Chimeric antigen receptor-macrophages targeting cx3cl1 and use in treatment of gjb2 mutation-associated genetic deafness

Abstract

The application discloses a chimeric antigen receptor-macrophage targeting CX3CL1 and application thereof in treatment of GJB2 mutation related genetic deafness, the chimeric antigen receptor-macrophage targeting CX3CL1 directly converts CX3CL1 mediated proinflammatory signals into anti-inflammatory signals, realizes directional polarization of macrophages to M2 type, plays an anti-inflammatory repair function, specifically responds to cochlea damage microenvironment, switches an anti-inflammatory repair phenotype and protects cochlea epithelial cells and spiral ganglion neurons, and can be used for precisely improving GJB2 mutation related genetic deafness inner ear local inflammation, promoting clearance of apoptotic cell fragments and not interfering with whole body immune homeostasis, and has a significant drug treatment application prospect.

Description

Technical Field

[0001] This invention relates to the field of deafness treatment technology, specifically to chimeric antigen receptor-macrophages targeting CX3CL1 and their application in the treatment of GJB2 mutation-related hereditary deafness. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved significant success in the treatment of tumors such as lymphoma. It primarily utilizes gene editing technology to equip the patient's own T cells with a CAR system, enabling them to precisely recognize specific antigens on the surface of cancer cells. The modified CAR-T cells are then infused into the patient, achieving a "precision strike" against cancer cells. In recent years, this technology has expanded to the fields of natural killer cells and macrophages. Chimeric antigen receptor macrophage (CAR-M) therapy, as an innovative treatment strategy, demonstrates unique therapeutic potential. It specifically recognizes tumor-associated antigens, mediating efficient phagocytosis to eliminate tumor cells. Macrophages are widely distributed throughout the body and can be divided into M1 and M2 types. In addition to the phagocytic function required for anti-tumor activity, they also possess regenerative and inflammatory regulatory functions, making them ideal candidates for cell therapy. M1 macrophages produce high levels of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, exhibiting strong phagocytic and tumor cell-killing capabilities—the main mechanism underlying the widely used CAR-M therapy for solid tumors. M2 macrophages, on the other hand, produce anti-inflammatory cytokines, such as IL-10 and TGF-β, participating in inflammation suppression, tissue repair, and angiogenesis promotion. Infusion of anti-inflammatory M2 macrophages has shown damage-relieving effects in various non-tumor disease models. However, traditional strategies for regulating macrophage phenotypes suffer from insufficient targeting and susceptibility to interference from the inflammatory microenvironment, making it difficult to precisely and stably exert anti-inflammatory effects. CAR-M, by targeting and activating "signal switch" antigens, can precisely regulate macrophage polarization towards M2 anti-inflammatory macrophages. This therapeutic approach has been explored and applied in non-tumor fields such as autoimmune diseases and fibrotic diseases with promising results.

[0003] Damage to the inner ear sensory epithelium and auditory nerve is irreversible, and current clinical interventions primarily rely on hearing aids or cochlear implants to assist hearing. On the one hand, these assistive devices struggle to accurately reproduce the natural listening experience and cannot meet the auditory needs in complex scenarios; on the other hand, children with congenital deafness are highly susceptible to missing the critical window for speech development, impacting their subsequent language abilities and social function. Therefore, targeted treatment in the early stages of damage has become an important direction for improving patient prognosis and filling clinical gaps. Recent studies have shown that inner ear inflammation and immunity are key mechanisms for maintaining auditory and balance homeostasis. The inner ear immune response to stresses such as acoustic trauma and ototoxic drugs directly determines the degree of damage and the effectiveness of repair.

