Shrna targeting human cd64 gene and application thereof in preparation of medicine for treating intestinal barrier dysfunction disease

Abstract

The application belongs to the field of medicine and particularly relates to a shRNA targeting a human CD64 gene and application thereof in preparation of a drug for treating intestinal barrier dysfunction diseases. A DNA sequence encoding the shRNA is shown as SEQ ID NO. 1. The shRNA can precisely and efficiently knock down the expression of CD64 in human macrophages. The shRNA is cloned into a lentivirus vector to obtain a macrophage strain stably knocking down CD64. Experiments prove that M1 type macrophages with knocked-down CD64 can significantly reduce the secretion of pro-inflammatory factors, increase the trans-epithelial electrical resistance value of intestinal epithelial cells, reduce the FITC-dextran permeability, and restore the expression of tight junction proteins. The application solves the problems of the prior art intestinal inflammation treatment system, such as large systematic side effects, short action time, and inability to block the pro-inflammatory pathway from the genetic level, and can be used for preparing a drug for treating intestinal barrier dysfunction diseases such as ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a shRNA that targets the human CD64 gene and its application in the preparation of drugs for treating intestinal barrier dysfunction. Background Technology

[0002] Inflammatory bowel disease (IBD), especially ulcerative colitis (UC), is a group of chronic, relapsing inflammatory bowel diseases, characterized by impaired intestinal epithelial barrier function. [1] Imbalance in gut immune homeostasis is a core mechanism in the pathogenesis of IBD. [2] Macrophages, as key cells of the intestinal immune system, play multiple roles in maintaining intestinal homeostasis, driving inflammatory responses, and promoting tissue repair. [3,4] In an inflammatory state, intestinal macrophages polarize into M1 type (pro-inflammatory type), further damaging the intestinal epithelial barrier by secreting large amounts of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. This manifests as a decrease in transepithelial electrical resistance (TEER) and an increase in FITC-glucan permeability, forming a vicious cycle of "inflammation-barrier damage-more severe inflammation." Meanwhile, M2 type macrophages (associated with IL-10) promote tissue repair. [5,6] .

[0003] CD64, as an Fcγ receptor I, is abnormally highly expressed on the surface of activated M1 macrophages. The CD64-mediated signaling pathway plays a key role in the pro-inflammatory polarization and inflammatory amplification of macrophages. Therefore, regulating CD64 expression to intervene in macrophage function has become a cutting-edge strategy for the treatment of inflammatory bowel disease.

[0004] Current treatment options for intestinal inflammation have the following main shortcomings:

[0005] 1. Systemic broad-spectrum anti-inflammatory drugs or anti-TNF-α monoclonal antibody therapy: This has problems such as large systemic side effects, easy development of drug resistance, high treatment costs, and some patients do not respond to the treatment.

[0006] 2. Neutralizing antibodies or nucleic acid aptamers targeting CD64: These can only block the interaction between CD64 and IgG by binding to the CD64 receptor on the cell surface, but cannot completely shut down the CD64-mediated pro-inflammatory signaling pathway at the gene transcription level. The therapeutic effect is limited and the duration is short.

[0007] 3. Transient transfection technology of conventional siRNA: Chemically synthesized siRNA is easily degraded by nucleases in vivo, with an action time of only 24-48 hours. It also has low transfection efficiency, no resistance selection marker, and cannot obtain stable CD64 knockdown cell lines, making it difficult to use for long-term intestinal barrier function research or cell therapy.

[0008] Based on the foregoing, the technical solution of the present invention is provided.

[0009] The cited prior art is as follows: [1]Patel A, Jain P, Ajazuddin. Recent advances in the therapeuticsand modes of action of a range of agents used to treat ulcerative colitis andrelated inflammatory conditions[J]. Inflammopharmacology, 2025, 33(9): 4965-4996. [2]Hausmann A, Steenholdt C, Nielsen O H, et al. Immune cell-derivedsignals governing epithelial phenotypes in homeostasis and inflammation[J].Trends Mol Med, 2024, 30(3): 239-251. [3]Li T, Li Q, Liu S, et al. Targeted V-type peptide-decoratednanoparticles prevent colitis by inhibiting endosomal TLR signaling andmodulating intestinal macrophage polarization[J]. Biomaterials, 2025, 314:122843. [4]Hegarty L M, Jones G R, Biram A, et al. Tissue resident colonicmacrophages persist through acute inflammation and adapt to aid tissue repair[J]. Mucosal Immunol, 2026, 19(1): 1624-1635. [5]Xu M, Cui Y, Wei S, et al. Emerging nanomaterials targeting macrophage adapted to abnormal metabolism in cancer and atherosclerosistherapy (Review)[J]. Int J Mol Med, 2024, 53(2). [6] Yuan S, Liu BH, Cheng WW, et al. Polyphyllin VI modulates macrophage polarization through autophagy-NLRP3 inflammasome to alleviate inflammatory bowel disease[J]. Phytomedicine, 2025, 143: 156640. Summary of the Invention The purpose of this invention is to overcome the shortcomings of the prior art and provide a shRNA targeting the human CD64 gene and its application in the preparation of drugs for treating intestinal barrier dysfunction, thereby solving the problems of existing intestinal inflammation treatments being unable to completely block the CD64-mediated pro-inflammatory pathway at the gene level, having a short duration of action, and having significant systemic side effects.

