construction of DDX4 protein expression system: Using CRISPR / Cas9-mediated homology-directed repair technology, the coding sequence of enhanced green fluorescent protein (EGFP) was precisely knocked into the C-terminus of the endogenous DDX4 gene in the human cervical cancer cell line HeLa, before the stop codon, to obtain the HeLa DDX4-EGFP knock-in cell line that stably expresses the DDX4-EGFP fusion protein.

[0050] S2. Initial screening of candidate compounds: HeLa DDX4-EGFP cells were screened at a density of 1×10⁶ cells per well. 3 Cells were seeded at a density of 1,000 cells per well in 384-well plates with a black transparent bottom and incubated overnight at 37°C in a 5% CO2 incubator. The next day, using an automated liquid handling workstation, different candidate small molecule compounds (numbered C-037, C-158, C-1024, C-005, C-017, C-056, and C-089) were added to each well to a final concentration of 10 μmol / L. Each compound was used in triplicate. A solvent control group (0.1% DMSO) was also included. The cells were incubated for another 12 hours.

[0051] S3. High-throughput detection of DDX4 protein expression level: After culture, the fluorescence intensity of each well was detected using a multi-functional microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 507 nm. The percentage of fluorescence intensity of each treatment group relative to the solvent control group was calculated, and compounds that reduced fluorescence intensity by more than 50% were screened as positive candidates for the initial screening.

[0052] S4. DDX4 Protein Expression Validation and Cell Viability Detection: Positive candidates from the initial screening were rescreened in wild-type HeLa cells. Wild-type HeLa cells were seeded in 24-well plates and treated with candidate small molecule compounds at a final concentration of 10 μmol / L for 48 hours. Cells were collected, lysed, and total protein was extracted. After separation by SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. Western blot analysis was performed using an anti-DDX4 antibody (1:1000 dilution). The relative expression level of DDX4 protein was calculated using GAPDH as an internal control. The results are as follows: Figure 1 As shown in Figure A, compound C-1024, which exhibited the most significant downregulation effect, was selected as a representative candidate molecule for subsequent synergistic effect verification experiments (A1). Simultaneously, cell viability in each treatment group was assessed using the CCK-8 assay. Figure 1 As shown in B, cell viability is greater than 85%, indicating that the downregulation of the screened molecules is not due to false positives caused by cytotoxicity.

[0053] S5. Validation of the synergistic effect of gene therapy: The candidate molecule (A1) that significantly downregulated DDX4 expression was combined with adeno-associated virus (AAV-GFP) carrying the green fluorescent protein (GFP) reporter gene for synergistic effect validation. HEK293T cells were divided into four groups: blank control group, AAV-GFP single treatment group, A1 single treatment group, and AAV-GFP+A1 combined treatment group. After 48 hours of treatment, cells were collected, and the percentage of GFP-positive cells and the average fluorescence intensity were detected by flow cytometry to evaluate the enhancing effect of A1 on AAV-mediated gene transduction efficiency. The results are as follows: Figure 2 As shown, the proportion of GFP-positive cells in the combined treatment group was significantly higher than that in the single treatment group, and the fluorescence intensity peak of the AAV-GFP+A1 combined treatment group shifted to the right as a whole compared with the AAV-GFP single treatment group. This indicates that the synergistic effect of A1 does not only act on a few cells, but also generally increases the gene expression level of the entire cell population, thus verifying the feasibility of the method of screening gene therapy synergists with DDX4 as the target.

[0054] The candidate small molecule compounds include FDA-approved drugs, most of which have entered clinical trials and have been proven to have good safety and stability, natural products, and structurally diverse artificial synthetic compounds, totaling 200,000 compounds.

[0055] Example 2: Screening of nucleic acid molecular potentiators based on DDX4 protein activity

[0056] This embodiment illustrates a method for screening siRNAs targeting the DDX4 encoding gene as gene therapy enhancers, using DDX4 protein ATPase activity as a detection indicator:

[0057] X1. Cell transfection and grouping: Human glioblastoma cell line U87-MG was transfected into cells at 2 × 10⁶ cells / well. 5 Cells were seeded at a density of 10000000 in 6-well plates and cultured until confluence reached approximately 60-70%. Then, liposome transfection reagent was used to transfect the negative control siRNA (siNC, 50 nmol / L) and three siRNAs targeting the human DDX4 gene (siDDX4-1, siDDX4-2, siDDX4-3, 50 nmol / L) into the cells. The cells were cultured for another 48 hours after transfection.

