Extracellular vesicle loaded with WNT and r-spondin proteins, and use thereof

Abstract

The present invention relates to an extracellular vesicle loaded with WNT and RSPO proteins and a use thereof. Specifically, provided are an extracellular vesicle loaded with both WNT and RSPO proteins, and a preparation method and use of the extracellular vesicle.

Description

Extracellular vesicles loaded with WNT and R-spondin proteins and their applications Technical Field

[0001] This disclosure belongs to the field of molecular biology technology, specifically relating to extracellular vesicles simultaneously loaded with WNT and R-spondin proteins, as well as the preparation method and application of such extracellular vesicles. Background Technology

[0002] Many tissues and organs in the adult body, including the small intestine, skin, brain, mammary glands, and liver, still possess strong self-renewal and regeneration capabilities because they still contain some stem cell populations. Research has found that stem cell-dependent tissue regeneration is generally regulated by the WNT signaling pathway, including the classical WNT signaling pathway (WNT / β-catenin signaling pathway) and non-classical signaling pathways (WNT / PCP signaling pathway and WNT / Ca2+ signaling pathway). 2+ (Signaling pathway). Once the WNT signaling pathway is inhibited, tissue regeneration will also stop. Therefore, regulating the activation level of endogenous tissue stem cells through the WNT signaling pathway is one of the strategies to promote tissue regeneration, combat organ aging, and treat diseases.

[0003] In the absence of WNT ligands, membrane receptor zinc finger protein 3 (ZNRF3) and ring finger protein 43 (RNF43) induce the internalization and degradation of the coiled receptor (FZD), while LRP5 / 6 receptors are also degraded via ubiquitination. Intracellular β-catenin is recruited by Axin, CK1α, APC, GSK3β, etc., to form a complex, which is then phosphorylated and degraded by the proteasome. At this time, typical WNT signaling activity is maintained at a basal level. In the presence of WNT ligand proteins, WNT ligands bind to FZD and LRP5 / 6 receptors, recruiting DVL, CK1γ, Axin, and GSK3β to form a complex, releasing β-catenin which accumulates in the cytoplasm. β-catenin can further be transported to the nucleus and bind to transcription factors such as TCF / LEF, HIFα, and FOXO, regulating the expression of downstream genes. In addition, WNT ligand proteins can also regulate WNT / PCP and WNT / Ca2+ through mechanisms such as JNK. 2+ Signaling pathway. Therefore, the WNT signaling pathway can regulate stem cell proliferation and differentiation, promote tissue regeneration, delay aging, and prevent or treat diseases.

[0004] Furthermore, WNT ligand proteins undergo lipidation modification after synthesis on the endoplasmic reticulum, and this modification is an important prerequisite for WNT activity. However, the hydrophobic nature of lipidation modification leads to difficulties in purification and maintaining activity of WNT ligand proteins during use.

[0005] Even in the presence of WNT ligand proteins, the internalization and degradation of the WNT receptor protein FZD induced by ZNRF3 and RNF43 cannot be completely prevented. R-spondin protein, by binding to its receptors Lgr4 / 5 / 6, recruits RNF43 and ZNRF3, making the binding of the WNT receptor protein to Frizzled more stable, thereby enhancing the WNT / β-catenin signaling pathway and is considered an important activator of the WNT signaling pathway. In certain tissues and organs, WNT ligand proteins alone are often insufficient to effectively regulate the activation level of endogenous stem cells, requiring the combined action of appropriate R-spondin ligand proteins.

[0006] However, there is currently no effective vector or recombinant that can simultaneously load active WNT protein and R-spondin protein to efficiently activate and regulate specific WNT signaling pathways and exert corresponding effects. Summary of the Invention

[0007] This disclosure provides an extracellular vesicle simultaneously loaded with WNT ligand protein and R-spondin ligand protein. The extracellular vesicle of this disclosure achieves the effects of promoting the proliferation and differentiation of tissue and organ cells, promoting the regeneration of multiple tissues (including internal organs such as skin, hair follicles, lungs, intestines, liver, and the nervous system), delaying / reversing aging, and preventing or treating diseases by efficiently activating the WNT signaling pathway.

[0008] According to one aspect of this disclosure, an engineered extracellular vesicle is provided, the engineered extracellular vesicle being loaded with WNT ligand protein and R-spondin ligand protein.

[0009] 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 functional variants or fragments thereof. In some embodiments, the WNT ligand protein is selected from one or more of WNT3A, WNT5A, and WNT7A, or functional variants or fragments thereof.

[0010] In some embodiments, the R-spondin ligand protein is selected from one or more of R-spondin1, R-spondin2, R-spondin3, and R-spondin4 proteins, or functional variants or fragments thereof.

[0011] In some embodiments, the engineered extravesicles include: a WNT ligand protein selected from WNT3, WNT5A, WNT7A, or functional variants or fragments thereof; and an R-spondin ligand protein selected from R-spondin1, R-spondin2, R-spondin3, or functional variants or fragments thereof.

[0012] In some embodiments, the engineered exovesicles may include: WNT3A or a functional variant or fragment thereof; and R-spondin1 or a functional variant or fragment thereof. In some embodiments, the engineered exovesicles may include: WNT5A or a functional variant or fragment thereof; and R-spondin2 or a functional variant or fragment thereof. In some embodiments, the engineered exovesicles may include: WNT7A or a functional variant or fragment thereof; and R-spondin3 or a functional variant or fragment thereof.

[0013] In some embodiments, the WNT ligand protein is displayed on the surface of the engineered extracellular vesicles. In some embodiments, the WNT ligand protein can be displayed on the surface of the engineered extracellular vesicles via a WNT carrier protein (e.g., WLS protein). In some embodiments, the WNT ligand protein binds to the WNT carrier protein (e.g., WLS protein), for example, to form a protein complex. In some embodiments, the WNT ligand protein is passively loaded onto the extracellular vesicles via a WNT carrier protein (e.g., WLS protein). Those skilled in the art will understand that the WNT carrier protein can be naturally expressed or exogenously expressed in the cells that produce the engineered extracellular vesicles.

[0014] In some embodiments, the WLS protein is selected from the full-length WLS protein or a combination of one or more transmembrane domains of the WLS protein, namely M1, M2, M3, M4, M5, M6, M7 or M8.

[0015] In some embodiments, the R-spondin ligand protein is displayed on the surface or inside the engineered extracellular vesicles. In some embodiments, the R-spondin ligand protein can be passively loaded via extracellular vesicles or actively loaded via cytoskeletal proteins.

[0016] In some embodiments, the scaffold protein may include transmembrane proteins or membrane anchoring proteins. In some embodiments, the scaffold protein may include any extravesicular vesicle scaffold protein known in the art, such as lysosome-associated membrane protein 2 (LAMP2), LAMP2B, CD63, lactobacin C1 / C2, CD9, CD81, PDGFR, GPI anchoring protein, CD20, Claudin18.2, Claudin6, TM4SF, LAPTM4B, Tarp, CD133, GPRC5D, CXCR4, CCR5, CCR8, SSTR2, L1CAM, β-Klotho, CD147, PTGFRN, BASP1, WLS, or functional variants or fragments thereof.

[0017] In some embodiments, the density of the WNT ligand protein and / or the R-spondin ligand protein on the surface of the engineered extracellular vesicle is higher than that of the WNT ligand protein and / or R-spondin ligand protein on unengineered extracellular vesicles or other extracellular vesicles.

[0018] In some embodiments, the engineered extracellular vesicles may include microvesicles and exosomes. In a preferred embodiment, the engineered extracellular vesicles may be exosomes.

[0019] In some embodiments, the extracellular vesicles may also be loaded with exogenous nucleic acids, peptides, proteins, or small molecule drugs.

[0020] According to another aspect of this disclosure, a cell that produces the above-described extracellular vesicles is provided, wherein an exogenous sequence encoding the WNT ligand protein and / or the R-spondin ligand protein is inserted into its genome sequence.