[0010] The present invention provides an shRNA that targets the human CD64 gene, and the DNA sequence encoding the shRNA is shown in SEQ ID NO.1.

[0011] SEQ ID NO.1: GTCATGAGAAGAAGGTAATTTCTCGAGAAATTACCTTTCTTCTCATGACTTTTTT.

[0012] Based on the same technical concept, the present invention provides a recombinant expression vector containing the DNA coding sequence of the shRNA targeting the human CD64 gene.

[0013] Preferably, the recombinant expression vector is a viral vector or a non-viral plasmid vector; the viral vector is one of a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0014] Preferably, the recombinant expression vector contains a promoter that drives the transcription of the shRNA, and the promoter is one of the U6 promoter, CD68 promoter, and CD11b promoter.

[0015] Based on the same technical concept, the present invention further provides a nucleic acid delivery composition comprising an active nucleic acid component and a pharmaceutically acceptable delivery carrier; wherein the active nucleic acid component is the shRNA; and the delivery carrier is a liposome nanoparticle or an exosome.

[0016] Based on the same technical concept, the present invention further provides a genetically modified host cell with downregulated CD64 expression, wherein the recombinant expression vector or the nucleic acid delivery composition is transferred into the genetically modified host cell.

[0017] Preferably, the genetically modified host cell is a mononuclear cell line or an induced macrophage; the mononuclear cell line is one of the THP-1 cell line and the U937 cell line; the induced macrophage is a macrophage obtained by inducing differentiation from human peripheral blood mononuclear cells.

[0018] Based on the same technical concept, the present invention further provides the application of the aforementioned shRNA targeting the human CD64 gene, the aforementioned recombinant expression vector, the aforementioned nucleic acid delivery composition, and the aforementioned genetically modified host cells in the preparation of drugs for treating intestinal barrier dysfunction diseases.

[0019] Preferably, the intestinal barrier dysfunction includes inflammatory bowel disease; the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0020] The beneficial effects of this invention are as follows: 1. Precise targeting and high knockdown efficiency: The shRNA sequence obtained by this invention through multiple rounds of screening and verification can specifically recognize human CD64 mRNA and mediate its degradation, blocking the expression of CD64 protein from the source of gene transcription. This is completely different from the existing CD64 neutralizing antibody's mode of action, which only competitively blocks at the protein receptor level. It avoids the problems of insufficient drug efficacy caused by incomplete receptor occupancy and activation of signal bypass. The knockdown specificity and inhibition efficiency are significantly better than protein-level intervention methods.

[0021] 2. Long-lasting and stable effects, supporting long-term research and therapeutic applications: Compared with chemically synthesized siRNA, which is easily degraded by nucleases and has a short-term intervention effect of only 24-48 hours, this invention uses lentiviral vectors to mediate the integration of shRNA into the host cell genome. Combined with puromycin resistance screening, stable CD64 knockdown cell lines can be obtained, which can inhibit CD64 expression in a long-term and stable manner. This not only meets the experimental needs of long-term intestinal inflammation mechanism research and drug screening, but also provides a technical basis for the development of long-acting cell therapy products.

[0022] 3. Combining visual and screening markers: The recombinant lentiviral vector carries both the copGFP fluorescent reporter gene and the puromycin resistance gene: GFP fluorescence allows for intuitive and real-time monitoring of viral transfection efficiency and cell viability using a fluorescence microscope without damaging cell viability, facilitating quality control during experiments; the puromycin resistance marker enables rapid pressure screening of stable cell lines, significantly reducing the time cost and operational difficulty of cell line construction.

