An engineered enucleated mesenchymal stem cell, its preparation method and application

Abstract

An engineered enucleated mesenchymal stem cell (EMC) and its preparation method and application are disclosed, which can effectively solve the problem of preparing engineered enucleated EMC and realize its application in the preparation of drugs for treating herpesvirus infections. The steps are as follows: Lentiviral transfection is used to obtain EMC overexpressing Nectin-1, the overexpression being achieved through genetic engineering, including transfection of EMC using a lentiviral vector or adenovirus vector; then, enucleation is performed using discontinuous density gradient centrifugation to obtain the engineered enucleated EMC. The engineered enucleated EMC of this invention acts as "bait" to competitively capture viruses; the enucleation design fundamentally eliminates the risk of viral replication and leakage within cells; and the captured viruses are subsequently cleared along with the engineered enucleated EMC's own programmed apoptosis and subsequent macrophage phagocytosis, thereby achieving safe and efficient virus capture and physical clearance.

Description

Technical Field

[0001] This invention relates to the fields of cell engineering and antiviral therapy, and in particular to an engineered enucleated mesenchymal stem cell, its preparation method, and its application. Background Technology

[0002] Herpes simplex encephalitis (HSE) is the most common sporadic, acute viral encephalitis caused by herpes simplex virus type 1 infection, with a mortality rate as high as 20%-30%. More than half of the survivors suffer from severe neurological sequelae, such as cognitive impairment and epilepsy. Currently, the first-line clinical treatment for herpes simplex encephalitis is the nucleoside analog acyclovir (ACV). However, this drug faces serious challenges: firstly, ACV requires activation by viral thymidine kinase (TK) phosphorylation to exert its therapeutic effect, and long-term use can easily induce mutations in the TK gene or DNA polymerase gene, leading to the emergence of drug-resistant strains and significantly reducing the treatment effect; secondly, to cross the blood-brain barrier and achieve an effective therapeutic concentration, high-dose intravenous administration is required, which can cause serious adverse reactions such as nephrotoxicity and bone marrow suppression. To overcome these problems, in recent years, antiviral strategies based on "decoy" receptors have provided new ideas for antiviral drugs. One promising approach is to use vesicles that highly express viral receptors as "bait" to competitively capture viruses and prevent them from infecting host cells. However, nanovesicles have significant limitations: they can only bind to viruses through membrane surface receptors and cannot completely internalize and isolate the virus, leaving viral glycoproteins exposed and potentially leading to secondary infection; furthermore, they lack active inflammatory chemotaxis, and their targeting ability in vivo is still insufficient for clinical application. Therefore, there is an urgent clinical need to develop novel antiviral drugs that are highly effective against viruses while also possessing precise targeting and good safety profiles.

[0003] Mesenchymal stem cells (MSCs) possess advantages such as low immunogenicity, inflammatory chemotaxis, and ease of engineering, and have been used to treat various diseases. Nectin-1 is a key receptor for herpes simplex virus type 1 (HSV-1) to invade host cells, mediating viral adhesion and membrane fusion. It is envisioned that MSCs highly expressing Nectin-1 could serve as ideal viral "bait." However, directly using intact, nucleated MSCs carries potential risks: captured viruses may complete their replication cycle intracellularly, leading to viral progeny production and leakage, which could exacerbate infection. Simultaneously, the long-term survival and uncontrolled differentiation of allogeneic MSCs raise safety concerns, limiting the expansion of stem cell applications in antiviral therapy. Therefore, how to construct and obtain novel stem cell-based antiviral drugs remains a pressing technical problem to be solved in this field. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide an engineered enucleated mesenchymal stem cell, its preparation method and application, which can effectively solve the problem of preparing engineered enucleated mesenchymal stem cells and realize their application in the preparation of drugs for treating herpesvirus infections.

