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US-20260124151-A1 - OGA-OVEREXPRESSED EXOSOME SYSTEM, PREPARATION METHOD THEREFOR AND USE THEREOF

US20260124151A1US 20260124151 A1US20260124151 A1US 20260124151A1US-20260124151-A1

Abstract

A bionic OGA-overexpressed exosome system for treating fatty liver-related liver cancer based on glucose metabolism and a preparation method therefor are provided. The O-GlcNAcase (OGA) sequence is transfected into mesenchymal stem cells by lentivector transfection, and exosomes are then obtained by ultracentrifugation for the treatment of fatty liver-related liver cancer. The exosome biomimetic system of the invention have the ability to target migration, low immunogenicity and improved ability to transport bioactive substances, normalize the interrupted glucose metabolism, reduce the endoplasmic reticulum stress and inhibit the epithelial-mesenchymal transition signal transduction. In vitro and in vivo experiments proved that MSCOGA-EXOs can effectively target cancer cells in vitro, reduce the abnormal O-GlcNAc modifications, and inhibit the malignant behavior of tumor. After reaching fatty liver-related hepatocellular carcinoma in vivo, the MSCOGA-EXOs can reduce the O-GlcNAc modification level and restore the damaged metabolism, thus inhibiting the progression of cancer.

Inventors

  • Jinglin WANG
  • Haozhen REN

Assignees

  • NANJING DRUM TOWER HOSPITAL

Dates

Publication Date
20260507
Application Date
20241227
Priority Date
20241107

Claims (8)

  1. 1 . An O-GlcNAcase (OGA)-overexpressed exosome system, wherein the OGA-overexpressed exosome system is obtained by a lentivector transfection for transfecting an OGA sequence into human mesenchymal stem cells (MSCs), followed by an ultracentrifugation, wherein exosomes are spherical in shape, with an average diameter of 120 nm-125 nm.
  2. 2 . A preparation method for the OGA-overexpressed exosome system according to claim 1 , comprising the following steps: 1) a preparation of the human MSCs; 2) a preparation of OGA-overexpressed human MSCs (MSC OGA ): diluting OGA-overexpressed lentivector particles with a medium by the lentivector transfection, adding the human MSCs into the medium for a co-incubation in an incubator, and adding purinomycin to select infected cells expressing resistance genes until no cells die; and 3) a preparation of OGA-overexpressed exosomes: culturing the human MSCs in a medium lacking the exosomes, and performing a primary centrifugation and a secondary centrifugation on the medium lacking the exosomes to obtain the OGA-overexpressed exosome system.
  3. 3 . The preparation method according to claim 2 , wherein a titer of the OGA-overexpressed lentivector particles in the step 2) is 10 9 copies/mL.
  4. 4 . The preparation method according to claim 2 , wherein the medium in the step 2) is a Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) medium, and a volume ratio of the OGA-overexpressed lentivector particles to the DMEM/F12 medium is 1:1000.
  5. 5 . The preparation method according to claim 2 , wherein a concentration of the human MSCs in the step 2) in the medium is 10 6 cells/mL.
  6. 6 . The preparation method according to claim 2 , wherein a concentration of the purinomycin added to the medium in the step 2) is 2 μg/mL.
  7. 7 . The preparation method according to claim 2 , wherein the primary centrifugation in the step 3) is: at a room temperature, centrifuging the medium lacking the exosomes at 300×g for 10 min, then at 2000×g for 10 min, and then at 10,000×g for 30 min, and resuspending pellet particles in phosphate buffer saline (PBS).
  8. 8 . The preparation method according to claim 2 , wherein the secondary centrifugation in the step 3) is: centrifuging pellet particles obtained from the primary centrifugation at 100000×g, 4° C. for 70 min, and removing a supernatant and obtaining pellets, wherein the pellets are the exosomes.