[0023] 4. Clear Mechanism and Significant Efficacy: This invention not only confirms at the cellular level that CD64 knockdown can significantly inhibit the secretion of pro-inflammatory factors such as IL-6, IL-8, and TNF-α by M1 macrophages, but also upregulates the expression of the anti-inflammatory factor IL-10, achieving bidirectional regulation of pro-inflammatory and anti-inflammatory immune homeostasis; furthermore, through transepithelial electrical resistance (TEER) detection and FITC-glucan permeability experiments, it verifies at the functional level its protective effect on the intestinal epithelial physical barrier, and further clarifies that it can maintain the expression of tight junction and adhesion junction proteins such as ZO-1, occludin, and E-cadherin, confirming the effect of intestinal barrier structure repair at the molecular mechanism level, breaking the vicious cycle of "inflammation-barrier damage-inflammation aggravation".

[0024] 5. Flexible delivery and application: The technical solution of this invention can be extended to various product forms: it can be developed into nucleic acid drugs targeting macrophages through delivery carriers such as liposomes and exosomes, or cell therapy products can be developed based on in vitro modification of autologous mononuclear / macrophages; at the same time, the carrier can be driven by macrophage-specific promoters such as CD68 and CD11b to achieve targeted expression of immune cells, reduce the off-target risk and systemic side effects of systemic administration, and improve the safety of treatment. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a flow cytometry result showing the expression of CD64 on the surface of THP-1 cells induced into M1 macrophages after sh-control and sh-CD64 were detected.

[0027] Figure 2 This is a graph showing the results of Western blot analysis of CD64 expression on the surface of THP-1 cells induced into M1 macrophages after the untreated group, sh-control, and sh-CD64 were converted into M1 macrophages.

[0028] Figure 3 This is a graph showing the results of the intestinal epithelial barrier "density" (transepithelial resistance TEER) test. P <0.001).

[0029] Figure 4 This is a graph showing the results of the intestinal epithelial barrier "leakage" (fluorescein permeability) test. P <0.001).

[0030] Figure 5 This is a graph showing the results of quantitative detection of the pro-inflammatory cytokine IL-6 in the supernatant of M1 macrophage-Caco-2 cell co-culture using the ELISA method. P <0.001).

[0031] Figure 6 This is a graph showing the results of quantitative detection of the pro-inflammatory cytokine IL-8 in the supernatant of M1 macrophage-Caco-2 cell co-culture using the ELISA method. P <0.001).

[0032] Figure 7 This is a graph showing the results of quantitative detection of the pro-inflammatory cytokine TNF-α in the supernatant of M1 macrophage-Caco-2 cell co-culture using the ELISA method. P <0.01).

[0033] Figure 8 This is a graph showing the results of quantitative detection of the anti-inflammatory cytokine IL-10 in the supernatant of M1 macrophage-Caco-2 cell co-culture using the ELISA method. P <0.01).

[0034] Figure 9 This is a graph showing the protein expression results.

[0035] Figure 10 This is a graph showing the protein content of tight junction protein ZO-1 in Caco-2 cells co-cultured with sh-control or sh-CD64 M1 macrophages (Western blot method). P <0.05).

[0036] Figure 11 This is a graph showing the protein content of the tight junction protein occludin in Caco-2 cells co-cultured with sh-control or sh-CD64 M1 macrophages (Western blot method). P <0.05).

[0037] Figure 12 This is a graph showing the protein content of the adhesion junction protein E-cadherin in Caco-2 cells co-cultured with sh-control or sh-CD64 M1 macrophages (Western blot method). P <0.05). Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Example 1: Design of shRNA sequence targeting human CD64 and construction of lentiviral vector 1. shRNA sequence design: Based on the mRNA sequence of the human CD64 gene, a specific shRNA sequence was designed. After multiple rounds of screening and verification, the optimal sequence was determined to be SEQ ID NO.1, which was described as "sh-CD64" in subsequent experiments.

[0040] 2. Lentiviral vector construction: (1) Anneal the sense and antisense strands of the synthesized shRNA to form a double-stranded DNA fragment; (2) The pLent-U6-shRNA-CMV-copGFP-P2A-Puro vector backbone was digested with restriction endonucleases BamHI and EcoRI, and the linearized vector was recovered. (3) The annealed shRNA double strands were ligated to the linearized vector at 16°C overnight using T4 DNA ligase; (4) Transform the ligation product into Escherichia coli DH5α competent cells, spread them on LB plates containing ampicillin, and incubate overnight at 37°C; (5) Select a single colony for PCR identification and sequencing verification to confirm that the shRNA sequence is correctly inserted into the vector and obtain the recombinant lentiviral vector pLent-U6-shCD64-CMV-copGFP-P2A-Puro.