[0058] X2. DDX4 mRNA protein expression level and activity detection: After culture, cells from each group were collected, and total RNA was extracted from each group of cells. cDNA was synthesized by reverse transcription. Using GAPDH as an internal reference gene, the relative expression level of DDX4 mRNA was detected by real-time quantitative PCR. The reference sequences used were: DDX4-F: 5'-CAAGAGGAGTTGGTGAAGGTG-3', DDX4-R: 5'-CTGGTGATGAAGGTGGTGATG-3', GAPDH-F: 5'-GAGTCAACGGATTTGGTCGT-3', GAPDH-R: 5'-GACAAGCTTCCCGTTCTCAG-3'. Simultaneously, total protein was extracted and DDX4 protein levels were detected by Western blot. The ATP hydrolysis activity of DDX4 protein was measured using an ATPase activity assay kit. The results are as follows: Figure 3 As shown, compared with the siNC control group, all three siDDX4 lines could downregulate the expression levels of DDX4 mRNA and protein to varying degrees. Among them, siDDX4-2 had the best effect, and after transfection with siDDX4-2, the ATPase activity of DDX4 decreased to 28%±5% of the control group, which was consistent with the downregulation trend of protein expression. Therefore, the most effective siRNA (siDDX4-2) was selected for subsequent experiments.

[0059] X3. Enhancement Effect Verification: The selected effective siRNAs were combined with a lentiviral vector carrying the therapeutic gene (LV-TK, expressing herpes simplex virus thymidine kinase) to observe its enhancing effect on tumor cell killing. U87-MG cells were divided into four groups: blank control group, LV-TK alone treatment group, siDDX4-2 alone treatment group, and LV-TK and siDDX4-2 combined treatment group. After 48 hours of treatment, ganciclovir (GCV, 5 μmol / L) was added to induce cell killing. After 72 hours of further culture, cell viability was detected using the CCK-8 assay. The results are as follows: Figure 4 As shown.

[0060] analyze Figure 4 The results showed that the cell viability of the LV-TK+siDDX4 combined treatment group was the most significantly reduced, indicating that siDDX4 can significantly enhance the killing effect of the LV-TK / GCV system on tumor cells, and confirming that nucleic acid molecules targeting DDX4 can serve as effective gene therapy enhancers.

[0061] Example 3: Anti-DDX4 antibody as a synergist for CRISPR-Cas9 gene editing

[0062] This embodiment illustrates a method for preparing a monoclonal antibody that specifically binds to the DDX4 protein and inhibits its ATPase activity as a gene therapy enhancer, and its application in enhancing the therapeutic effect of CRISPR-Cas9 gene editing:

[0063] Y1. Preparation and screening of anti-DDX4 monoclonal antibodies: Recombinant human DDX4 protein (prokaryotic expression and purification, containing the N-terminal domain of DDX4) was used as the antigen. After being fully emulsified with Freund's adjuvant, 6-8 week old female BALB / c mice were immunized by subcutaneous injection at multiple sites on the back. The immunization dose for each mouse was 50 μg, and a total of 3 immunizations were performed, with an interval of 2 weeks between each immunization. Three days after the last immunization, mouse spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1 and fused with polyethylene glycol (PEG). After fusion, the cells were seeded in 96-well plates and screened using HAT medium. Positive hybridoma cell lines were screened by ELISA. After three subclonings using the limiting dilution method, a monoclonal hybridoma cell line that stably secretes anti-DDX4 antibody was obtained (named 4D5).

[0064] Y2. Antibody Purification and Activity Identification: Ascites fluid was prepared by inoculating monoclonal hybridoma cell lines into the peritoneal cavity of mice. The ascites fluid was purified using a Protein G affinity chromatography column to obtain anti-DDX4 monoclonal antibody. The antibody affinity was then determined by ELISA, showing that the anti-DDX4 monoclonal antibody had high affinity for recombinant DDX4 protein (EC50 value of 0.23 μg / mL). The inhibitory activity was then evaluated using an immunoprecipitation-ATPase activity assay: Lysate of the human non-small cell lung cancer cell line A549 was incubated with different concentrations of antibody (0, 0.1, 1, 10 μg / mL). The DDX4-antibody complex was precipitated using Protein G agarose beads, and the ATPase activity of DDX4 in the precipitate was measured. The results are as follows: Figure 5 As shown, this antibody can inhibit the ATPase activity of DDX4 in a dose-dependent manner;