[0004] Our team's previous research found that inner ear inflammation and immunity are also important pathological drivers of hereditary deafness. This was achieved through two specific... Gjb2 Systematic analysis of the knockout mouse model confirmed that macrophage-related immune responses play a crucial role in cochlear epithelial damage in hereditary deafness, and that their activation pattern is highly synchronized with the cochlear cell degeneration process. Notably, unlike the inflammatory responses induced by exogenous damage such as noise and ototoxic drugs, Gjb2 In relevant models of hereditary deafness, only CX3CL1 chemokine expression was significantly upregulated, while classic inflammatory factors such as TNF-α and IL-1β showed no significant changes. Immunofluorescence confirmed that after the death of outer hair cells, Deiters cells coupled with them and surviving spiral ganglion neurons highly expressed CX3CL1, regulating macrophage recruitment and antigen presentation through the CX3CL1-CX3CR1 axis. The discovery of this specific inflammatory regulatory mechanism provides a new target for immunomodulatory intervention in hereditary deafness and lays an experimental foundation for developing precision therapeutic strategies targeting the CX3CL1 signaling pathway. Summary of the Invention

[0005] This invention provides a "signal-switching" CAR-M cell therapy targeting CX3CL1, a target of GJB2-related hereditary deafness, which converts inflammatory signals into anti-inflammatory signals and aims to salvage GJB2-related hereditary deafness through systemic infusion of modified CAR-M.

[0006] In view of this, the solution of the present invention is as follows:

[0007] A first aspect of the present invention is to provide a chimeric antigen receptor targeting CX3CL1, comprising a single-chain antibody scFv that recognizes human CX3CL1 antigen, a CD8α hinge region and a transmembrane domain, and an IL-4Rα intracellular co-stimulatory signal transduction domain; the amino acid sequence of the single-chain antibody scFv that recognizes human CX3CL1 antigen is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequences of the CD8α hinge region and transmembrane domain are shown in SEQ ID NO.2.

[0009] Furthermore, the amino acid sequence of the intracellular co-stimulatory signal transduction structure of the IL-4Rα is shown in SEQ ID NO.3.

[0010] Furthermore, the chimeric antigen receptor also includes a fluorescent tag, preferably a green fluorescent protein (GFP) tag.

[0011] Furthermore, the chimeric antigen receptor also includes a signal peptide, which is a granulocyte-macrophage colony-stimulating factor.

[0012] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.6.

[0013] A second aspect of the invention is to propose a gene encoding the chimeric antigen receptor described in the first aspect.

[0014] A third aspect of the present invention is to provide a recombinant vector comprising the gene described in the second aspect; preferably, the recombinant vector is a lentiviral vector.

[0015] A fourth aspect of the invention is to provide a chimeric antigen receptor-macrophage (CX3CL1-CAR-M) that expresses a chimeric antigen receptor targeting CX3CL1 as described in the first aspect.

[0016] Furthermore, the chimeric antigen receptor-macrophage is an M2 type macrophage.

[0017] A fifth aspect of the invention is the application of the chimeric antigen receptor-macrophage described in the fourth aspect in the preparation of a medicament for the treatment of noise-induced hearing loss.

[0018] Furthermore, the drug is an injectable preparation, preferably for infusion into the inner ear.

[0019] Furthermore, the drug includes any one of the following uses:

[0020] 1) Improve hearing frequency thresholds;

[0021] 2) Improve the ABR threshold.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The chimeric antigen receptor targeting CX3CL1 provided by this invention contains an HSP70-specific binding fragment in its extracellular domain and an IL-4 signaling pathway functional element in its intracellular domain, forming a "signal switch" CAR structure targeting CX3CL1. This structure can be used to prepare chimeric antigen receptor-macrophages targeting CX3CL1 and to mediate macrophage polarization towards the M2 type and exert anti-inflammatory functions.

[0024] The chimeric antigen receptor-macrophage (CX3CL1-CAR-M) targeting CX3CL1 described in this invention can mediate macrophage polarization towards the M2 type, exert anti-inflammatory and repair functions, specifically respond to the cochlear injury microenvironment (areas of outer hair cell loss, Deiters cells, and spiral ganglion neurons degenerate), switch to an anti-inflammatory and repair phenotype, and protect cochlear epithelial cells and spiral ganglion neurons. Therefore, CX3CL1-CAR-M cells can be used to precisely improve local inflammation in the inner ear of GJB2 mutation-related hereditary deafness, promote the clearance of apoptotic cell debris, and not interfere with systemic immune homeostasis, showing significant potential for drug therapeutic applications.