[0041] Example 2: Packaging and titer determination of recombinant lentivirus 1. Lentiviral packaging: (1) Seed 293T cells in 10cm culture dishes and cultured until the cell confluence reached 70%-80%; (2) Following the instructions of the Lipofectamine 3000 transfection reagent, the recombinant lentiviral vector and packaging plasmids (pMD2.G and psPAX2) were co-transfected into 293T cells; (3) Replace with fresh culture medium 6 hours after transfection, and continue culturing for 48 hours and 72 hours, and collect cell supernatant containing virus respectively; (4) After filtering the collected supernatant through a 0.45 μm filter membrane, the virus was concentrated by ultracentrifugation and stored at 4°C for later use.

[0042] 2. Virus titer determination: Lentiviral titers were determined using a serial dilution method. 293T cells were seeded in 96-well plates and cultured until 50% confluence. Different dilutions of viral solution were added, and after 72 hours of incubation, the number of GFP-positive cells was counted using a fluorescence microscope to calculate the viral titer. In this example, the recombinant lentivirus titer obtained was 6 × 10⁻⁶. 8 TU / mL.

[0043] Example 3: Construction and validation of a THP-1 cell line with stable CD64 knockdown 1. Lentiviral infection of THP-1 cells: (1) THP-1 mononuclear cells were seeded into 6-well plates, with 2 × 10⁶ cells per well. 5 indivual; (2) Recombinant lentivirus was added at a ratio of multiple of infection (MOI) = 50, and Polybrene was added at a final concentration of 8 μg / mL to improve infection efficiency. (3) After 72 hours of infection, replace the culture medium with fresh culture medium and add puromycin at a final concentration of 10 μg / mL for pressure screening. Replace the culture medium containing puromycin every 2-3 days. After 2 weeks of screening, obtain a stable transfected cell line.

[0044] 2. Induction of M1 macrophage differentiation: (1) Collect stably transfected THP-1 cells in the logarithmic growth phase by centrifugation, resuspend them in RPMI-1640 complete medium, and adjust the cell density to 1×10⁻⁶. 6Cells / mL; (2) Add PMA (Sigma, P1585) to a final concentration of 50 ng / mL, culture for 48 hours, and induce differentiation into M0 macrophages; (3) Discard the culture medium and replace it with fresh RPMI-1640 complete culture medium, and let it stand for 24 hours; (4) Add complete culture medium containing 50 ng / mL LPS (Sigma, L3024) and 20 ng / mL IFN-γ (Peprotech, AF-300-02) and treat for 24 hours to induce M0 macrophages to polarize into M1 macrophages.

[0045] 3. CD64 knockdown efficiency verification: (1) Flow cytometry detection: Induced M1 macrophages were collected, washed twice with PBS, and CD64-APC antibody was added. The cells were incubated at 4°C in the dark for 30 minutes. After washing, the proportion of CD64-positive cells was detected by flow cytometry. Results are as follows: Figure 1 As shown, at the cell surface expression level, compared with the control group (sh-control), the proportion of CD64-positive cells transfected with sh-CD64 (after induction into M1 macrophages) was significantly reduced (from 55.0% to 1.11%). This demonstrates that sh-CD64 effectively knocks down CD64 expression on the cell surface.

[0046] (2) Western blot detection: Total cellular protein was extracted, subjected to SDS-PAGE electrophoresis and membrane transfer, incubated with CD64 antibody and GAPDH internal control antibody, and then subjected to ECL chemiluminescence staining. The results are as follows: Figure 2 As shown, at the total protein expression level, with GAPDH as an internal control (ensuring consistent loading amounts across groups), the CD64 protein band was darker in the untreated group and the control group (sh-control); while the CD64 protein band was significantly weaker in the experimental group transfected with sh-CD64. This demonstrates that sh-CD64 successfully reduced CD64 protein expression at the total protein level.

[0047] The above results demonstrate that, at both the cell surface and total protein levels, the expression level of CD64 in M1 macrophages decreased significantly after using the sequence (sh-CD64) of this invention. This proves that the gene silencing tool designed in this invention is precise and efficient.

[0048] Example 4: In vitro validation of intestinal barrier repair effect 1. Construction of Transwell co-culture system: (1) Caco-2 intestinal epithelial cells were seeded into the upper chamber of a Transwell chamber (pore size 0.4 μm), with 1 × 10 cells per well. 5 One cell was cultured until a dense monolayer of cells was formed. (2) Use an epithelial resistance meter to measure the TEER value. When the TEER value is more than 5 times higher than that of the blank well, the intestinal epithelial barrier is considered to have been successfully constructed. (3) Induced sh-control M1 macrophages or sh-CD64 M1 macrophages were seeded into the lower chamber of the Transwell chamber, with 5 × 10⁶ cells per well. 4 indivual.