[0005] The technical solution provided by this invention is: an engineered enucleated mesenchymal stem cell, which is an enucleated mesenchymal stem cell with high expression of Nectin-1, with an average particle size of 8-12 μm, and retains complete cell membrane structure and various organelles such as lysosomes, Golgi apparatus and endoplasmic reticulum; the high expression of Nectin-1 is achieved by capturing viruses through the high expression of Nectin-1 on its surface and promoting viral clearance through programmed apoptosis; Its preparation method is achieved by the following steps: S1. Mesenchymal stem cells overexpressing Nectin-1 were obtained through lentiviral transfection. Overexpression was achieved through genetic engineering, including transfection of mesenchymal stem cells using a lentiviral vector or adenovirus vector. The lentiviral transfection involved transfecting mesenchymal stem cells at a density of 3-5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 cells / well in six-well plates. When the confluence reached 30%-50%, culture medium containing lentiviral vector and 1-10 μg / mL polybrene or other infection-promoting agents was added. After 16-24 h of transfection, the medium was replaced with fresh medium and cultured for another 48-72 h. Stable clones were then further screened using antibiotics. The antibiotic used was puromycin, with a screening concentration of 1-10 μg / mL and a screening time of 72-120 h. S2. The mesenchymal stem cells obtained in S1 are enucleated by discontinuous density gradient centrifugation to obtain the engineered enucleated mesenchymal stem cells. The application of engineered enucleated mesenchymal stem cells prepared by the above method in the preparation of drugs for treating herpesvirus infections, wherein the herpesvirus is herpes simplex virus type 1.

[0006] This invention constructs engineered enucleated mesenchymal stem cells that highly express the herpes simplex virus type 1 receptor Nectin-1, enabling them to competitively capture the virus as "bait." The enucleation design fundamentally eliminates the risk of viral replication and leakage within cells. The captured virus is then eliminated along with the engineered enucleated mesenchymal stem cells through programmed apoptosis and subsequent macrophage phagocytosis, thus achieving safe and efficient virus capture and physical clearance. This is a major innovation in the fields of cell engineering and antiviral therapy, and has practical clinical and promotional application value. Attached Figure Description

[0007] Figure 1This is a Western Blot result of Nectin-1 protein expression in different cells in Experiment 1 of this invention; Figure 2 These are laser confocal microscopy images of the cell morphology and Nectin-1 expression of engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells in Experiment 1 of this invention. Figure 3 This is an immunofluorescence assay result of the ability of engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells to capture HSV-1 viral particles in Experiment 2 of this invention. Figure 4 These are confocal microscopy images of virus competitive capture in the co-culture system of engineered enucleated mesenchymal stem cells and HT-22 neuronal cells in Experiment 2 of this invention. Figure 5 This is a graph showing the qPCR results of viral DNA copy number detection in the supernatant of engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells infected with HSV-1 at different time points in Experiment 3 of this invention. Figure 6 These are confocal microscopy images and flow cytometry results of the cell morphology and apoptosis of engineered enucleated mesenchymal stem cells that captured HSV-1 at different time points in Experiment 4 of this invention. Figure 7 These are confocal microscopy images of the phagocytic activity of macrophages on engineered enucleated mesenchymal stem cells or microvesicles that capture HSV-1 in Experiment 4 of this invention. Figure 8 These are images showing the results of immunofluorescence and pathological H&E staining of virus distribution in the brain tissue of HSE model mice in different treatment groups in Experiment 5 of this invention. Detailed Implementation