[0025] The chimeric antigen receptor-macrophage targeting CX3CL1 described in this invention directly converts CX3CL1-mediated pro-inflammatory signals into anti-inflammatory signals, achieving macrophage directional polarization towards the M2 type. After cochlear damage is repaired, CX3CL1 expression naturally declines, and CX3CL1-CAR-M automatically terminates its function due to the loss of target binding signals, avoiding long-term intervention that could lead to an imbalance in the cochlear immune microenvironment or abnormal tissue repair.

[0026] The chimeric antigen receptor-macrophage targeting CX3CL1 described in this invention can be infused into the inner ear as a drug, precisely targeting damaged sites such as the spiral ligament and basilar membrane, protecting hair cells and the auditory nerve, and blocking the progression of damage in the early stages of hearing loss. Addressing the pathological characteristics of noise-induced neuroimmune responses and elevated systemic inflammatory markers, systemic infusion achieves simultaneous improvement of local and systemic inflammation in the inner ear, overcoming the limitations of traditional treatments that only focus on the inner ear and expanding the application boundaries of CAR-M therapy. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the chimeric antigen receptor structure targeting CX3CL1 described in one embodiment of the present invention.

[0028] Figure 2 This is the result of verifying the polarization phenotype of macrophages after transfection with the CAR-M vector targeting CX3CL1 in Example 1 of the present invention.

[0029] Figure 3 This is the verification result of the GJB2-related hereditary deafness model in Example 2 of the present invention.

[0030] Figure 4 This document describes the verification process and results of the efficacy of CX3CL1-CAR-M targeting a GJB2 mutation-related hereditary deafness model in Example 2 of this invention. Detailed Implementation

[0031] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0032] In one embodiment, a CX3CL1-targeting CAR (CX3CL1-CAR) is proposed. The core design of this CAR utilizes a "signal transduction" mechanism to convert inflammatory signals into anti-inflammatory signals, thereby regulating macrophage phenotype. Its structure comprises three key functional domains that synergistically achieve targeted recognition and signal transduction, as detailed below:

[0033] 1) Extracellular recognition domain: Utilizing an anti-CX3CL1 single-chain variable fragment (scFv), it specifically binds to CX3CL1 in the inflammatory microenvironment, ensuring that CAR is activated only at the site of inflammation. The anti-CX3CL1 scFv is constructed based on the heavy chain (VH) and light chain (VL) variable regions of the CX3CL1-binding antibody, forming a VH-(G4S)3-VL structure via a (G4S)3 linker, ensuring its specific binding ability to CX3CL1. The anti-CX3CL1 scFv sequence (SEQ ID NO.1) is as follows:

[0034] QVQLQSGPELVKPGASVKMSCKASGYTFTNYYIHWVKQRPGQGLEWIGWIYPGDGSPKFNERFKGKTTLTADKSSNTAYMLLSSLTSEDSAIYFCATGPTDGDYFDYWGQGTTLTVSSGG GGSGGGGSGGGGSDIQMTQSPASLSSASVGEYVTITCRASGNIHNFLAWYQQKRGKSPQFLVYNEKTLADGVPSRFSGSGSGTOYSLKINSLOPEDFGIYFCQQFWSTPYTFGGGTKLEIK

[0035] 2) Transmembrane connection domain: Using the hinge region and transmembrane domain of mouse or human CD8α, it is responsible for connecting the extracellular domain and the intracellular domain to maintain the stability of the CAR structure.

[0036] The transmembrane linker domain sequence (SEQ ID NO.2) is: GLDFACDIYIWAPLAG.

[0037] 3) Intracellular signaling domain: Integrates the intracellular domain of interleukin-4 receptor α (IL-4Rα). When scFv binds to CX3CL1, it activates downstream IL-4 signaling pathways (such as p-STAT6 and p-AKT) through this domain, inducing macrophages to transform into an anti-inflammatory phenotype (M2 type) instead of initiating conventional pro-inflammatory signals.

[0038] The intracellular signal domain sequence (SEQ ID NO.3) is as follows:

[0039] .

[0040] The CX3CL1-CAR is constructed as follows:

[0041] 1. Design and synthesis of scFv: The anti-CX3CL1 scFv is constructed based on the heavy chain (VH) and light chain (VL) variable regions of the CX3CL1 binding antibody. It forms a VH-(G4S)3-VL structure through the (G4S)3 linker to ensure its specific binding ability to CX3CL1.