[0021] In some embodiments, the cells may include cells derived from humans or non-human mammals. In some embodiments, the non-human mammal may be a mouse, rat, monkey, cow, sheep, pig, rabbit, or horse. In some embodiments, the cells may be selected from immortalized cell lines or primary cells.

[0022] In some embodiments, the cells may be selected from HEK293 cells, HEK293T cells, Chinese hamster ovary (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 (MSCs).

[0023] According to another aspect of this disclosure, a method for preparing engineered extracellular vesicles of this disclosure is provided, the method comprising the steps 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.

[0024] 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 functional variants or fragments thereof. In some embodiments, the WNT ligand protein is selected from one or more of WNT3A, WNT5A, and WNT7A, or functional variants or fragments thereof.

[0025] In some embodiments, the R-spondin ligand protein is selected from one or more of R-spondin1, R-spondin2, R-spondin3, and R-spondin4 proteins, or functional variants or fragments thereof.

[0026] In some embodiments, the method further includes the step of separating the outer vesicles.

[0027] In some embodiments, the step of inserting the exogenous sequences encoding WNT ligand proteins and R-spondin ligand proteins into the genome of a cell includes: transfecting the cells with a recombinant vector carrying the exogenous sequences encoding WNT ligand proteins and / or R-spondin ligand proteins.

[0028] In some embodiments, the recombinant vector includes a viral vector or a non-viral vector.

[0029] In some embodiments, the recombinant vector may include at least one selected from lentiviral vectors, adenovirus vectors, baculovirus vectors, retroviral vectors, poxvirus vectors, Sendai virus vectors, and herpes simplex virus vectors. In specific embodiments, the recombinant vector may include at least one selected from pLVX, pCDH, pQCXIP, pLNCX, pLenti, pRRLSIN, and pTRIP.

[0030] In some embodiments, the exogenous sequence encoding the WNT ligand protein and the exogenous sequence encoding the R-spondin ligand protein are located in different recombinant vectors. In some embodiments, the exogenous sequence encoding the WNT ligand protein and the exogenous sequence encoding the R-spondin ligand protein are located in the same recombinant vector.

[0031] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the external vesicles described above and a pharmaceutically acceptable carrier.

[0032] According to another aspect of this disclosure, the engineered extracellular vesicles, cells, or pharmaceutical compositions described above are provided for use in one or more of the following: (1) promoting differentiation of stem cells, progenitor cells, or pluripotent stem cells; (2) preventing, treating, or improving diseases or conditions related to WNT signaling; (3) delivering drugs; (4) promoting tissue differentiation; (5) promoting tissue regeneration; (7) culturing organoids; (8) preventing or reversing aging, including weight regulation, prevention of spontaneous tumors and fatty liver; (9) promoting liver regeneration after acute injury, reducing fibrotic areas of the liver, improving the granularity and texture of the liver surface, or improving liver function.

[0033] In some embodiments, the engineered extracellular vesicles, cells, or pharmaceutical compositions disclosed herein can be used to promote the differentiation of stem cells, progenitor cells, or pluripotent stem cells into, for example, but not limited to, liver cells, skin cells, hair follicle cells, lung cells, intestinal cells, nerve cells, endometrial cells, etc.

[0034] In some embodiments, the diseases or conditions associated with WNT signaling may include liver diseases such as liver injury, liver fibrosis, acute liver failure, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of all causes, cirrhosis, liver fibrosis of all causes, portal hypertension, chronic liver failure of all causes, end-stage liver disease (ESLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic hepatitis, hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small forsize" syndrome in liver surgery and transplantation, congenital liver diseases and conditions, and any other liver conditions or defects caused by hereditary diseases, degeneration, aging, drugs, or injury.

[0035] In some embodiments, the engineered extracellular vesicles, cells, or pharmaceutical compositions disclosed herein can be used to promote liver regeneration after injury.

[0036] In some embodiments, the organoids include organoids derived from humans or non-human mammals. In some embodiments, the non-human mammals may include mice, rats, guinea pigs, sheep, pigs, cattle, dogs, cats, rabbits, alpacas, horses, etc. In some embodiments, the organoids may include, but are not limited to, intestinal organoids (e.g., colonic epithelium, intestinal epithelium, small intestinal organoids), heart organoids, stomach organoids, retinal organoids, mammary gland organoids, prostate organoids, pancreatic organoids, endometrial organoids, neural organoids, lung organoids (e.g., alveolar organoids), and liver organoids.

[0037] In specific implementations, engineered exovesicles including WNT3 or its functional variants or fragments and R-spondin1 or its functional variants or fragments can be used for one or more of the following purposes: (1) promoting the differentiation of stem cells, progenitor cells or pluripotent stem cells into hepatocytes; (2) preventing or reversing aging, including weight regulation, prevention of spontaneous tumors and fatty liver; (3) promoting liver regeneration, reducing fibrotic areas of the liver, improving the granularity and texture of the liver surface, or improving liver function in the case of acutely injured liver; (4) preventing or treating chronic liver fibrosis.

[0038] In specific implementations, engineered exovesicles including WNT3A or its functional variants or fragments and R-spondin1 or its functional variants or fragments can be used for one or more of the following purposes: (1) promoting the differentiation of stem cells, progenitor cells or pluripotent stem cells into hepatocytes; (2) preventing or reversing aging, including weight regulation, prevention of spontaneous tumors and fatty liver; (3) for acutely injured liver, promoting liver regeneration, reducing fibrotic areas of the liver, improving the granularity and texture of the liver surface, or improving liver function; (4) preventing or treating chronic liver fibrosis.

[0039] In specific implementations, engineered exovesicles including WNT5A or a functional variant or fragment thereof and R-spondin2 or a functional variant or fragment thereof can be used to maintain or promote the growth of alveolar organoids.

[0040] In specific implementations, engineered extravesicles including WNT7A or a functional variant or fragment thereof and R-spondin3 or a functional variant or fragment thereof may be used for one or more of the following purposes: (1) maintaining or promoting the (in vitro) growth, proliferation, number and / or size of endometrial organoids; (2) promoting regeneration after endometrial injury; and (3) promoting the recovery of endometrial thickness and / or glandular number after endometrial injury.

[0041] According to another aspect of this disclosure, a method for preventing, treating, or improving diseases or conditions related to WNT signaling is provided, the method comprising administering the engineered extracellular vesicles, cells, or pharmaceutical compositions of the present disclosure to a subject in need.

[0042] In some embodiments, the engineered extracellular vesicles, the cells, or the pharmaceutical composition can be administered orally, parenterally, intravenously, topically, by inhalation, or intramuscularly. Attached Figure Description

[0043] Figure 1 shows a schematic diagram of engineered exosomes simultaneously loaded with WNT ligand protein and R-spondin protein.

[0044] Figure 2 shows the identification of WNT3A / R-spondin1 engineered cells and exosomes. Figure 2A shows the expression of WNT3A, R-spondin1, and β-actin in WNT3A-engineered cells, R-spondin1-engineered cells, and WNT3A / R-spondin1-engineered cells; Figure 2B shows the expression of WNT3A, R-spondin1, CD63, and HSP70 in WNT3A-engineered exosomes (exoWNT3A), R-spondin1-engineered exosomes (exoRSPO1), and WNT3A / R-spondin1-engineered exosomes (exoWNT3A / RSPO1); Figure 2C shows an immunogold electron microscope image of exoWNT3A / RSPO1 showing the presence of the same exosome containing WNT3A and R-spondin1; Figure 2D shows the TOPFlash luciferase activity of HEK293FT cells after treatment with exoWNT3A, exoRSPO1, and exoWNT3A / RSPO1.