[0008] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0009] The present invention is illustrated in the following specific embodiments: An engineered enucleated mesenchymal stem cell is an enucleated mesenchymal stem cell with high expression of Nectin-1, with an average particle size of 8-12 μm, and retains complete cell membrane structure and various organelles such as lysosomes, Golgi apparatus and endoplasmic reticulum; the high expression of Nectin-1 is to capture viruses through the high expression of Nectin-1 on its surface and promote viral clearance through programmed apoptosis. Its preparation method is achieved by the following steps: S1. Mesenchymal stem cells overexpressing Nectin-1 were obtained through lentiviral transfection. Overexpression was achieved through genetic engineering, including transfection of mesenchymal stem cells using a lentiviral vector or adenovirus vector. The lentiviral transfection involved transfecting mesenchymal stem cells at a density of 3-5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 cells / well in six-well plates. When the confluence reached 30%-50%, culture medium containing lentiviral vector and 1-10 μg / mL polybrene or other infection-promoting agents was added. After 16-24 h of transfection, the medium was replaced with fresh medium and cultured for another 48-72 h. Stable clones were then further screened using antibiotics. The antibiotic used was puromycin, with a screening concentration of 1-10 μg / mL and a screening time of 72-120 h. S2. The mesenchymal stem cells obtained in S1 are enucleated using discontinuous density gradient centrifugation to obtain the engineered enucleated mesenchymal stem cells; the discontinuous density gradient centrifugation method is as follows: a) Prepare a density gradient system containing polysucrose solutions of different concentrations, the concentration range of which is 10% to 50%; the density gradient system includes at least 4-6 gradients from bottom to top, including 0-5 mL layers of polysucrose solutions with concentrations of 20%-30%, 15%-20%, and 12%-17%, respectively, and cytochalasin B, the working concentration of which is 5-20 μg / mL, and the incubation time is 0-30 min; The refractive index of the sucrose solution layer with a concentration of 20%-30% ranges from 1.370 to 1.391, the refractive index of the sucrose solution layer with a concentration of 15%-20% ranges from 1.360 to 1.370, and the refractive index of the sucrose solution layer with a concentration of 12%-17% ranges from 1.354 to 1.365. b) Add the cell suspension to the top of the density gradient system and ultracentrifuge at 20,000-30,000 rpm for 60-120 min at 25-37 ℃; the cell density of the cell suspension is 0.5–2.0 × 10⁻⁶. 7 cells / mL; c) After centrifugation, collect the enucleated cells located at the interface layer of 12%-17% sucrose solution and wash them 3-5 times with serum-free culture medium.

[0010] The application of engineered enucleated mesenchymal stem cells prepared by the above method in the preparation of drugs for treating herpesvirus infections, wherein the herpesvirus is herpes simplex virus type 1.

[0011] The infections caused by herpes simplex virus type 1 include, but are not limited to, herpes simplex encephalitis (HSE), and also cover HSV-1 related infectious diseases such as oral herpes (OH), herpetic gingivostomatitis (HG), herpetic keratitis (HK), genital herpes (GH), herpetic eczema (HE), and neonatal herpes (NH).

[0012] The preparation method of this invention is scientifically sound and reasonable, with abundant raw materials, resulting in high-quality and stable products. It constructs engineered enucleated mesenchymal stem cells that highly express the herpes simplex virus type 1 receptor Nectin-1, using these cells as "bait" to competitively capture the virus. The enucleation design fundamentally eliminates the risk of intracellular viral replication and leakage. The captured virus is subsequently eliminated through programmed apoptosis of the engineered enucleated mesenchymal stem cells and subsequent phagocytosis by macrophages, thus achieving safe and efficient virus capture and physical clearance. Repeated experiments have yielded consistent results, demonstrating that the product is stable and reliable, possessing strong practical application value. Relevant experimental data are as follows: Experiment 1: Preparation of engineered enucleated mesenchymal stem cells and Nectin-1 expression experiment This experiment aimed to construct engineered mesenchymal stem cells stably overexpressing Nectin-1 and to prepare their enucleated cells. Healthy mouse mesenchymal stem cells were harvested and processed at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 g / well in 6-well plates. When the cell confluence reached 30%-50%, the medium was replaced with medium containing the lentiviral vector pCDH-CAG-Nectin1-EF1-Puro (MOI=20) and 8 μg / mL polygel for transfection. Sixteen h after transfection, the medium was replaced with complete medium, and the cells were cultured for another 48 h. Subsequently, selection medium containing 4 μg / mL puromycin was added for stress selection until the untransfected control group cells died completely, yielding engineered mesenchymal stem cells stably overexpressing Nectin-1.

[0013] Engineered enucleated mesenchymal stem cells (MSCs) were prepared using a discontinuous density gradient centrifugation method. Different concentrations of sucrose solutions were prepared using a culture medium containing 10 μg / mL cytochalasin B. In ultracentrifuge tubes, 2 mL of 25%, 2 mL of 17%, 0.5 mL of 16%, 0.5 mL of 15%, and 2 mL of 12.5% ​​sucrose solutions were added sequentially to create a density gradient, and the mixture was incubated overnight at 37°C. Engineered MSCs that had grown to 90% confluence were digested and collected, resuspended in a 12.5% ​​sucrose solution containing cytochalasin B, and the cell concentration was adjusted to 1.0 × 10⁻⁶ cells / mL. 7Cells / mL; 3.2 mL of cell suspension was added to the top of the gradient solution, and after replenishing the culture medium, the cells were centrifuged at 26,000 rpm for 60 min at 31℃. After centrifugation, engineered enucleated mesenchymal stem cells with a 15%-17% polysucrose interface layer were collected, washed three times with serum-free culture medium, and then resuspended in DMEM high glucose culture medium containing 10% serum for later use.