[0049] 2. Intestinal barrier function testing: (1) TEER value determination: The TEER values ​​of each group were measured using an epithelial osmotic pressure meter at 0 hours and 24 hours of co-culture. The results are as follows: Figure 3 As shown, it indicates that M1 pro-inflammatory macrophages (sh-control M1) disrupt the intestinal barrier, leading to a decrease in electrical resistance; however, after co-culturing for 24 hours with macrophages treated according to this invention (sh-CD64) in the lower layer, the intestinal barrier electrical resistance (TEER) was significantly higher than that of the control group. P <0.001). This proves that the present invention can effectively resist inflammatory damage and maintain the integrity of the intestinal physical barrier.

[0050] (2) FITC-glucan permeability determination: After co-culturing for 24 hours, FITC-glucan with a final concentration of 80 μg / mL was added to the upper chamber and incubated at 37℃ for 1 hour. 200 μL of culture medium from the lower chamber was collected, and the fluorescence intensity was detected using a fluorescence microplate reader (excitation wavelength 490 nm, emission wavelength 520 nm). The fluorescein permeability was calculated. The results are as follows: Figure 4 As shown, the amount of fluorescent substance leaked was significantly lower in the group using the present invention (sh-CD64) than in the control group. P <0.001). Combined with... Figure 3 From the reverse perspective (leakage prevention), this further proves that the present invention effectively protects the intestinal barrier function and prevents "intestinal leakage".

[0051] 3. Inflammatory factor detection: Cell supernatant was collected after 24 hours of co-culture, and the levels of pro-inflammatory factors IL-6, IL-8, TNF-α, and anti-inflammatory factor IL-10 were measured according to the ELISA kit instructions. Results are as follows: Figures 5-8As shown, it demonstrates that the present invention (sh-CD64) can significantly reduce the release of pro-inflammatory factors (IL-6, IL-8, TNF-α), while significantly increasing the level of anti-inflammatory factor (IL-10). P <0.01, P <0.001).

[0052] 4. Detection of epithelial junction proteins: Total protein was extracted from Caco-2 cells in the upper chamber of Transwell cells and analyzed by Western blot to detect the expression of tight junction proteins ZO-1, occludin, and adhesion junction protein E-cadherin. GAPDH was used as an internal control. Results are as follows: Figures 9-12 As shown, the control group showed less damage to the aforementioned cell junction proteins due to inflammation, while the use of the present invention (sh-CD64) resulted in significant protection and preservation of these key junction proteins. This explains the mechanism by which the present invention protects the intestinal barrier at the molecular structural level.

[0053] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A shRNA targeting the human CD64 gene, characterized in that, The DNA sequence encoding the shRNA is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The DNA coding sequence of the shRNA targeting the human CD64 gene as described in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that, The recombinant expression vector is a viral vector or a non-viral plasmid vector; the viral vector is one of a lentiviral vector, adenovirus vector, or adeno-associated virus vector.

4. The recombinant expression vector according to claim 2, characterized in that, The recombinant expression vector contains a promoter that drives the transcription of the shRNA, and the promoter is one of the U6 promoter, CD68 promoter, and CD11b promoter.

5. A nucleic acid delivery composition, characterized in that, It comprises an active nucleic acid component and a pharmaceutically acceptable delivery carrier; the active nucleic acid component is the shRNA of claim 1; the delivery carrier is a liposome nanoparticle or an exosome.

6. A genetically modified host cell with downregulated CD64 expression, characterized in that, The genetically modified host cell is transferred with the recombinant expression vector of any one of claims 2-4 or the nucleic acid delivery composition of claim 5.

7. The genetically modified host cell according to claim 6, characterized in that, The genetically modified host cell is a mononuclear cell line or an induced macrophage; the mononuclear cell line is either the THP-1 cell line or the U937 cell line; the induced macrophage is a macrophage obtained by inducing differentiation from human peripheral blood mononuclear cells.

8. The use of the shRNA targeting the human CD64 gene as described in claim 1, the recombinant expression vector as described in any one of claims 2-4, the nucleic acid delivery composition as described in claim 5, and the genetically modified host cell as described in any one of claims 6-7 in the preparation of a medicament for treating intestinal barrier dysfunction.

9. The application according to claim 8, characterized in that, The intestinal barrier dysfunction diseases include inflammatory bowel disease; the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

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