[0045] Figure 3 illustrates how WNT / R-spondin1 exosomes promote the differentiation of hepatic endoderm cells into hepatocytes. Figure 3A shows a light microscopic image of the morphological changes in hepatic endoderm cells; Figure 3B shows real-time PCR detection of the expression of WNT downstream genes (R-spondin (RSPO)-leucine-rich repeat G protein-coupled receptor (LGR)5 and cMYC) and hepatocyte genes (alpha-fetoprotein (AFP), albumin (ALB), and hepatocyte nuclear factor (HNF) 4α); Figure 3C shows the results of immunofluorescence detection.

[0046] Figure 4 illustrates how WNT3A / R-spondin1 exosomes promote liver regeneration in mice after acute carbon tetrachloride (CCl4)-induced liver injury. Figure 4A shows the experimental procedure and histopathological results for mouse treatment; Figure 4B shows the statistical results of necrotic areas; Figure 4C shows the detection results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities; Figure 4D shows the results of immunofluorescence detection.

[0047] Figure 5 illustrates how WNT3A / R-spondin1 exosomes promote liver regeneration in mice following acute acetaminophen (APAP)-induced injury. Figure 5A shows the experimental procedure and histopathological results for mouse treatment; Figure 5B shows the statistical results of necrotic areas; Figure 5C shows the results of AST and ALT activity detection; and Figure 5D shows the results of immunofluorescence detection.

[0048] Figure 6 illustrates the liver injury and regeneration repair at different time points following acute liver injury induced by WNT3A / R-spondin1 exosomes in mice induced by acetaminophen (APAP). Figure 6A shows the experimental procedure for mouse treatment; Figure 6B shows the changes in gene expression levels associated with cell necrosis; Figure 6C shows the fluorescence levels of apoptotic cells; Figure 6D shows the changes in the proportion of apoptotic cells; Figure 6E shows the expression level of the cell proliferation marker Ki67 gene; Figure 6F shows Ki67-positive cells; and Figure 6G shows the changes in the proportion of Ki67-positive cells.

[0049] Figure 7 illustrates how WNT3A / R-spondin1 exosomes promote the improvement of CCl4-induced chronic liver fibrosis in mice. Figure 7A shows the experimental procedure for mouse treatment, as well as the results of Masson staining and Sirius red staining; Figure 7B shows the quantitative results of Masson staining and Sirius red staining; Figure 7C shows the detection results of AST and ALT activities.

[0050] Figure 8 illustrates the pathological changes in WNT3A / R-spondin1 exosomes in mice that reversed aging. Figure 8A shows the experimental procedure for mouse treatment, as well as the results of HE staining and β-galactosidase staining; Figure 8B shows the results of immunofluorescence labeling of hepatocyte proliferation and aging markers.

[0051] Figure 9 illustrates the identification of WNT5A / R-spondin2 exosomes and their role in promoting the in vitro growth of human respiratory system organoids. Figure 9A shows transmission electron microscopy images of WNT5A / R-spondin2 engineered exosomes; Figure 9B shows the expression of WNT5A, R-spondin2, and HSP70 in WNT5A engineered exosomes (exoWNT5A), R-spondin2 engineered exosomes (exoRSPO2), and WNT5A / R-spondin2 engineered exosomes (exoWNT5A / RSPO2); Figure 9C shows the growth status of human alveolar organoids under different engineered exosome culture conditions (same number of particles) (only the exoWNT5A / RSPO2 treatment resulted in the best organoid state).

[0052] Figure 10 illustrates the identification of WNT7A / R-spondin3 exosomes and their role in promoting the in vitro growth of human endometrial organoids and the regeneration of rat endometrial tissue after injury. Figure 10A shows transmission electron microscopy images of WNT7A / R-spondin3 engineered exosomes; Figure 10B shows the expression of WNT7A, R-spondin3, and HSP70 in WNT7A engineered exosomes (exoWNT7A), R-spondin3 engineered exosomes (exoRSPO3), and WNT7A / R-spondin3 engineered exosomes (exoWNT7A / RSPO3); Figure 10C shows the growth status of human endometrial organoids under different engineered exosome culture conditions (same number of particles) (only exoWNT7A / RSPO3 treatment resulted in the best organoid status); Figure 10D shows the endometrial repair in rats after 14 days of treatment with different engineered exosomes (same number of particles) following endometrial injury (only exoWNT7A / RSPO3 treatment resulted in the best endometrial regeneration status).

[0053] Figure 11 illustrates the application of WNT3A / R-spondin1 exosomes in weight regulation, inhibition of spontaneous tumors, and fatty liver in aging mice. Figure 11A shows the procedure of treating aging mice with WNT3A / R-spondin1 exosomes; Figure 11B shows the coat condition and frailty index scores of aging mice after different treatments; Figure 11C shows the weight changes of aging mice after different treatments; Figure 11D shows the statistical results of spontaneous tumors in aging mice after different treatments; Figure 11E shows the spontaneous tumors in aging mice without WNT3A / R-spondin1 exosome treatment; Figure 11F shows the level of fatty liver in aging mice after different treatments. Detailed Implementation

[0054] The WNT signaling pathway is an important signaling pathway that is closely related to stem cell pluripotency and embryonic development.

[0055] A key step in the WNT signaling pathway is the stability of its key factor β-catenin and its entry into the nucleus, which determines the expression of downstream genes. When extracellular WNT ligand proteins are absent, cytoplasmic β-catenin is recruited to a degradation complex composed of APC and Axin. β-catenin is phosphorylated by CK1α and GSK3β, leading to its binding to the E3 ubiquitin ligase β-TrCP and entry into the ubiquitination and proteasome-dependent degradation pathway. Therefore, in the inactive state, cytoplasmic β-catenin protein levels remain low, while LEF and TCF in the nucleus bind to Groucho, inhibiting the expression of downstream genes regulated by WNT. When WNT proteins bind to the receptor complex, the corresponding signaling pathway is activated. The co-receptor LRP can be phosphorylated by CK1α and GSK3β, and Axin is recruited to the cell membrane, thus preventing β-catenin from phosphorylation and its transport into the nucleus. In the cell nucleus, β-catenin and TCF / LEF form a transcription complex, thereby activating the expression of downstream genes in the WNT signaling pathway, such as c-Myc and cyclin D1.

[0056] WNT proteins contain approximately 350 amino acid residues and have a molecular weight of about 40 kDa. They are secreted glycoproteins, rich in cysteine, and relatively conserved in evolution. To date, 19 human WNT genes have been discovered: WNT1, WNT2, WNT2B (WNT13), WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A (WNT14), WNT9B (WNT14B, WNT15), WNT10A, WNT10B (WNT12), WNT11, and WNT16.

[0057] R-spondins are secreted cysteine-rich glycoproteins belonging to the protein superfamily containing prothrombin type 1 repeats.

[0058] This disclosure provides extracellular vesicles simultaneously loaded with one or more combinations of WNT and R-spondin ligand proteins. Compared to extracellular vesicles loaded with WNT ligand protein alone or R-spondin ligand protein alone, the engineered extracellular vesicles of this disclosure significantly enhance the activation level of the WNT signaling pathway.

[0059] This disclosure also selected the optimal combination of WNT ligand protein and R-spondin ligand protein through screening, and loaded them together in the same extracellular vesicle, which significantly improved the activation level of the WNT signaling pathway by WNT ligand protein or R-spondin ligand protein alone.

[0060] The engineered extracellular vesicles disclosed herein can efficiently activate the WNT signaling pathway in target cells, regulate stem cell proliferation and differentiation, promote tissue regeneration, delay aging, and prevent or treat diseases.