[0014] To verify Nectin-1 expression, Western blotting was used. Microvascular endothelial cells from bEnd.3 mice, BV2 microglia, HT-22 hippocampal neurons, and engineered mesenchymal stem cells were collected, and total protein was extracted using RIPA lysis buffer. After quantification using the dicaprolactone assay, SDS-PAGE electrophoresis was performed, followed by membrane transfer. The membrane was blocked with 5% skim milk, and then incubated overnight (12-18 h) at 4°C with Nectin-1 primary antibody and β-actin internal control antibody, respectively. After washing, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h, followed by chemiluminescence enhancement. (See attached image.) Figure 1 As shown, Western blotting results confirmed that the gray value of the Nectin-1 protein band in engineered mesenchymal stem cells was significantly higher than that in bEnd.3, BV2, and HT-22 cells, making them suitable as primitive cell vectors for engineered modification.

[0015] Morphological characterization of engineered enucleated mesenchymal stem cells was performed using laser confocal microscopy: Engineered enucleated mesenchymal stem cells were seeded into confocal culture dishes and cultured for 4-6 hours until adherence. After fixation, they were fixed with 4% paraformaldehyde and incubated overnight at 4°C with anti-Nectin-1 primary antibody. Then, they were incubated for 1 hour at room temperature in the dark with Alexa Fluor 488-labeled secondary antibody. Simultaneously, the cytoskeleton was stained with Actin-Tracker Red, and the nuclei were stained with DAPI, as shown in the attached image. Figure 2 As shown, the confocal images show that the engineered enucleated mesenchymal stem cells only exhibit a red cytoskeleton structure, with no blue nuclear fluorescence signal, confirming that the cell nucleus has been successfully removed and the cell morphology is intact. More importantly, the enucleation process does not affect the stability of Nectin-1 expression on the membrane surface.

[0016] Experiment 2: In vitro virus capture capacity experiment of engineered enucleated mesenchymal stem cells This experiment evaluated the in vitro virus capture ability of engineered enucleated mesenchymal stem cells. A separate virus capture experiment was conducted: engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells were seeded separately into 24-well plates and cultured for 24 h. The culture medium was then discarded, and 100 TCID⁻¹ was added to each well. 50HSV-1 viral suspension was adsorbed at 37°C for 2 h. Unbound virus was removed by washing three times with pre-cooled PBS buffer (pH 7.4), fixed with 4% paraformaldehyde for 15 min, and incubated overnight at 4°C with anti-HSV-1 gD primary antibody. Then, it was incubated at room temperature in the dark with Alexa Fluor 488-labeled secondary antibody for 1 h. The cytoskeleton and nucleus were labeled with Actin-Tracker Red and DAPI, respectively. After mounting, the samples were observed under a laser confocal microscope, as shown in the attached figure. Figure 3 As shown, immunofluorescence images revealed strong green viral fluorescence signals in both engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells, confirming that engineered enucleated mesenchymal stem cells retained the same viral capture capacity as engineered mesenchymal stem cells.

[0017] Competitive virus capture experiment: HT-22 neurons and engineered enucleated mesenchymal stem cells were co-cultured at a 1:1 ratio, and 100 TCID45 was added. 50 HSV-1 infection for 2 hours; washing, fixation, and immunofluorescence staining were performed using the same method as above. (See attached image) Figure 4 As shown, confocal images reveal that viral signals are mainly distributed in engineered enucleated mesenchymal stem cells without nuclei, while viral signals are significantly reduced in HT-22 neurons, demonstrating that engineered enucleated mesenchymal stem cells have competitive virus capture capabilities.

