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CN-121991896-A - Extracellular vesicles loaded with WNT and R-spondin proteins and application thereof

CN121991896ACN 121991896 ACN121991896 ACN 121991896ACN-121991896-A

Abstract

The present disclosure relates to extracellular vesicles loaded with WNT and RSPO proteins and uses thereof. Specifically, the present disclosure provides extracellular vesicles loaded with WNT and RSPO proteins simultaneously, and a preparation method and application of the extracellular vesicles.

Inventors

  • YANG LINGYAN
  • ZENG YONGSHENG

Assignees

  • 广州国家实验室

Dates

Publication Date
20260508
Application Date
20241101

Claims (13)

  1. 1. An engineered extracellular vesicle, wherein the engineered extracellular vesicle is loaded with both WNT ligand protein and R-spondin ligand protein.
  2. 2. The extracellular vesicle according to claim 1, wherein said WNT ligand protein is selected from one or more of WNT1、WNT2、WNT2B、WNT3、WNT3A、WNT4、WNT5A、WNT5B、WNT6、WNT7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9B、WNT10A、WNT10B、WNT11 and WNT16, or a functional variant or fragment thereof, preferably said WNT ligand protein is selected from one or more of WNT3, WNT3A, WNT a and WNT7A, or a functional variant or fragment thereof, and/or The R-spondin ligand protein is selected from one or more of R-spondin1 protein, R-spondin2 protein, R-spondin3 protein and R-spondin4 protein, or functional variants or fragments thereof, preferably the R-spondin ligand protein is selected from one or more of R-spondin1 protein, R-spondin2 protein and R-spondin3 protein, or functional variants or fragments thereof.
  3. 3. The extracellular vesicle according to claim 2, wherein the engineered extracellular vesicle comprises a WNT ligand protein selected from the group consisting of WNT3, WNT3A, WNT A, WNT A, and functional variants or fragments thereof, and an R-spondin ligand protein selected from the group consisting of R-spondin1 protein, R-spondin2 protein, R-spondin3 protein, and functional variants or fragments thereof; Preferably, the engineered extracellular vesicles comprise: WNT3 or a functional variant or fragment thereof, and R-spondin1 or a functional variant or fragment thereof, and/or WNT3A or a functional variant or fragment thereof, and R-spondin1 or a functional variant or fragment thereof, and/or WNT5A or a functional variant or fragment thereof, and R-spondin2 or a functional variant or fragment thereof, and/or WNT7A or a functional variant or fragment thereof, and R-spondin3 or a functional variant or fragment thereof.
  4. 4. The extracellular vesicle according to claim 1, wherein said WNT ligand protein is displayed on the surface of said engineered extracellular vesicle, Preferably, the WNT ligand protein binds to WNT carrier protein WLS protein; Preferably, the WLS protein is selected from the group consisting of a WLS full-length protein or a combination of one or more transmembrane domains of M1, M2, M3, M4, M5, M6, M7 or M8 of a WLS protein.
  5. 5. The extracellular vesicle according to claim 1, wherein the R-spondin ligand protein is displayed on the surface or inside of the engineered extracellular vesicle, Preferably, the R-spondin ligand protein is actively loaded to the engineered extracellular vesicle by passive loading or by a scaffold protein; preferably, the R-spondin ligand protein is linked to extracellular vesicle skeletal protein either directly or via a linker, Preferably, the extracellular vesicle scaffold protein comprises a transmembrane protein or a membrane anchoring protein, Preferably, the extracellular vesicle scaffold protein is selected from lysosomal associated membrane protein 2 (LAMP 2), LAMP2B, CD63, TETRASPANIN, lactadherin C1/C2, CD9, CD81, PDGFR, GPI ankyrin 、CD20、Claudin18.2、Claudin6、TM4SF、LAPTM4B、Tarp、CD133、GPRC5D、CXCR4、CCR5、CCR8、SSTR2、L1CAM、β-Klotho、CD147、PTGFRN、BASP1、WLS,, or a functional variant or fragment thereof.
  6. 6. The extracellular vesicles according to claim 1, wherein the engineered extracellular vesicles comprise microvesicles and exosomes, preferably exosomes, Preferably, the extracellular vesicles are further loaded with exogenous nucleic acids, polypeptides, proteins or small molecule drugs.
  7. 7. A cell producing the extracellular vesicle of any one of claims 1 to 6, wherein the genomic sequence of the cell is inserted with an exogenous sequence encoding the WNT ligand protein and/or the R-spondin ligand protein.
  8. 8. The cell of claim 7, wherein the cell comprises a cell derived from a human or non-human mammal, Preferably, the non-human mammal is a mouse, rat, monkey, cow, sheep, pig, rabbit or horse, Preferably, the cells are selected from the group consisting of immortalized cell lines or primary cells, Preferably, the cell is selected from the group consisting of HEK293 cells, HEK293T cells, CHO cells, vero cells, COS-7 cells, BHK cells, MDCK cells, tumor cells, embryonic stem cells, induced pluripotent stem cells, induced totipotent stem cells, or mesenchymal stem cells.