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0062] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0063] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0064] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0065] Extracellular vesicles (EVs) are small, double-membrane vesicles that detach from the cell membrane or are secreted by the cell, ranging in diameter from 30 nm to 1,000 nm. EVs mainly include microvesicles (MVs) and exosomes (Exos). Microvesicles are small vesicles that detach from the cell membrane after cell activation, damage, or apoptosis, with a diameter of approximately 200–1000 nm. Exosomes are released extracellularly in the form of secretions from multivesicular bodies fused to the cell membrane, with a diameter of approximately 30–200 nm. EVs are widely distributed in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, breast milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. Extracellular vesicles possess natural biocompatibility, high delivery efficiency, low toxicity, and low immunogenicity, making them an emerging drug delivery vector. Engineered extracellular vesicles are typically designed through genetic modification of their donor cells. In some embodiments, the extracellular vesicles described in this disclosure also carry payloads of interest, i.e., the extracellular vesicles encapsulate exogenous or endogenous molecules such as proteins, nucleic acids, peptides, and small molecules.

[0066] The engineered exovesicles disclosed herein have exovesicles whose composition has been modified or altered. For example, the composition of the exovesicles, particularly the membrane composition, can be modified or altered by changing the content of proteins, lipids, or glycans on the membrane.

[0067] In some embodiments, the engineered extravesicles of this disclosure are generated by genetic engineering. In a specific embodiment, the engineered extravesicles of this disclosure are generated by genetically modified cells.

[0068] TOPFlash (reporter plasmid) is a reporter gene plasmid used to detect the level of TCF / LEF transcriptional activity mediated by β-catenin in the WNT signaling pathway. Inserting a TCF / LEF binding site sequence into its multiple cloning site allows for highly sensitive detection of TCF / LEF transcriptional activity levels.

[0069] As used herein, the term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. Domains in the fusion peptides of the present invention can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two peptides or domains. Linkers should have a length suitable for linking two or more monomeric domains in this manner, and the linker should ensure that the different domains to which it is linked fold correctly and are properly presented to perform their biological activities. In this disclosure, linkers ensure that the linked R-spondin ligand protein and backbone protein fold correctly and are properly presented to perform their respective biological activities. In various embodiments, the linkers have a flexible conformation. Those skilled in the art can select appropriate, conventionally used linkers as needed. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker may be selected from (Glycine, glutamine, and / or serine residues). n S) m (G) n (EAAAK) n Or (XP) n , where n and m are each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4, or 5, and m is selected from 1, 2, 3, 4, or 5. In some embodiments, the connector may also include, for example, KESGSVSSEQLAQFRSLD (SEQ ID NO:1), EGKSSGSGSESKST (SEQ ID NO:2), (Gly)8 (SEQ ID NO:3), GSAGSAAGSGEF (SEQ ID NO:4), etc.

[0070] As used in this article, "organoid" refers to an artificial in vitro construct derived from adult stem cells, which is created to mimic or resemble the function and / or histological structure of an organ or part thereof.

[0071] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0072] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0073] Example

[0074] reagents

[0075] Anti-WNT3A antibody, WNT5A antibody, WNT7A antibody, anti-RSPO1 antibody, anti-RSPO2 antibody, anti-RSPO3 antibody, anti-β-actin antibody, anti-CD63 antibody, anti-HSP70 antibody, secondary antibodies used in the examples, gold particles conjugated to anti-mouse secondary antibody, gold particles conjugated to anti-rabbit secondary antibody, P21 antibody, and Ki67 antibody were purchased from Cell Signal.

[0076] The transfection reagent Lipo8000 was purchased from Beyotime Biotechnology Co., Ltd.

[0077] TOPFlash report plasmids and lentiviral packaging aid plasmids were purchased from Addgene.

[0078] Matrigel, Collagen I, Collagenase P, and cell culture medium were purchased from Gibco.

[0079] The luciferase assay kit was purchased from Promega.

[0080] The hindgut differentiation medium, liver endoderm cell differentiation medium, hepatocyte differentiation medium, alveolar organoid culture medium, and endometrial organoid culture medium were purchased from STEMCELL.

[0081] CCl4 (carbon tetrachloride) and APAP (acetaminophen) were purchased from Selleck.

[0082] The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits, erythrocyte lysis buffer, and β-galactosidase staining reagents were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0083] Example 1. Construction of WNT and RSPO expression vectors

[0084] Gene sequences encoding the full-length proteins WNT3 (NP_110380.1) and WNT3A (NP_149122.1) were synthesized and constructed into the pLVX-IRES-tdTomato lentiviral vector to obtain lentiviral expression plasmids for WNT3 and WNT3A proteins, respectively. The gene sequence encoding the full-length human R-spondin protein (also known as RSPO1 or RSPO) (NP_001033722.1) was synthesized and constructed into the pLVX-IRES-EGFP lentiviral vector to obtain the RSPO1 lentiviral expression plasmid.

[0085] The expression plasmid was added to DH5α competent cells, and the cells were transformed by incubation on ice for 30 min, followed by heat shock at 42°C for 90 s, and then incubation on ice for 2 min. The transformed competent cells were transferred to 500 μL of LB liquid medium and incubated at 37°C for 1 h. After removing some of the supernatant, the cells were thoroughly mixed and spread onto LB solid medium containing ampicillin. The plates were inverted and incubated at 37°C for 12–18 h, and colonies were selected for verification. Positive colonies were added to 20 mL of LB liquid medium containing ampicillin and incubated at 37°C and 180 rpm for 12–18 h. The plasmid was extracted and purified, and its concentration was determined. After Sanger sequencing confirmed its accuracy, it was used in subsequent experiments.

[0086] Example 2. Preparation of engineered exosomes of this disclosure

[0087] HEK293T cells were cultured until the cell confluence reached 70%–80%. Then, WNT3, WNT3A, and RSPO1 protein expression plasmids, lentiviral packaging helper plasmids (psPAX2 and pCMV-VSV-G), and transfection reagent Lipo8000 were mixed and co-incubated with HEK293T cells. Virus solutions were collected at 48 h and 72 h, centrifuged at 1200 rpm for 5 min, and the supernatant was collected and filtered through a 0.45 μm filter to obtain WNT3, WNT3A, and RSPO1 protein expression virus solutions. These solutions can be stored at 4°C for one week or at -80°C for one year.

[0088] After mixing the WNT3 and WNT3A protein expression virus solutions with the infection reagent, they were incubated with HEK293 cells for 24 hours and then replaced with fresh culture medium. After 72 hours, the intensity and proportion of red fluorescence expression were observed. Red fluorescence positive cells, i.e., cells stably overexpressing WNT3 and WNT3A proteins, were obtained by flow cytometry cell sorting.

[0089] After mixing the RSPO1 protein expression virus solution with the infection reagent, the mixture was incubated with WNT3 and WNT3A protein stably overexpressing cells for 24 hours, followed by fresh culture medium. The intensity and proportion of green fluorescence expression were observed after 72 hours. Green fluorescence-positive cells, i.e., WNT3 / RSPO1 and WNT3A / RSPO1 stably overexpressing cells, could be obtained by flow cytometry cell sorting. These stably overexpressing cells could be expanded and cultured, and then cryopreserved in liquid nitrogen.

[0090] The above-mentioned stable expression cells were expanded and cultured to obtain culture supernatant. Dead cells were removed by centrifugation at 300g for 5 min at 4℃, cell debris was removed by centrifugation at 2,000g for 10 min, microvesicles were collected by centrifugation at 10,000g for 20 min, and WNT3 / RSPO1 and WNT3A / RSPO1 engineered extracellular vesicles / exosomes were obtained by centrifugation at 100,000g for 10 min.