[0042] 2. Assembly of the full-length CAR sequence: The CAR was assembled in the order of "labeled peptide-extracellular domain-transmembrane domain-intracellular domain", sequentially linking the hinge region and transmembrane domain of anti-CX3CL1scFv and CD8α (connection function), and the intracellular domain of IL-4Rα (signal transduction); a flag tag was introduced as a marker for detecting transduction efficiency (SEQ ID NO.4). The labeled peptide is granulocyte-macrophage colony-stimulating factor GM-CSF-2A, which is used to maintain the functional plasticity of macrophages.

[0043] A schematic diagram of the assembled CX3CL1-CAR is shown below. Figure 1 As shown, the full-length amino acid sequence is shown in SEQ ID NO.5.

[0044] Example 1: Construction of CX3CL1-CAR-M

[0045] I. Design and Construction of CAR

[0046] The core design of CARs (autoimmune chemoradiotransfer) is to convert inflammatory signals into anti-inflammatory signals through a "signal transduction" mechanism, thereby regulating the macrophage phenotype. Its structure contains three key functional domains that work synergistically to achieve target recognition and signal transduction, as detailed below:

[0047] 1. CX3CL1-CAR Construction Scheme

[0048] (1) CAR sequence design: The construction of the extracellular recognition domain anti-CX3CL1 single-chain variable fragment (scFv) is based on the heavy chain variable region (VH) and light chain variable region (VL) of the immunogen sequence of the CX3CL1 binding antibody. The anti-CX3CL1 scFv sequence shown in SEQ ID NO.1 was constructed by synthesizing a gene fragment containing the VH-(G4S)3-VL structure through Priscilla Technologies.

[0049] Transmembrane connection domain: Using the hinge region and transmembrane domain of mouse CD8α, it is responsible for connecting the extracellular domain and the intracellular domain, maintaining the stability of the CAR structure.

[0050] Intracellular signaling domain: Integrates the intracellular domain of interleukin-4 receptor α (IL-4Rα). When scFv binds to CX3CL1, it activates downstream IL-4 signaling pathways (such as p-STAT6 and p-AKT) through this domain, inducing macrophages to transform into an anti-inflammatory phenotype (M2 type) instead of initiating conventional pro-inflammatory signals.

[0051] A truncated CAR (CARΔ) was constructed as a control, lacking the overexpressed fragment, to verify the necessity of the signal domain.

[0052] The constructed CX3CL1-CAR amino acid sequence is shown in SEQ ID NO.5.

[0053] 2. Lentiviral Packaging

[0054] (1) Plasmid preparation: After the construction of the recombinant lentiviral vector is completed, a large amount of plasmid is extracted to prepare for subsequent transfection.

[0055] (2) 293T cell culture and transfection: Seed 293T cells into 10cm dishes and control the cell density to 70-80% the next day for transfection (ensure that the cells are passaged no more than 20 times and are in good condition). Remove the culture dish 1 hour before transfection, remove the original culture medium, and add 10ml of Opti-MEM medium.

[0056] (3) Preparation of transfection complex: Dissolve 32 μg of plasmid to be transfected (packaging plasmid: shuttle plasmid = 1:1) in Opti-MEM medium, total volume 500 μl, mix gently and let stand for 5 min; dissolve the transfection reagent in Opti-MEM medium, total volume 500 μl, mix gently and let stand for 5 min; add the dilution of the transfection reagent dropwise to the dilution of the plasmid, mixing gently while adding, and let stand at room temperature for 20 min to allow the DNA to fully combine with the transfection reagent to form a stable transfection complex.

[0057] (4) Transfection and medium change: Take out the culture dish and add the prepared DNA-transfection reagent mixture; after 6 hours of transfection, remove the culture medium, wash once with PBS, and add 10 ml of fresh complete culture medium to continue culturing.

[0058] (5) Lentiviral harvest: Collect the supernatant of 293T cells 48h and 72h after transfection, and aliquot them into 50ml centrifuge tubes; centrifuge at 3500rpm at room temperature for 10min to remove cells and large debris; filter the supernatant into an ultracentrifuge tube using a 0.45μm filter membrane.