[0018] Experiment 3: Risk Detection Experiment for Replication and Leakage of Engineered Enucleated Mesenchymal Stem Cells After Viral Capture This experiment investigated the replication and leakage risks of engineered enucleated mesenchymal stem cells after viral capture. Engineered mesenchymal stem cells and engineered enucleated mesenchymal stem cells were cultured at a ratio of 6 × 10⁻⁶. 5 Seeds were planted at a density of cells / well in 6-well plates, and after attachment to the walls, were sprayed with 100 TCID50. 50 HSV-1 infection was performed 2 h post-infection, followed by fresh culture medium and continued culturing. Cell supernatant was collected at 12, 24, 36, and 48 h post-infection, and total viral DNA was extracted using a viral DNA extraction kit. Using the extracted DNA as a template, qPCR was performed to detect the α0, α27, and UL47 genes of HSV-1. GAPDH was used as an internal control, and the viral genome copy number was calculated using the 2^–ΔΔCt method. The primer sequences for detecting these genes using quantitative real-time PCR are shown in Table 1, and the results are attached. Figure 5 As shown, qPCR results indicated that the viral DNA copy number in the supernatant of the engineered enucleated mesenchymal stem cell group at 24, 36, and 48 h was significantly lower than that in the engineered mesenchymal stem cell control group, confirming that engineered enucleated mesenchymal stem cells can effectively block viral replication and release.

[0019] Experiment 4: Experiment on the apoptosis and macrophage phagocytosis mechanisms of engineered enucleated mesenchymal stem cells This experiment investigated apoptosis and its clearance mechanism in engineered enucleated mesenchymal stem cells. Apoptotic morphology was observed: engineered enucleated mesenchymal stem cells were seeded in 24-well plates and inoculated with 100 TCID45 solution. 50 Cells were cultured for 2 hours after HSV-1 infection. Cells were fixed at 24, 48, 60, 72, and 80 hours post-infection and immunofluorescence stained with Actin-Tracker Red and anti-HSV-1 gD antibody; see attached... Figure 6 As shown in Figure A, under a laser confocal microscope, engineered enucleated mesenchymal stem cells 60 h post-infection exhibit apoptotic morphology such as cell body contraction and membrane bubbling, and viral signals remain confined to the intracellular space until 80 h.

[0020] Apoptosis was quantitatively detected by flow cytometry: engineered enucleated mesenchymal stem cells were cultured at 5 × 10⁻⁶ cells / mL. 5 HSV-1 infected cells were seeded at a density of 1 cell / well in 6-well plates, with separate HSV-1 infected and uninfected control groups. Sixty h after infection, cells were digested and collected, and analyzed by flow cytometry according to the Annexin V-FITC / PI apoptosis detection kit instructions. See attached... Figure 6 As shown in Figure B, flow cytometry results showed that the proportion of Annexin V-positive cells in the infected group reached 64.35%, which was similar to the positive proportion of engineered enucleated mesenchymal stem cells without virus (60.04%). This preliminarily confirms that virus capture in engineered enucleated mesenchymal stem cells does not affect the occurrence of programmed apoptosis.

[0021] Macrophage phagocytosis assay: Engineered mesenchymal stem cells were resuspended in PBS and sequentially passed through polycarbonate membranes with pore sizes of 5 μm, 1 μm, and 400 nm for two cycles of compression. The cells were then purified and concentrated using a 100 kDa ultrafiltration centrifuge tube, and finally washed three times with PBS to obtain vesicles. 100 TCID⁻¹ was added to engineered enucleated mesenchymal stem cells and vesicles containing equal amounts of protein. 50 Viral suspensions were incubated for 2 hours. Virus conjugates were directly collected from the microvesicle group, while engineered enucleated mesenchymal stem cell group was cultured for 60 hours to simulate cellular dynamics. Subsequently, engineered enucleated mesenchymal stem cells and microvesicles with captured viruses were labeled with DiD, and BV-2 microglia were labeled with DiI. The labeled engineered enucleated mesenchymal stem cells or microvesicles were co-cultured with BV-2 cells for 4 hours. After fixation, the virus was visualized using anti-HSV-1 gD antibody and Alexa Fluor488-labeled secondary antibody. Cell nuclei were counterstained with DAPI, and observed under a laser confocal microscope. (See attached image). Figure 7 As shown, confocal images reveal that macrophages have a significantly higher phagocytic efficiency for enucleated mesenchymal stem cells than microvesicles, and viral signals from the enucleated engineered enucleated mesenchymal stem cells also enter the macrophages.