[0091] Engineered cells, microvesicles, and exosomes were collected separately, and protein lysis buffer was added. After lysis on ice for 30 minutes, the cells were centrifuged at 12,000 rpm for 20 minutes, and the protein supernatant was collected. The total protein content of cells, microvesicles, and exosomes was detected using a BCA kit. 40 μL of total protein was added to 10 μL of 5× protein loading buffer and boiled at 100°C for 10 minutes to promote protein denaturation. SDS-PAGE gels were prepared, and 10 μL of each sample was added. Electrophoresis was performed at 100V for 100 minutes. Proteins on the gel were transferred to a PVDF membrane using a wet transfer method, with a crossflow of 250 mA for 2 hours. The membrane was blocked with 5% skim milk powder and incubated with the corresponding primary antibody dilution overnight at 4°C with shaking. The membrane was washed three times with TBST buffer for 10 minutes each time, and incubated with the corresponding HRP-labeled secondary antibody dilution at room temperature with shaking for 22 hours. The membrane was washed three times with TBST buffer for 10 minutes each time, and the corresponding protein levels were detected using ECL substrate reaction solution. The results are shown in Figures 2A and 2B.

[0092] As shown in Figures 2A and 2B, WNT3A / RSPO1 engineered cells simultaneously highly express WNT3A and RSPO1 proteins (Figure 2A), and their derived exosomes exoWNT3A / RSPO1 also enrich WNT3A and RSPO1 proteins (Figure 2B).

[0093] ExoWNT3A / RSPO1 was added to the copper mesh surface and allowed to stand for 2 minutes. Residual liquid was then blotted away with filter paper, and the mesh was allowed to dry at room temperature for 1 hour. Diluted buffer containing WNT3A protein antibody (mouse anti) and RSPO1 protein antibody (rabbit anti) was added to the copper mesh surface and incubated overnight at 4°C. The mesh was washed three times with PBS for 10 minutes each time. Then, 10 nm gold particles conjugated with anti-mouse secondary antibody and 6 nm gold particles conjugated with anti-rabbit secondary antibody were added and incubated at room temperature for 2 hours. The mesh was washed three times with PBS for 10 minutes each time. The morphology of the exosomes and the location of the gold nanoparticles were observed using a transmission electron microscope. As shown in Figure 2C, the same exosome was simultaneously labeled with 10 nm gold particles (indicating WNT3A protein) and 6 nm gold particles (indicating RSPO1 protein). This indicates that the same exosome was simultaneously loaded with WNT3A and RSPO1 proteins.

[0094] Example 3. Effects of engineered exosomes of this disclosure on the WNT signaling pathway

[0095] HEK293 cells were transfected with the TOPFlash reporter plasmid to obtain lentiviral solution. HEK293FT cells were then infected with the lentiviral solution, and stable HEK293FT-TOPFlash luciferase reporter gene cells were obtained by selection with puromycin.

[0096] The same number of particles (10) 9WNT3A protein-engineered exosomes, RSPO1 protein-engineered exosomes, and WNT3A / RSPO1 protein-engineered exosomes were added to HEK293FT-TOPFlash luciferase reporter cells and incubated overnight. TOPFlash luciferase activity in different treatment groups was detected using a luciferase assay kit. Untreated refers to the untreated cell group, and exoWT refers to exosomes derived from wild-type HEK293 cells. The results showed that the activation activity of WNT3A / RSPO1 protein-engineered exosomes (exoWNT3A / RSPO1) was tens of times higher than that of the other exosomes alone (exoWNT3A and exoRSPO1) treatment groups (Figure 2D).

[0097] Example 4. Effects of engineered exosomes of this disclosure on pluripotent stem cell differentiation

[0098] 4×10 5 100 pluripotent stem cell (iPS) cells were seeded in 24-well Matrigel plates. After complete cell adhesion, the cells were treated with an endoderm shaping kit (STEMCELL, Cat#05110) for 3 days. Subsequently, the cells were treated with hindgut-foregut differentiation medium for 4 days. Finally, the cells were cultured at 4 × 10⁻⁶ cells / well. 5 Cells were passaged at a density of 4 × 10⁴ wells in pre-coated Collagen I plates and treated with hepatic endoderm (HE) cell differentiation medium for 7 days. Purified HE cells were then obtained through successive passages (5 generations) or cell clone picking. 5 Cells were passaged at a density in 24-well plates pre-coated with Collagen I. Hepatic endoderm cell differentiation media containing exoWT, exoWNT3, exoWNT3A, exoRSPO1, exoWNT3 / RSPO1, or exoWNT3A / RSPO1 were added for 7 days, with the corresponding media changed every 2 days. On day 7, cell morphology changes were observed and total RNA was collected. Real-time quantitative PCR was used to detect the expression levels of WNT downstream genes (LGR5 and cMYC), as well as liver markers and stem cell markers (AFP, ALB, and HNF4α). The results showed that only the exoWNT3 / RSPO1 and exoWNT3A / RSPO1 treatment groups significantly altered the morphological changes of hepatic endoderm cells (Figure 3A), and promoted the gene expression levels of WNT downstream genes, liver markers, and stem cell markers (Figure 3B). The effects of exoWNT3, exoWNT3A, and exoRSPO1 alone were similar to those of exoWT, and they could not effectively promote the differentiation of hepatic endoderm cells into hepatocytes.

[0099] Immunofluorescence labeling was used to detect the protein expression of some biomarkers. Cells were first fixed with 4% paraformaldehyde, blocked with 1% BSA for 1 h, and then incubated with the corresponding primary antibody overnight at 4°C. Cells were washed three times with TPBS for 10 min each time, then incubated with the corresponding fluorescently labeled secondary antibody for 2 h at room temperature. Cells were then washed three times with TPBS for 10 min each time, incubated with DAPI dye for 10 min, and the fluorescence expression level was observed using a laser confocal microscope. Immunofluorescence labeling showed that only the exoWNT3 / RSPO1 and exoWNT3A / RSPO1 treatment groups showed significant expression of hepatocyte markers ALB, AFP, and HNF4α (Figure 3C), while exoWNT3, exoWNT3A, and exoRSPO1 alone showed no significant changes, consistent with exoWT.

[0100] In addition, the inventors also examined the effects of engineered exovesicles containing combinations of other WNT proteins (such as WNT1, WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16) with RSPO1 on pluripotent stem cell differentiation, and found that these combinations could not effectively promote the differentiation of hepatic endodermal cells into hepatocytes (results not shown).

[0101] Example 5. Effects of engineered exosomes of this disclosure on liver regeneration after acute injury.

[0102] Olive oil was used to prepare 25% v / v CCl4 (carbon tetrachloride), and 2.5 μL / g was injected intraperitoneally into C57 mice (n=5). Mice were then treated with exoWT or exoWNT3A / RSPO1 via the tail vein at 24 and 48 hours (2 × 10⁻⁶). 10 / mouse), 24 hours after the second treatment, peripheral blood was collected from the orbital cavity of the mice and liver tissue was collected. Part of the tissue was fixed in 4% paraformaldehyde solution and the rest was frozen in liquid nitrogen. The experimental procedure is shown in Figure 4A.

[0103] Peripheral blood was centrifuged and serum was collected. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were used to detect AST and ALT activities according to the kit instructions to assess liver function damage and repair.

[0104] Liver tissue fixed in paraformaldehyde solution was dehydrated, embedded, sectioned, and then hepatocyte protein markers were labeled by HE staining and immunofluorescence.

[0105] HE staining and ImageJ software analysis showed that exoWNT3A / RSPO1 treatment significantly reduced CCl4-induced fibrosis and necrosis in the central vein of the mouse liver (Figure 4A, Figure 4B). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels indicated effective improvement in liver function in mice (Figure 4C). Immunofluorescence showed that exoWNT3A / RSPO1 treatment significantly increased the proportion of glutamine synthetase (GS) and HNF4-positive hepatocytes around the central vein (CV), and exoWNT3A / RSPO1 promoted liver regeneration after CCl4-induced acute injury (Figure 4D).

[0106] Example 6. Effects of engineered exosomes of this disclosure on APAP-induced acute liver injury.