[0065] Y3. Validation of the synergistic effect: The anti-DDX4 monoclonal antibody was combined with a CRISPR-Cas9 plasmid system targeting the PD-L1 gene (pX459-sgPD-L1). Human non-small cell lung cancer cell line A549 was divided into four groups: a blank control group, a pX459-sgPD-L1 transfection group, an anti-DDX4 antibody treatment group (10 μg / mL), and a pX459-sgPD-L1 + anti-DDX4 antibody combined treatment group. Cells were collected 48 hours after transfection. Flow cytometry was used to detect the expression level of PD-L1 on the cell membrane surface, and the proportion of PD-L1 positive cells was calculated. The mutation frequency of the PD-L1 gene locus was detected using the T7E1 restriction enzyme method. Genomic DNA was extracted, and the PD-L1 target site amplification products were subjected to Sanger sequencing. The sequencing peaks were deconvolved using the TIDE online analysis tool to quantitatively analyze the indel type and total editing efficiency. Total cellular protein was extracted and analyzed using Western spectroscopy. The PD-L1 knockout effect was verified at the protein level using blot (with GAPDH as an internal control). Flow cytometry was used to detect the fluorescence intensity of cell populations in each treatment group. The results are as follows: Figure 6 and Figure 7 As shown.

[0066] like Figure 6 The results showed that the proportion of PD-L1 positive cells in the pX459-sgPD-L1 + anti-DDX4 antibody co-treatment group was significantly lower than that in the single transfection group. T7E1 restriction enzyme digestion and Sanger sequencing analysis showed that the gene editing efficiency of the co-treatment group was significantly improved. Figure 7As shown, Western blot analysis confirmed that the PD-L1 protein expression level in the combined treatment group was significantly lower than that in the single transfection group, indicating a significant enhancement in PD-L1 gene knockout. Flow cytometry histograms showed that the PD-L1 fluorescence intensity peak in the combined treatment group was shifted to the left compared to the single transfection group, indicating that the synergistic effect of the anti-DDX4 antibody was not limited to a few cells, but rather caused a general downregulation of PD-L1 expression levels throughout the entire cell population. These results confirm that the anti-DDX4 monoclonal antibody can significantly enhance the efficiency of CRISPR-Cas9-mediated gene editing.

[0067] Example 4: Artificial intelligence-aided design of synergists based on the three-dimensional structure of DDX4 protein

[0068] This embodiment provides a method for obtaining novel small molecule inhibitors and verifying their synergistic effects by utilizing the three-dimensional structure of the DDX4 protein, combined with computer-aided drug design and artificial intelligence technology:

[0069] A1. Acquisition and processing of the three-dimensional structure of DDX4 protein: The three-dimensional structure of DDX4 protein was predicted using AlphaFold2 or ProteinX tools. The structural model with the highest confidence was selected, and the energy minimization optimization of the model was performed using Rosetta Relax software. Then, the protein surface was analyzed using CavityPlus software to identify the ATP binding pocket as the key active site and extract the pocket coordinates and key amino acid residue information.

[0070] A2. Virtual Screening and Molecular Generation: Based on the identified active sites, a pharmacophore model was established. Drug-like compounds were then screened from the ZINC database. AutoDock Vina software was used to perform molecular docking between the compound library and the DDX4 protein. Nine conformations were generated for each compound. Screening was based on binding free energy (ΔG) scores. Compound D4-IN-1 achieved the best score (-9.8 kcal / mol), and its docking mode is as follows: Figure 8 As shown, D4-IN-1 can form hydrogen bonds and hydrophobic interactions with key residues (such as Lys and Glu) in the ATP binding pocket of the DDX4 protein. Then, compounds with binding free energy less than -8.0 kcal / mol were selected for structural cluster analysis, and the top 50 compounds with good structural diversity were selected as candidate compounds.

[0071] A3. Molecular Dynamics Simulation and Optimization: The selected D4-IN-1 and DDX4 protein complex with the best score was subjected to a 100 ns molecular dynamics simulation using the GROMACS software package under a visible water model. The root mean square deviation (RMSD) of the complex was analyzed. The results showed that the RMSD of the complex tended to stabilize after about 30 ns, maintaining at around 2.0 Å, indicating that the complex structure was stable.