[0022] Experiment 5: Therapeutic effect of engineered enucleated mesenchymal stem cells in a primary HSE mouse model This experiment evaluated the therapeutic effect of engineered enucleated mesenchymal stem cells on primary herpes encephalitis. Six- to eight-week-old SPF-grade female BALB / c mice were anesthetized with isoflurane and injected with 40 μL of a solution containing 1 × 10³ TCID2 into the right side of the skull. 50 HSE model was established using HSV-1 viral suspension. Twenty-four hours after modeling, mice were randomly divided into four groups: PBS group, Vesicles group, Acyclovir (ACV) group (150 mg / kg / day), and engineered enucleated mesenchymal stem cell (eMSCs) group (3 × 10⁻⁶ mcg / kg / day). 6 (6 animals per group). The engineered enucleated mesenchymal stem cell group and the microvesicle group were injected via tail vein on days 1 and 3 post-infection, the acyclovir group was injected intraperitoneally for 7 consecutive days, and the PBS group was injected with an equal volume of PBS. Mice were sacrificed on day 14 post-infection, and brain tissue was collected for evaluation. Partial brain tissue was fixed in 4% paraformaldehyde and prepared into paraffin sections. Immunofluorescence staining with anti-HSV-1 gD antibody was performed, and cell nuclei were counterstained with DAPI, as shown in the attached image. Figure 8 As shown in Figure A, immunofluorescence images revealed the lowest viral signal intensity in the brain of the engineered enucleated mesenchymal stem cell group, significantly lower than that of the PBS group and the microvesicle group. Simultaneously, H&E staining was performed on the corresponding brain tissue sections, and the results are shown in the attached figure. Figure 8 As shown in Figure B, H&E staining results indicate that the pathological damage and inflammatory infiltration of brain tissue in the engineered enucleated mesenchymal stem cell group were significantly reduced compared to the PBS group and the microvesicle group.

[0023] In summary, the beneficial effects of this invention are: 1. This invention utilizes enucleated mesenchymal stem cells that highly express Nectin-1 as "bait," which has high specificity and high virus capture efficiency, enabling the elimination of existing viruses; the enucleation design fundamentally eliminates the risk of viruses using the host cell nucleus for replication and spread, achieving safe isolation. 2. The engineered enucleated mesenchymal stem cells of the present invention, while maintaining the inherent inflammatory homing of mesenchymal stem cells, have a stronger "inflammatory enrichment" ability in the inflammatory microenvironment of herpes simplex virus type 1 encephalitis due to their smaller size and preservation of the original membrane structure. This enables active and efficient targeting at the lesion site, thereby significantly improving local antiviral efficacy and significantly reducing the risk of exposure to peripheral organs. 3. This invention provides a clear and optimized operating range for the preparation method of engineered enucleated mesenchymal stem cells, especially for parameters of lentiviral transfection (such as cell density, polybrene concentration, and puromycin screening window) and key steps of enucleation (such as sucrose gradient composition, refractive index range, centrifugation conditions, and cytochalasin B pretreatment), ensuring the reproducibility, stability, and high cell yield of the preparation process. 4. After completing their task, the engineered enucleated mesenchymal stem cells of this invention can be naturally cleared by the body's macrophages through programmed apoptosis, avoiding the risk of long-term retention that may exist in traditional cell therapy, and simultaneously achieving the final degradation of the captured virus. More importantly, this natural macrophage clearance pathway theoretically does not cause the risk of drug resistance caused by current antiviral drugs (nucleotide analogs, such as acyclovir).

[0024] 5. This invention clearly defines the use of engineered enucleated mesenchymal stem cells to prepare a treatment for infections caused by herpes simplex virus type 1, providing a novel and promising biological therapeutic drug for solving this clinical problem. The application target is clear, the market orientation is obvious, and the application value is great.

[0025] As can be clearly seen from the above, this invention constructs engineered enucleated mesenchymal stem cells that highly express the herpes simplex virus type 1 receptor Nectin-1, enabling them to competitively capture the virus as "bait." The enucleation design fundamentally eliminates the risk of viral replication and leakage within cells. The captured virus is then cleared along with the engineered enucleated mesenchymal stem cells through programmed apoptosis and subsequent macrophage phagocytosis, thereby achieving safe and efficient virus capture and physical clearance. This provides drug-based technical support for the treatment of encephalitis caused by herpes simplex virus type 1, representing a major innovation in the fields of cell engineering and antiviral therapy, and possessing practical clinical and promotional application value.