[0107] c57 mice (n=5) were fasted for 16 hours prior to treatment and then injected intraperitoneally with 600 mg / kg APAP (acetaminophen) to induce an acute liver injury model. At 24 and 48 hours, the mice were treated intravenously with exoWT or exoWNT3A / RSPO1 (2 × 10⁻⁶ mg / kg). 10 / mouse). Twenty-four hours after the second treatment, peripheral blood was collected from the orbital cavity of the mice and liver tissue was collected. Part of the liver tissue was fixed in 4% paraformaldehyde solution, and the rest was frozen in liquid nitrogen. The experimental procedure is shown in Figure 5A.

[0108] Peripheral blood was centrifuged and serum was collected. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were used to detect AST and ALT activities according to the kit instructions to assess liver function damage and repair.

[0109] Liver tissue fixed in paraformaldehyde solution was dehydrated, embedded, sectioned, and then hepatocyte protein markers were labeled by HE staining and immunofluorescence.

[0110] Histopathological results, analyzed using ImageJ software, showed that exoWNT3A / RSPO1 treatment significantly reduced APAP-induced central vein and necrotic areas in mouse livers (Figure 5A, Figure 5B). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels indicated effective improvement in liver function in mice (Figure 5C). Immunofluorescence showed that exoWNT3A / RSPO1 treatment significantly increased the proportion of GS and HNF4α-positive hepatocytes around the central vein, and that exoWNT3A / RSPO1 promoted liver regeneration after APAP-induced acute injury (Figure 5D).

[0111] Furthermore, mouse livers were collected 24 and 48 hours after APAP injury and exosome treatment for evaluation (Figure 6A). Total RNA was extracted from liver tissue, and real-time PCR analysis showed that the cell necrosis marker gene Cxcl1 was significantly elevated 24 hours after injury, but decreased after 48 hours of self-repair; however, the decrease was more significant in the exosome-treated group (Figure 6B). TUNEL staining for apoptosis showed that the exosome-treated group significantly reduced the proportion of apoptotic cells in the injured liver (Figure 6C, Figure 6D). Similarly, the cell proliferation marker gene Ki67 was significantly decreased 24 hours after injury, with no significant self-repair level; however, the exosome-treated group significantly increased the level of proliferating cells (Figure 6E). Ki67 immunofluorescence staining showed that the exosome-treated group significantly increased the proportion of proliferating cells in the injured liver (Figure 6F, Figure 6G). This indicates that exoWNT3A / RSPO1 effectively promotes hepatocyte proliferation while inhibiting apoptosis, thereby promoting liver tissue repair and regeneration after injury.

[0112] Example 7. Effects of engineered exosomes of this disclosure on chronic liver fibrosis

[0113] Olive oil was used to prepare 25% v / v CCl4 (carbon tetrachloride), which was injected intraperitoneally into c57bl / 6 mice at a dose of 2.5 μL / g twice a week to induce a mouse model of chronic liver fibrosis. This treatment was continued for 3 weeks. From weeks 4 to 6, mice (n=5) were treated intraperitoneally with CCl4 twice a week to maintain liver fibrosis, while simultaneously receiving weekly treatment via the tail vein with exoWT or exoWNT3A / RSPO1. After 6 weeks, peripheral blood was collected from the orbital cavity, and liver tissue was harvested. A portion was fixed in 4% paraformaldehyde solution, and another portion was cryopreserved in liquid nitrogen. An illustrative experimental procedure is shown in Figure 7A.

[0114] Peripheral blood was centrifuged and serum was collected. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were used to detect AST and ALT activities according to the kit instructions to assess liver function impairment.

[0115] Liver tissue fixed in paraformaldehyde solution was dehydrated, embedded, sectioned, and the degree of liver fibrosis was marked by Masson staining and Sirius red staining.

[0116] Histopathological results showed that exoWNT3A / RSPO1 treatment significantly reduced CCl4-induced liver fibrosis in mice, manifested in improved liver surface granularity and texture, and a significant reduction in fibrotic areas (Figure 7A, Figure 7B). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assays showed effective improvement in liver function in mice (Figure 7C).

[0117] Example 8. Effects of engineered exosomes of this disclosure on aging

[0118] 18-month-old senescent c57bl / 6 mice were treated via tail vein with exoWT or exoWNT3A / RSPO1 (2×10⁻⁶). 10 / each), twice a week, for a total of 2 weeks. An illustrative experimental procedure is shown in Figure 8A.

[0119] Peripheral blood was collected from the eye sockets of mice, and liver tissue was also collected. Part of the tissue was fixed in 4% paraformaldehyde solution, and the rest was frozen in liquid nitrogen.

[0120] Liver tissue fixed in paraformaldehyde solution was dehydrated, embedded, sectioned, and analyzed by HE staining, β-galactosidase staining, and immunofluorescence labeling of hepatocyte proliferation marker Ki67 and senescence marker p21. Results showed that exoWNT3A / RSPO1 treatment significantly reduced pathological damage and the proportion of β-galactosidase-positive areas in aged mice (Figure 8A), while increasing the number of Ki67-labeled proliferating cells and decreasing the number of p21-labeled senescent cells (Figure 8B).

[0121] Example 9. Effects of engineered exosomes of this disclosure on the growth of alveolar organoids

[0122] Following a method similar to that in Example 1, the gene sequence encoding the full-length WNT5A protein (NP_003383.4) was synthesized and constructed into the pLVX-IRES-tdTomato lentiviral vector to obtain a WNT5A protein lentiviral expression plasmid. The gene sequence encoding the full-length human RSPO2 protein (NP_848660.3) was synthesized and constructed into the pLVX-IRES-EGFP lentiviral vector to obtain an RSPO2 protein lentiviral expression plasmid. After confirming correct Sanger sequencing, the plasmid was used for further experiments.

[0123] The expression plasmid was added to DH5α competent cells, and the cells were transformed by incubation on ice for 30 min, followed by heat shock at 42°C for 90 s and incubation on ice for 2 min. The transformed competent cells were transferred to 500 μL of LB liquid medium and incubated at 37°C for 1 h. After removing some of the supernatant, the cells were thoroughly mixed and spread onto LB solid medium containing ampicillin. The plates were inverted and incubated at 37°C for 12–18 h, and colonies were selected for verification. Positive colonies were added to 20 mL of LB liquid medium containing ampicillin and incubated at 37°C and 180 rpm for 12–18 h. The plasmid was extracted and purified, and its concentration was determined. After confirming accuracy using Sanger sequencing, the cells were used for subsequent procedures.

[0124] HEK293T cells were cultured until the cell confluence reached 70%–80%. WNT5A and RSPO2 protein expression plasmids, lentiviral packaging helper plasmids, and transfection reagents were mixed and co-incubated with HEK293T cells. Virus was collected at 48 h and 72 h, respectively. The cells were centrifuged at 1200 rpm for 5 min, and the supernatant was collected and filtered through a 0.45 μm filter to obtain the WNT5A protein expression virus solution. The solution can be stored at 4 °C for one week or at -80 °C for one year.

[0125] After mixing the WNT5A protein expression virus solution with the infection reagent, it was incubated with HEK293 cells for 24 hours and then replaced with fresh culture medium. After 72 hours, the intensity and proportion of red fluorescence expression were observed. Red fluorescent positive cells, i.e., WNT5A protein stably overexpressing cells, could be obtained by flow cytometry cell sorting.

[0126] After mixing the RSPO2 protein expression virus solution with the infection reagent, the mixture was incubated with WNT5A protein stably overexpressing cells for 24 hours, followed by fresh culture medium. The intensity and ratio of green fluorescence expression were observed after 72 hours. Green fluorescence-positive cells, i.e., WNT5A / RSPO2 protein stably overexpressing cells, could be obtained by flow cytometry cell sorting. These stably overexpressing cells could be expanded and cultured and then cryopreserved in liquid nitrogen.