[0059] (6) Lentiviral concentration: Centrifuge the above supernatant at 30,000 rpm and 4°C for 2 h. A white virus precipitate will be visible on one side of the tube wall. Discard the supernatant and invert the centrifuge tube onto sterile absorbent paper to remove any remaining supernatant. Add 80 μl to 120 μl of DPBS according to the amount of precipitate. Seal the tube opening with sealing film and dissolve the precipitate overnight at 4°C. Aliquot the virus as needed and store it in a -80°C refrigerator.

[0060] (7) Lentiviral titer determination:

[0061] ① The day before infection, administer 1×10 mg per well. 5 24-well plates were seeded at a density of 1,000 cells.

[0062] ② Viral infection: Add 10 μl, 1 μl, and 0.1 μl of virus to different cell culture wells (without adding Polybrene). The virus can be diluted before adding.

[0063] ③ Change the culture medium: After 24 hours of infection, replace the culture medium with fresh medium and continue culturing.

[0064] ④ Cell treatment: After 72-96 hours, discard the cell supernatant, collect the cells and extract genomic DNA from 293T cells.

[0065] ⑤qPCR detection: Using the extracted genome as a template, the internal reference gene or viral sequence WPRE is quantitatively detected.

[0066] ⑥ Plotting the standard curve: The amplification efficiency of the standard curve should be between 0.9 and 1.1, and R0 should be within the range of 0.9-1.1. 2 If the value is >0.990, primer specificity needs to be tested and the standard rediluted.

[0067] ⑦ Titer Calculation: Calculate the lentivirus titer (TU / ml) using the following formula:

[0068] Lentiviral titer (TU / ml) = (q-PCR copies / ul × M × gDNA dilution factor) × 1000ml / virus volume of infected cell (ul).

[0069] M represents the elution volume after gDNA extraction, and 1000 indicates that the viral units are converted from μl to ml.

[0070] II. Extraction, differentiation, polarization, and CAR lentiviral transduction of BMDMs

[0071] 1. Bone marrow-derived macrophages (BMDMs) were prepared using existing methods. Cell differentiation was detected by F4 / 80 staining on day 6 to confirm successful differentiation of BMDMs.

[0072] 2. Transduction and culture: Four days after BMDMs differentiated, the cells were transduced with concentrated CAR lentivirus at a multiplicity of infection of 500 infection units / cell. After transduction, 20 ng / ml macrophage colony-stimulating factor (M-CSF) was added and the cells were cultured for another 2 days to complete the preparation of CAR-modified macrophages.

[0073] III. Flow cytometry analysis of post-polarized macrophage function

[0074] 1. Wash once with pre-cooled PBS containing 2% FBS, add 300ul of trypsin (6-well plate) and digest at 37°C for 5min. Add 500ul of PBS containing 2% FBS to stop digestion. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000rpm for 5min.

[0075] 2. Wash once with 2% FBS in PBS buffer, centrifuge at 1500 rpm for 5 min. Resuspend cells in 2% FBS PBS and count them, adjusting the cell concentration to 1-2 × 10⁻⁶ cells / mL. 7cells / mL (make sure the concentration is appropriate).

[0076] 3. Take 100 µL of cell suspension (approximately 1-2 × 10⁻⁶ cells / mL). 6 Add cells to a flow cytometry tube. Add pre-diluted LIVE / DEAD dye (dilute according to instructions), mix gently, incubate on ice in the dark for 10 minutes, add 500 μL of 2% FBS PBS to stop staining, centrifuge and discard the supernatant.

[0077] 4. Fc receptor blocking: Resuspend cells in 100 μL PBS, add 0.5 μL Fc Block (CD16 / 32 antibody, 0.5-1 μg / 100 μL), mix gently, and incubate on ice for 15 minutes.

[0078] 5. Surface antibody incubation: No washing is required. Directly add fluorescent antibodies against the surface markers CD11b / CD38 / CD86 / EGR2. Gently mix and incubate on ice in the dark for 30 minutes. After incubation, wash the cells with 1 mL of PBS buffer, centrifuge, and discard the supernatant.