Description

CROSS REFERENCE TO THE RELATED APPLICATIONS This application is based upon and claims priority to Chinese Patent Application No. 2024115844035, filed on Nov. 7, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD The present invention belongs to the field of biomedical materials and, in particular, relates to a bionic OGA-overexpressed exosome system for treating fatty liver-related liver cancer based on glucose metabolism and a preparation method therefor and use thereof. BACKGROUND As the prevalence of obesity increases, the proportion of fatty liver-related liver cancer in hepatocellular carcinoma also increase. Surgical resection is the main clinical treatment for this type of tumor, and drug therapy is also used as an intervention strategy to prevent tumor recurrence and improve patient survival. Drug therapies often target multiple signaling pathways to promote the death of tumor cells. Specifically, intervention through metabolic pathways plays an important role in inducing tumor cell apoptosis and inhibiting tumor metastasis. In these pathways, the regulation of glucose metabolism, especially in the context of fatty liver and overnutrition, is essential to inhibit tumor growth. As a result, many anti-tumor treatments targeting glucose metabolism have been developed. However, despite significant advances, mechanism of glucose metabolism in fatty liver-related liver cancer is still poorly understood. Therefore, the development of effective treatment strategies based on glucose metabolism is still worth looking forward to. By sequencing the clinical excision samples of fatty liver-related liver cancer, it was found that the O-GlcNAcase (OGA) in these tumor tissues was significantly reduced. Previous studies have shown that a sharp increase in O-linked N-acetylglucosamine (O-GlcNAc) modification plays a key role in abnormal tumor glucose metabolism. However, the underlying mechanism of O-GlcNAc modification remains unexplored to great extent, leading to unsatisfactory results when targeting O-GlcNAc with drugs alone. It was found that OGA, a key enzyme that dynamically and temporarily regulates O-GlcNAc, was down-regulated in fatty liver-related liver cancer. By increasing the OGA level, abnormal O-GlcNAc modifications can be reduced, thereby restoring disrupted energy metabolism and alleviating endoplasmic reticulum (ER) stress. It is believed that the regulation of OGA simultaneously regulates the function of metabolic reprogramming and energy-related organelles, providing a promising pathway for targeted therapy of liver cancers. Compared with conventional mesenchymal stem cells, exosomes have the ability to target migration, low immunogenicity and efficient transport of bioactive substances, so they are adaptable and controllable in therapeutic applications. Therefore, to confirm this hypothesis, an OGA-overexpressed mesenchymal stem cell (MSCOGA) exosome system (MSCOGA-EXOs) is developed for the treatment of fatty liver-related liver cancer. SUMMARY Objective of invention: In order to solve the above technical problems, the invention provides a bionic OGA-overexpressed exosome system (MSCOGA-EXOs) for the treatment of fatty liver-related liver cancer based on glucose metabolism. The exosome system is prepared by lentivector transfection and ultracentrifugation. The method is simple, versatile and convenient for large-scale production. The technical solution: An OGA-overexpressed exosome system (MSCOGA-EXOs) of the invention is obtained by lentivector transfection for transfecting an O-GlcNAcase (OGA) sequence into human mesenchymal stem cells (MSCs), followed by ultracentrifugation, wherein the exosomes are spherical in shape, with an average diameter of 120 nm-125 nm. The specific steps are as follows: 1) preparation of human MSCs;2) preparation of OGA-overexpressed human mesenchymal stem cells (MSCOGA): diluting OGA-overexpressed lentivector particles with a medium by lentivector transfection, adding the MSCs into the medium for co-incubation in an incubator, and adding purinomycin to select infected cells expressing resistance genes until no cells die; and3) preparation of OGA-overexpressed exosomes (MSCOGA-EXOs): culturing the human MSCs in a medium lacking exosomes, and performing primary centrifugation and secondary centrifugation on the medium to obtain the exosomes system. Preferably, the titer of the OGA-overexpressed lentivector particles in step 2) is 109 copies/mL. Preferably, the medium in step 2) is Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) medium and a volume ratio of the OGA-overexpressed lentivector particles to the DMEM/F12 medium is 1:1000. Preferably, the concentration of the human MSCs in the medium is 106 cells/mL. Preferably, the concentration of the purinomycin added to the medium in step 2 is 2 μg/mL. Preferably, the primary centrifugation in step 3 is: at room temperature, centrifuging the medium at 300×g for 10 min, then