[0127] The stably expressing cells were expanded and cultured to obtain culture supernatant. The supernatant was then centrifuged at 4°C for 5 min to remove dead cells, at 2,000 g for 10 min to remove cell debris, at 10,000 g for 20 min to collect microvesicles, and at 100,000 g for 10 min to obtain engineered extracellular vesicles / exosomes of WNT5A and RSPO2 proteins. Figure 9A shows a transmission electron microscope image of the engineered WNT5A / RSPO2 exosomes (exoWNT5A / RSPO2).

[0128] Engineered cells, microvesicles, and exosomes were collected, and protein lysis buffer was added. After lysis on ice for 30 minutes, the cells were centrifuged at 12,000 rpm for 20 minutes, and the protein supernatant was collected. The total protein content of cells, microvesicles, and exosomes was detected using a BCA kit. 40 μL of total protein was added to 10 μL of 5× protein loading buffer and boiled at 100°C for 10 minutes to promote protein denaturation. SDS-PAGE gels were prepared, and 10 μL of each sample was added. Electrophoresis was performed at 100V for 100 minutes. Proteins on the gel were transferred to a PVDF membrane using a wet transfer method, and incubated at 250 mA for 2 hours. The membrane was blocked with 5% skim milk powder and incubated with the corresponding primary antibody dilution overnight at 4°C with shaking. The membrane was washed three times with TBST buffer for 10 minutes each time, and incubated with the corresponding HRP-labeled secondary antibody dilution at room temperature with shaking for 22 hours. The membrane was washed three times with TBST buffer for 10 minutes each time, and ECL substrate reaction solution was added to detect the corresponding protein levels (Figure 9B).

[0129] Fresh mouse lung tissue was rinsed 2-3 times with PBS to remove residual blood. Then, alveolar tissue from the lung margin was excised with scissors and immersed in AdDMEM / F12 medium to maintain cell viability. Next, the alveolar tissue was cut into small pieces with a blade and transferred to centrifuge tubes. The remaining tissue pieces were rinsed with AdDMEM / F12 medium and transferred to the centrifuge tubes. Then, an appropriate amount of Collagenase P was added, and the alveolar tissue was digested into single cells at 37°C. The digestion reaction was then terminated by adding 1% BSA in 3 times the volume of the digestion solution. The cells were then centrifuged at 4°C, 1000 rpm for 5 min. The supernatant was carefully aspirated, and the cell pellet was collected. 1 mL of erythrocyte lysis buffer was added to lyse the erythrocytes for 2 min. The cells were centrifuged again at 4°C, 1000 rpm for 5 min to remove the erythrocyte lysis buffer. Then, 1 mL of cell washing buffer (2x Pen / Strep) was added to treat the cells for 2 min. The cells were then centrifuged again at 4°C, 1000 rpm for 5 min to remove the cell washing buffer. Finally, resuspend the cell pellet with an appropriate amount of Matrigel, seed the cell suspension in the center of a 24-well plate, invert it to allow the droplets to solidify, and then add 500 μL of alveolar organoid culture medium. Change the culture medium every 2–3 days, and passage the cells after the organoids have confluently grown into the droplets.

[0130] After passage, mouse alveolar organoids were treated with exoWT, exoWNT5A, exoRSPO2, and exoWNT5A / RSPO2, respectively, and their in vitro growth was observed. The results showed that only the alveolar organoids treated with exoWNT5A / RSPO2 could grow normally and maintain a large organoid size and growth rate (Figure 9C).

[0131] In addition, the inventors also examined the effects of engineered exovesicles containing combinations of other WNT proteins (such as WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16) with RSPO2 on the growth of alveolar organoids, and found that these combinations did not maintain the normal growth of alveolar organoids (results not shown).

[0132] Example 10. Effects of engineered exosomes of this disclosure on endometrial organoid growth and regeneration after endometrial injury.

[0133] Following a method similar to that in Example 1, the gene sequence encoding the full-length WNT7A (NP_004616.2) protein was synthesized, and the WNT7A gene sequence was constructed into the pLVX-IRES-tdTomato lentiviral vector to obtain a WNT7A protein lentiviral expression plasmid. The full-length gene sequence encoding the human RSPO3 (NP_116173.2) protein was synthesized, constructed into the pLVX-IRES-EGFP lentiviral vector to obtain an RSPO3 protein lentiviral expression plasmid. After confirming that the Sanger sequencing was correct, it was used for the next step of the experiment.

[0134] The expression plasmid was added to DH5α competent cells, and the cells were transformed by incubation on ice for 30 min, followed by heat shock at 42°C for 90 s and incubation on ice for 2 min. The transformed competent cells were transferred to 500 μL of LB liquid medium and incubated at 37°C for 1 h. After removing some of the supernatant, the cells were thoroughly mixed and spread onto LB solid medium containing ampicillin. The plates were inverted and incubated at 37°C for 12–18 h, and colonies were selected for verification. Positive colonies were added to 20 mL of LB liquid medium containing ampicillin and incubated at 37°C and 180 rpm for 12–18 h. The plasmid was extracted and purified, and its concentration was determined. After confirming accuracy using Sanger sequencing, the cells were used for subsequent procedures.

[0135] HEK293T cells were cultured until the cell confluence reached 70%–80%. WNT7A and RSPO3 protein expression plasmids, lentiviral packaging helper plasmids, and transfection reagents were mixed and co-incubated with HEK293T cells. Virus was collected at 48 h and 72 h, respectively. The cells were centrifuged at 1200 rpm for 5 min, and the supernatant was collected and filtered through a 0.45 μm filter to obtain the WNT7A protein expression virus solution. The solution can be stored at 4 °C for one week or at -80 °C for one year.

[0136] After mixing the WNT7A protein expression virus solution with the infection reagent, it was incubated with HEK293 cells and other receptors for 24 hours, and then the culture medium was replaced with fresh medium. After 72 hours, the intensity and proportion of red fluorescence expression were observed. Red fluorescent positive cells, i.e., WNT7A protein stably overexpressing cells, could be obtained by flow cytometry cell sorting.

[0137] After mixing the RSPO3 protein expression virus solution with the infection reagent, the mixture was incubated with WNT7A protein stably overexpressing cells for 24 hours, followed by fresh culture medium. The intensity and ratio of green fluorescence expression were observed after 72 hours. Green fluorescence-positive cells, i.e., WNT7A / RSPO3 protein stably overexpressing cells, could be obtained by flow cytometry cell sorting. These stably overexpressing cells could be expanded and cultured and then cryopreserved in liquid nitrogen.

[0138] The stably expressing cells were expanded and cultured to obtain culture supernatant. The supernatant was then subjected to centrifugation at 300g for 5 min at 4°C to remove dead cells, centrifugation at 2000g for 10 min to remove cell debris, centrifugation at 10000g for 20 min to harvest microvesicles, and centrifugation at 10000g for 10 min to obtain WNT7A / RSPO3 protein-engineered extracellular vesicles / exosomes. Figure 10A shows a transmission electron microscope image of the WNT7A / RSPO3 engineered exosomes.

[0139] Engineered cells, microvesicles, and exosomes were collected, and protein lysis buffer was added. After lysis on ice for 30 minutes, the cells were centrifuged at 12,000 rpm for 20 minutes, and the protein supernatant was collected. The total protein content of cells, microvesicles, and exosomes was detected using a BCA kit. 40 μL of total protein was taken, and 10 μL of 5× protein loading buffer was added. The mixture was boiled at 100°C for 10 minutes to promote protein denaturation. SDS-PAGE gels were prepared, and 10 μL of each sample was added. Electrophoresis was performed at 100V for 100 minutes. Proteins on the gel were transferred to a PVDF membrane using a wet transfer method, and the membrane was subjected to a crossflow of 250 mA for 2 hours. The membrane was blocked with 5% skim milk powder and incubated with the corresponding primary antibody dilution overnight at 4°C with shaking. The membrane was washed three times with TBST buffer for 10 minutes each time, and then incubated with the corresponding HRP-labeled secondary antibody dilution at room temperature with shaking for 22 hours. The membrane was washed three times with TBST buffer for 10 minutes each time, and the corresponding protein levels were detected using ECL substrate reaction solution (Figure 10B).