  9. 9. A method for preparing the extracellular vesicles according to any one of claims 1 to 6, comprising the step of inserting an exogenous sequence encoding a WNT ligand protein and an exogenous sequence encoding an R-spondin ligand protein into the genome of a cell.
  10. 10. The method of claim 9, wherein the step of inserting the exogenous sequence encoding the WNT ligand protein and the exogenous sequence encoding the R-spondin ligand protein into the genome of the cell comprises transfecting the cell with a recombinant vector carrying the exogenous sequence encoding the WNT ligand protein and/or the exogenous sequence encoding the R-spondin ligand protein, Preferably, the recombinant vector comprises a viral vector or a non-viral vector, Preferably, the recombinant vector comprises at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, sendai virus vector, herpes simplex virus vector, Preferably, the recombinant vector comprises at least one of pLVX, pCDH, pQCXIP, pLNCX, pLenti, pRRLSIN, pTRIP, Preferably, the exogenous sequence encoding the WNT ligand protein and the exogenous sequence encoding the R-spondin ligand protein are located in different recombinant vectors, or in the same recombinant vector, Preferably, the method further comprises the step of isolating the outer vesicle.
  11. 11. A pharmaceutical composition comprising an extracellular vesicle as described above of the present disclosure and a pharmaceutically acceptable carrier.
  12. 12. Use of an extracellular vesicle according to any one of claims 1 to 5, a cell according to claim 6 or 7 or a pharmaceutical composition according to claim 10 for one or more of the following: (1) Promoting differentiation of stem cells, progenitor cells or pluripotent stem cells, preferably to liver cells, skin cells, hair follicle cells, lung cells, intestinal cells, nerve cells, endometrial cells; (2) Preventing, treating or ameliorating a disease or disorder associated with WNT signaling; (3) Delivering the drug; (4) Promoting tissue differentiation; (5) Promoting tissue regeneration; (7) Culturing the organoids; (8) Preventing or reversing aging; (9) Promoting liver regeneration after acute injury, reducing liver fibrosis region, improving liver surface granule feeling and texture, or improving liver function, Preferably, the disease or condition associated with WNT signaling includes liver disease, preferably liver injury, liver fibrosis, acute liver failure, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure, liver cirrhosis, liver fibrosis, portal hypertension, chronic liver insufficiency, end-stage liver disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, alcoholic hepatitis, hepatitis C virus-induced liver disease, hepatitis B virus-induced liver disease, hepatitis A virus-induced liver disease, hepatitis D virus-induced liver disease, primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury and liver transplantation, and/or The extracellular vesicles, the cells or the pharmaceutical composition are used for promoting regeneration after liver injury, and/or The organoids include organoids of human origin or of non-human mammal origin, preferably including mice, rats, guinea pigs, sheep, pigs, cattle, dogs, cats, rabbits, alpacas, horses, Preferably, the organoids comprise intestinal organoids, cardiac organoids, gastric organoids, retinal organoids, breast organoids, prostate organoids, pancreatic organoids, endometrial organoids, neural organoids, pulmonary organoids, liver organoids.
  13. 13. The use according to claim 12, characterized in that, An engineered outer vesicle comprising WNT3 or a functional variant or fragment thereof and R-spondin1 or a functional variant or fragment thereof for use in one or more of (1) promoting differentiation of stem cells, progenitor cells or pluripotent stem cells into liver cells, (2) preventing or reversing aging, (3) promoting liver regeneration in an acutely injured liver, reducing fibrotic regions of the liver, improving graininess and texture of the liver surface, or improving liver function, (4) preventing or treating chronic liver fibrosis, and/or An engineered outer vesicle comprising WNT3A or a functional variant or fragment thereof and R-spondin1 or a functional variant or fragment thereof for one or more of (1) promoting differentiation of stem cells, progenitor cells or pluripotent stem cells into liver cells, (2) preventing or reversing aging, (3) promoting regeneration of liver for acute injury, reducing fibrotic regions of liver, improving graininess and texture of liver surface, or improving liver function, (4) preventing or treating chronic liver fibrosis, and/or Engineered outer vesicles comprising WNT5A or a functional variant or fragment thereof and R-spondin2 or a functional variant or fragment thereof for use in maintaining or promoting alveolar organoids, and/or An engineered outer vesicle comprising WNT7A or a functional variant or fragment thereof and R-spondin3 or a functional variant or fragment thereof is used for one or more of (1) maintaining or promoting the growth, proliferation, number and/or size of an endometrial organoid, (2) promoting regeneration after an endometrial injury, (3) promoting thickness recovery and/or restoration of the number of glands after an endometrial injury.