[0026] Finally, it should be noted that the above experiments are merely examples to intuitively illustrate the technical concept of the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that within the technical framework disclosed in the present invention, various forms of changes or equivalent substitutions can be made, and all such changes or substitutions are within the protection scope of the present invention.

Claims

1. An engineered enucleated mesenchymal stem cell, characterized in that, The enucleated mesenchymal stem cells with high expression of Nectin-1 have an average particle size of 8-12 μm and retain the complete cell membrane structure and various organelles such as lysosomes, Golgi apparatus and endoplasmic reticulum.

2. The engineered enucleated mesenchymal stem cell of claim 1, wherein, The high expression of Nectin-1 is that the virus is captured by Nectin-1 highly expressed on the surface of the enucleated mesenchymal stem cells, and the virus clearance is promoted by programmed cell death.

3. The method of producing the engineered enucleated mesenchymal stem cell of claim 1 or 2, characterized in that, The high expression of Nectin-1 is achieved by the following steps: S1, mesenchymal stem cells overexpressing Nectin-1 are obtained by lentiviral transfection, and the overexpression is achieved by genetic engineering, which includes transfecting mesenchymal stem cells using a lentiviral vector or an adenoviral vector; the lentiviral transfection is as follows: mesenchymal stem cells are seeded in a six-well plate at a density of 3-5 x 10 5 cells per well, and when the cell confluence reaches 30%-50%, a culture medium containing a lentiviral vector and 1-10 μg / mL polybrene or other infection-promoting reagents is added, and after 16-24 h of transfection, fresh culture medium is replaced for continued culture, and after 48-72 h of infection, stable clones are further selected by antibiotics; the antibiotics are puromycin, the selection concentration is 1-10 μg / mL, and the selection time is 72-120 h; S2, the mesenchymal stem cells obtained in S1 are subjected to enucleation treatment by a discontinuous density gradient centrifugation method to obtain the engineered enucleated mesenchymal stem cells.

4. The method of claim 3, wherein the engineered enucleated mesenchymal stem cell is prepared by, The discontinuous density gradient centrifugation method is as follows: a) preparing a density gradient system containing polyfructose solutions with different concentrations, the concentration range of the polyfructose solutions covering 10% to 50%; the density gradient system comprises 4-6 gradient layers from bottom to top, at least including 0-5 mL of polyfructose solution layers with concentrations of 20%-30%, 15%-20% and 12%-17%, and cytochalasin B, the working concentration of cytochalasin B being 5-20 μg / mL, and the incubation time being 0-30 min; wherein the refractive index of the polyfructose solution layer with a concentration of 20%-30% ranges from 1.370 to 1.391, the refractive index of the polyfructose solution layer with a concentration of 15%-20% ranges from 1.360 to 1.370, and the refractive index of the polyfructose solution layer with a concentration of 12%-17% ranges from 1.354 to 1.365; b) adding the cell suspension to the top of the density gradient system and performing ultracentrifugation at 20,000-30,000 rpm for 60-120 min at 25-37 °C; the cell density of the cell suspension is 0.5-2.0 x 10 7 cells / mL; c) after centrifugation, the enucleated cells located at the interface layer of the polyfructose solution with a concentration of 12%-17% are collected, and washed 3-5 times with a serum-free medium.

5. Use of the engineered enucleated mesenchymal stem cells of claim 1 or 2 in the preparation of a drug for treating an infection of a virus of the Herpesviridae family.

6. Use according to claim 5, characterized in that, The virus of the Herpesviridae family is herpes simplex virus type 1.

7. Use according to claim 5 or 6, characterized in that, The infection of the virus of the Herpesviridae family is an infection caused by herpes simplex virus type 1.

8. Use according to claim 7, characterized in that, The infection caused by herpes simplex virus type 1 is herpes simplex encephalitis (HSE), oral herpes (OH), herpes gingivostomatitis (HG), herpes keratitis (HK), genital herpes (GH), herpes eczema (HE) and neonatal herpes (NH).

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