[0079] 6. Cell fixation and permeabilization: Add 500 μL of 4% PFA to fix the cells, incubate at room temperature in the dark for 20 min, add 500 μL of PBS, centrifuge at 3000 rpm for 5 min and discard the supernatant. Add 500 μL of 0.1% Triton X-100 for permeabilization for 5 min, and gently vortex to mix.

[0080] 7. Wash the cells once with buffer, then stain the cells with anti-CD206 antibody diluted in PBS overnight.

[0081] 8. Washing and Analytical Processing: Wash cells twice with PBS buffer and resuspend cells in 100 μL of PBS buffer. Keep sample tubes on ice and protected from light before analysis.

[0082] like Figure 2 As shown, flow cytometry analysis of macrophage M2 phenotype polarization mediated by CX3CL1-CAR-M revealed that, compared with the control group, macrophages treated with CX3CL1-CAR-M exhibited a significantly increased mean fluorescence intensity (MFI) of CD206, the M2-specific marker; while the empty CAR-M (ΔCAR-M) treatment group showed no such effect, confirming that CX3CL1-CAR can effectively induce macrophage polarization towards the anti-inflammatory M2 phenotype. Figure 2 A, 2B).

[0083] Example 2: Validation of the effect of CX3CL1-CAR-M on a GJB2-related hereditary deafness model

[0084] 1. Model Construction

[0085] Experimental Model and Administration Method: This study adopted... Gjb2 fl / fl Sox2creER mouse construction Gjb2 Related hereditary deafness models, the experimental animals used include Gjb2 fl / fl Mice and Sox2creER mice, selected from sexually mature (2 months and older) mice. Gjb2 fl / fl Mice were crossed with Sox2-CreER mice, and then Sox2creER mice were... Gjb2 fl / wt The F1 generation mice were crossbred to obtain... Gjb2 fl / fl Sox2creER mice. To specifically activate Cre recombinase and achieve conditional knockout of Cx26 in cochlear supporting cells, mice were subcutaneously injected with tamoxifen (TMX) on the day of birth (P0) and the first day after birth (P1). TMX was prepared into a suspension with a final concentration of 7.5 mg / mL using 10% fat emulsion and heated in a 55°C water bath for 15 min before use. The total dose of TMX injected into the suckling mice was 1.5 mg / 10 g body weight, administered over two days. Gjb2 The knockout was performed. Immunofluorescence staining was conducted on frozen sections of the cochlea from mice 30 days after birth. Gjb2 The knockout of the protein Cx26 was investigated, and the results showed that, compared with wild-type mice, Cx26 was knocked out in all cochlear supporting cells (SCs) of the knockout group mice. Figure 3 , Figure 3 In this context, supporting cells (SC) and hair cells (HC) are used to represent hair cells.

[0086] 2. Cell infusion treatment

[0087] Intervention was performed by injecting CX3CL1-CAR-M via round window membrane. Figure 4A). For round window injection, mice should first be deeply anesthetized (0.2% pentobarbital solution, injection volume is 10 times the mouse's body weight in grams + 50ul). Hair should be removed and the skin prepared behind the ear, and the tongue should be pulled to one side to prevent suffocation. Then, make an incision of about 3mm at the edge of the root of the auricle. Use a curved vascular clamp to bluntly separate the adipose tissue and fix it with three hooks to form a triangular surgical field. Identify the transverse white facial nerve, Y-shaped blood vessels, and longitudinal muscle bundles. Expose the projection of the round window of the tympanic cavity wall at the angle between them. After pulling the muscle bundles apart, carefully break the surface mucosa. Drill a hole in the black corner area of ​​the upper right of the tympanic cavity with a 32G straight needle and enlarge it with a curved needle (avoiding the stapedius artery) until the stapedius artery, white ossification edge, and round window shadow are seen. Then, inject the round window obliquely to the upper right with a self-made micro-injection glass electrode. After feeling the breakthrough of the membrane, administer the drug slowly and evenly. After the injection is completed, slowly withdraw the electrode, fill the hole with a small piece of tissue, close the tissue layers one by one, and suture the wound with 6-0 sutures.

[0088] The specific operation timeline is shown in Table 1.

[0089] Table 1: Gjb2 Knockout mouse treatment timeline

[0090]

[0091] Note: P represents the number of days after the mouse is born, and the same applies below.