Description

Extracellular vesicles loaded with WNT and R-spondin proteins and application thereof Technical Field The present disclosure belongs to the technical field of molecular biology, and in particular relates to an extracellular vesicle loaded with WNT and R-spondin proteins simultaneously, and a preparation method and an application of the extracellular vesicle. Background Multiple tissue organs of the adult body, including the small intestine, skin, brain, breast, liver, etc., still have strong self-renewal and regeneration capacity because some stem cell populations still exist in these adult tissues. Tissue regeneration dependent on stem cells has been found to be commonly regulated by WNT signaling pathways, including classical WNT signaling pathway (WNT/β -catenin signaling pathway) and non-classical signaling pathway (WNT/PCP signaling pathway and WNT/Ca 2+ signaling pathway). Once WNT signaling pathways are inhibited, tissue regeneration will also cease. Therefore, modulation of endogenous tissue stem cell activation levels through WNT signaling pathway is one of the strategies to promote tissue regeneration, combat organ aging, and treat disease. In the absence of WNT ligand, the membrane receptors zinc finger protein 3 (ZNRF 3) and ring finger protein 43 (RNF 43) induce internalization and degradation of frizzled receptor (FZD), while LRP5/6 receptor is also degraded by the ubiquitinase pathway. Intracellular β -catenin is recruited to form complexes by Axin, ck1α, APC, gsk3β, etc., and is subsequently phosphorylated and degraded by proteasomes. At this point, typical WNT signaling activity is maintained at basal levels. When the WNT ligand protein is present, the WNT ligand binds to FZD receptor and LRP5/6 receptor, recruiting DVL, CK1 gamma, axin, gsk3β to form a complex, releasing β -catenin to aggregate in the cytoplasm. Beta-catenin can be further transported into cell nucleus to combine with transcription factors such as TCF/LEF, HIFα, FOXO and the like, and the expression of downstream genes is regulated. In addition, the WNT ligand protein can also regulate WNT/PCP and WNT/Ca 2+ signaling pathway through JNK and the like. Thus, WNT signaling pathway may regulate stem cell proliferation and differentiation, promote tissue regeneration, delay aging, prevent or treat disease. In addition, WNT ligand proteins undergo lipidation modifications after synthesis on the endoplasmic reticulum, an important prerequisite for WNT activity. However, the hydrophobic characteristic of the lipidation modified WNT ligand protein has the characteristics of difficult purification, difficult maintenance of activity and the like in use. In the presence of WNT ligand proteins, it is still not completely avoided that WNT receptor protein FZD is internalized and degraded by ZNRF3 and RNF 43. The R-spondin protein recruits RNF43 and ZNRF3 through binding with the receptor Lgr4/5/6, so that the WNT receptor protein is more stably bound with Frizzled, and the WNT/beta-catenin signal pathway is further enhanced, and is considered as an important activator in the WNT signal pathway. In specific tissues and organs, it is often difficult to effectively regulate endogenous stem cell activation levels using only WNT ligand proteins, and it is desirable to work with appropriate R-spondin ligand proteins. However, there is no effective vector or recombinant capable of simultaneously loading active WNT proteins and R-spondin proteins to efficiently activate and regulate specific WNT signaling pathways and exert corresponding effects. Disclosure of Invention The present disclosure provides an extracellular vesicle loaded with both WNT ligand protein and R-spondin ligand protein. The extracellular vesicles disclosed by the invention realize the effects of promoting proliferation and differentiation of tissue organ cells, promoting regeneration of multiple tissues (including internal organs such as skin, hair follicles, lung, intestines, liver and the like, nervous systems and the like), delaying/reversing aging and preventing or treating diseases by activating WNT signal pathways with high efficiency. According to one aspect of the present disclosure, an engineered extracellular vesicle is provided that is loaded with WNT ligand protein and R-spondin ligand protein. In some embodiments, the WNT ligand protein is selected from one or more of WNT1、WNT2、WNT2B、WNT3、WNT3A、WNT4、WNT5A、WNT5B、WNT6、WNT7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9B、WNT10A、WNT10B、WNT11 and WNT16, or a functional variant or fragment thereof. In some embodiments, the WNT ligand protein is selected from one or more of WNT3A, WNT a and WNT7A, or a functional variant or fragment thereof. In some embodiments, the R-spondin ligand protein is selected from one or more of R-spondin1 protein, R-spondin2 protein, R-spondin3 protein, and R-spondin4 protein, or a functional variant or fragment thereof. In some embodiments, the R-spondin ligand protein is selected from one or more of R-spo