[0072] A4. In vitro activity validation: The top 50 candidate compounds obtained through commercial channels or virtual screening were analyzed using an ATPase activity assay kit to detect their inhibitory effect on DDX4 protein ATPase activity. Results are as follows: Figure 9 As shown, among the 50 candidate compounds, 8 compounds inhibited DDX4 ATPase activity by more than 50% at a concentration of 10 μmol / L. Among them, D4-IN-1 had the highest inhibition rate, reaching 85% ± 4%, with an IC50 value of 1.2 μmol / L. Subsequently, the cytotoxicity of compound D4-IN-1 on HeLa cells was detected by the CCK-8 assay. The results showed that at a concentration of 10 μmol / L, the cell viability of D4-IN-1 was 92% ± 3%, with no significant cytotoxicity.

[0073] A5. Validation of Synergistic Effect: HEK293T cells were treated with compound D4-IN-1 in combination with adeno-associated virus (AAV-Luc) carrying the luciferase reporter gene. Cell lysis was performed to detect luciferase activity (RLU value). HEK293T cells were divided into four groups: blank control group, AAV-Luc single treatment group, D4-IN-1 single treatment group, and AAV-Luc + D4-IN-1 combined treatment group (final D4-IN-1 concentration 5 μmol / L). The results are as follows: Figure 10 As shown, compared with the AAV-Luc treatment group alone, the luciferase activity (RLU value) of the AAV-Luc+D4-IN-1 combined treatment group was significantly increased by about 4 times, which confirms that the DDX4 inhibitor D4-IN-1 obtained by AI-assisted design can significantly enhance the efficiency of AAV-mediated gene transduction.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0075] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for screening gene therapy synergists targeting DDX4 protein, characterized in that, The method includes the following steps: (1) Contact the candidate molecule with the system expressing DDX4 protein; (2) Detect the expression level or activity of DDX4 protein after contact with candidate molecules; (3) Verify the enhancing effect of candidate molecules on gene therapy; (4) Select candidate molecules that can significantly downregulate the expression level of DDX4 protein or significantly inhibit the activity of DDX4 protein and significantly enhance the gene therapy effect as gene therapy enhancers.

2. The method for screening gene therapy synergists targeting DDX4 protein according to claim 1, characterized in that, The system for expressing DDX4 protein includes cell, tissue, or cell-free in vitro expression systems that express DDX4 protein.

3. The method for screening gene therapy synergists targeting DDX4 protein according to claim 1, characterized in that, The detection of DDX4 protein expression level is performed by detecting the mRNA level or protein translation level of DDX4.

4. The method for screening gene therapy synergists targeting DDX4 protein according to claim 1, characterized in that, The detection of DDX4 protein activity is performed by detecting its ATPase activity, RNA helicase activity, or substrate specificity.

5. A gene therapy enhancer obtained by screening using the method described in any one of claims 1-4, characterized in that, The synergist is a regulator that targets the DDX4 protein or its encoding gene, and can downregulate the expression level of the DDX4 protein or inhibit the activity of the DDX4 protein.

6. The gene therapy enhancer according to claim 5, characterized in that, The synergist includes small molecule compounds, nucleic acid molecules, proteins, peptides, and combinations of peptides; the nucleic acid molecules include siRNA, shRNA, antisense oligonucleotides targeting the DDX4 encoding gene, as well as sgRNA and the CRISPR-Cas9 gene editing system targeting the DDX4 encoding gene; the proteins and peptides include antibodies or antigen-binding fragments thereof that specifically bind to the DDX4 protein and inhibit its activity.

7. The use of the gene therapy enhancer according to claim 6 in the preparation of a medicament for enhancing the effect of gene therapy.

8. The application according to claim 7, characterized in that, The gene therapy includes viral vector-mediated gene therapy, non-viral vector-mediated gene therapy, or gene editing therapy, wherein the viral vector is selected from adeno-associated virus vectors, lentiviral vectors, adenovirus vectors, or retroviral vectors.

9. A pharmaceutical composition prepared using the synergist of claim 6, characterized in that, The pharmaceutical composition includes gene therapy enhancers and gene therapy drugs.

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