[0092] 3. Setting detection time points: Accurately record the cell infusion time and set 1 day (early stage), 7 days (mid stage), and 28 days (long stage) after infusion as subsequent detection time points.

[0093] Auditory brainstem response (ABR) assay results showed that CX3CL1-CAR-M improved hearing function in model mice, compared with the wild-type (WT) group. Gjb2 fl / fl The model group mice showed a significant increase in hearing threshold across the entire frequency range from 8kHz to 40kHz; after CX3CL1-CAR-M injection intervention, Gjb2 fl / fl The ABR threshold in mice decreased at all detection frequencies, with more significant hearing recovery at low frequencies, achieving a recovery of 25-30 dB of hearing function. Figure 4 B).

[0094] 4. Site-specific distribution detection

[0095] Sample collection: Three mice were selected from each group at each testing time point and sacrificed by cervical dislocation. Cochlear tissue samples were collected from each mouse.

[0096] 5. Immunofluorescence detection: Collected cochlear and spleen tissues were prepared into frozen sections, incubated with flag fluorescent antibody, and stained. After staining, the sections were observed under a laser confocal microscope to detect flag-positive cells. The distribution of CAR-M in the spiral ligament, spiral ganglion, and peripheral tissues was analyzed. The results showed that positive cells were distributed at the base of the cochlear basilar membrane and in the spiral ligament. Figure 4 C).

[0097] The expression regulation of Cx3cl1 in cochlear tissue and the effects of qPCR detection on cochlear tissue RNA showed that: Gjb2 fl / fl The mRNA expression level of Cx3cl1 in the model group was significantly higher than that in the WT group; after intervention with CX3CL1-CAR-M injection, Gjb2 fl / fl The expression level of Cx3cl1 in the mouse cochlea was significantly downregulated. Figure 4 D).

[0098] The effect of CX3CL1-CAR-M on alleviating cochlear immune cell infiltration: Immunofluorescence staining (CD45 / F-actin / DAPI) results showed that there were fewer CD45-positive immune cells in the cochlea of ​​the WT group; Gjb2 fl / fl In the model group, CD45-positive cell infiltration in the cochlea was significantly increased and exhibited a protruding amoeboid appearance; while in the CX3CL1-CAR-M injection group, CD45-positive immune cell infiltration was reduced, and macrophage morphology was similar to that of anti-inflammatory M2 macrophages, suggesting that this intervention can alleviate the local immune inflammatory response in the cochlea. Figure 4 E).

[0099] 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.

Claims

1. A chimeric antigen receptor targeting CX3CL1, characterized in that, It includes a single-chain antibody scFv that recognizes the human CX3CL1 antigen, a CD8α hinge region and a transmembrane domain, and an IL-4Rα intracellular co-stimulatory signal transduction domain; the amino acid sequence of the single-chain antibody scFv that recognizes the human CX3CL1 antigen is shown in SEQ ID NO.

1.

2. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequences of the CD8α hinge region and transmembrane domain are shown in SEQ ID NO.2; And / or, the amino acid sequence of the intracellular co-stimulatory signal transduction structure of the IL-4Rα is shown in SEQ ID NO.3; And / or, the chimeric antigen receptor further includes a fluorescent tag.

3. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor also includes a signal peptide, which is a granulocyte-macrophage colony-stimulating factor.

4. The chimeric antigen receptor according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

6.

5. The gene encoding the chimeric antigen receptor as described in any one of claims 1-4.

6. A recombinant vector, characterized in that, It contains the gene described in claim 5.

7. A chimeric antigen receptor-macrophage, characterized in that, It expresses the chimeric antigen receptor targeting CX3CL1 as described in any one of claims 1-4.

8. The chimeric antigen receptor-macrophage according to claim 7, characterized in that, It is an M2 type macrophage.

9. The use of the chimeric antigen receptor-macrophage as described in claim 7 or 8 in the preparation of a therapeutic drug for GJB2 mutation-related hereditary deafness.

10. The application according to claim 9, characterized in that, The drug has any of the following uses: 1) Improve hearing frequency thresholds; 2) Improve the ABR threshold.

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