[0140] Fresh human endometrial tissue was rinsed 2–3 times with PBS to remove residual blood. The endometrial tissue was then dissected using scissors and immersed in AdDMEM / F12 medium to maintain cell viability. Collagenase P was added, and the tissue was digested into single cells at 37°C. The digestion was then terminated by adding 1% BSA in 3 times the volume of the digestion solution. The cells were then centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was carefully aspirated, and the cell pellet was collected. 1 mL of erythrocyte lysis buffer was added to lyse the erythrocytes for 2 min. The cells were centrifuged again at 1000 rpm for 5 min at 4°C to remove the erythrocyte lysis buffer. 1 mL of cell washing buffer (2x Pen / Strep) was added to treat the cells for 2 min. The cells were centrifuged again at 1000 rpm for 5 min at 4°C to remove the cell washing buffer. Finally, the cell pellet was resuspended in Matrigel, and the cell suspension was seeded in the center of a 24-well plate. The plate was inverted and allowed to solidify before adding 500 μL of endometrial organoid culture medium. The culture medium was changed every 2–3 days, and the cells were passaged after the organoids had grown into confluent droplets. After passage of human uterine organoids, exoWT, exoWNT7A, exoRSPO3, and exoWNT7A / RSPO3 were added respectively.

[0141] By observing the in vitro growth of endometrial organoids, the results showed that only exoWNT7A / RSPO3 supported the in vitro growth and proliferation of human organoids and maintained the number and size of organoids (Figure 10C).

[0142] Fifteen healthy female SD rats with normal estrous cycles were randomly divided into sham-operated and model groups. In the sham-operated group, only the abdomen was opened without any manipulation of the uterus. In the model group, after abdominal opening, a surgical incision was made on the left side of the uterus in each rat, followed by mechanical curettage of the endometrium. Then, 50 μL each of exoWT, exoWNT7A, exoRSPO3, and exoWNT7A / RSPO3 (4 × 10⁻⁶) were administered. 9 (Particle count) treatment was performed, and a surgical incision was made in the right uterus without any other treatment. After two estrous cycles, uterine tissue was collected from rats in both the sham-operated group and the model group during estrus and stained with hematoxylin and eosin (HE). The results showed that exoWNT7A / RSPO3 treatment significantly promoted the regeneration of the endometrium after injury in rats, as evidenced by the return of endometrial thickness to normal and a significant increase in the number of glands (Figure 10D).

[0143] In addition, the inventors also examined the effects of engineered extravesicles containing combinations of other WNT proteins (such as WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16) with RSPO3 on the endometrium. They found that these combinations could not effectively maintain the normal growth of endometrial organoids, nor did they significantly promote the regeneration of the endometrium after injury (results not shown).

[0144] Example 11. Effects of engineered exosomes of this disclosure on weight regulation, prevention of spontaneous tumors and fatty liver in aging mice.

[0145] exoWNT3A / RSPO1 was transplanted into 18-month-old aged mice via the tail vein, and injected 2×10⁶ mg / week. 10 Exosomes were injected continuously for 12 weeks (Figure 11A). After treatment, the mice's coat and frailty index were assessed, showing that exoWNT3A / RSPO1 effectively improved hair growth and the frailty index in aging mice (Figure 11B). While exoWNT3A / RSPO1 did not affect the overall body weight of the mice, the weight of mice in the treatment group was more uniform (Figure 11C). In aging mice not treated with exoWNT3A / RSPO1, 30% developed solid tumors in multiple organs, including the liver, lungs, small intestine, and lymph nodes; however, no significant tumor development was observed in mice treated with exoWNT3A / RSPO1, suggesting that exoWNT3A / RSPO1 treatment has the potential to inhibit spontaneous tumor development in aging organisms (Figure 11D, Figure 11E). Furthermore, exoWNT3A / RSPO1 treatment effectively improved the severity of fatty liver in aging mice (Figure 11F).

[0146] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles are simultaneously loaded with WNT ligand protein and R-spondin ligand protein.

2. The extracellular vesicle according to claim 1, characterized in that, 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 functional variants or fragments thereof. Preferably, the WNT ligand protein is selected from one or more of WNT3, WNT3A, WNT5A, and WNT7A, or functional variants or fragments 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. The extracellular vesicle according to claim 2, characterized in that, The engineered extracellular vesicles include: a WNT ligand protein selected from WNT3, WNT3A, WNT5A, WNT7A or functional variants or fragments thereof; and an R-spondin ligand protein selected from R-spondin1, R-spondin2, R-spondin3 or functional variants or fragments thereof. Preferably, the engineered extracellular vesicles comprise: WNT3 or its functional variants or fragments; and, R-spondin1 or its functional variants or fragments, and / or WNT3A or its functional variants or fragments; and, R-spondin1 or its functional variants or fragments, and / or WNT5A or its functional variants or fragments; and, R-spondin2 or its functional variants or fragments, and / or WNT7A or a functional variant or fragment thereof; and R-spondin3 or a functional variant or fragment thereof.

4. The extracellular vesicle according to claim 1, characterized in that, The WNT ligand protein is displayed on the surface of the engineered extracellular vesicles. Preferably, the WNT ligand protein binds to the WNT carrier protein WLS protein; Preferably, the WLS protein is selected from the full-length WLS protein or a combination of one or more transmembrane domains of the WLS protein, namely M1, M2, M3, M4, M5, M6, M7 or M8.

5. The extracellular vesicle according to claim 1, characterized in that, The R-spondin ligand protein is displayed on the surface or inside the engineered extracellular vesicles. Preferably, the R-spondin ligand protein is passively loaded or actively loaded onto the engineered extracellular vesicles via a cytoskeletal protein. Preferably, the R-spondin ligand protein is directly linked to the extracellular vesicle cytoskeleton protein or linked through a linker; Preferably, the extracellular vesicle cytoskeleton proteins include transmembrane proteins or membrane anchoring proteins; Preferably, the extracellular vesicle cytoskeleton protein is selected from lysosome-associated membrane protein 2 (LAMP2), LAMP2B, CD63, Tetraspanin, lactobacin C1 / C2, CD9, CD81, PDGFR, GPI ankylosing protein, CD20, Claudin18.2, Claudin6, TM4SF, LAPTM4B, Tarp, CD133, GPRC5D, CXCR4, CCR5, CCR8, SSTR2, L1CAM, β-Klotho, CD147, PTGFRN, BASP1, WLS, or functional variants or fragments thereof.

6. The extracellular vesicle according to claim 1, characterized in that, The engineered extracellular vesicles include microvesicles and exosomes, preferably exosomes. Preferably, the extracellular vesicles are further loaded with exogenous nucleic acids, peptides, proteins, or small molecule drugs.

7. A cell that produces extracellular vesicles according to any one of claims 1 to 6, characterized in that, An exogenous sequence encoding the WNT ligand protein and / or the R-spondin ligand protein is inserted into the genomic sequence of the cell.

8. The cell according to claim 7, characterized in that, The cells include cells derived from human or non-human mammals. Preferably, the non-human mammal is a mouse, rat, monkey, cow, sheep, pig, rabbit, or horse; Preferably, the cells are selected from immortalized cell lines or primary cells; Preferably, the cells are selected from 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. A method for preparing extracellular vesicles according to any one of claims 1 to 6, characterized in that, The method includes the following steps: inserting exogenous sequences encoding WNT ligand proteins and exogenous sequences encoding R-spondin ligand proteins into the genome of a cell.

10. The method according to claim 9, characterized in that, The step of inserting exogenous sequences encoding WNT ligand proteins and R-spondin ligand proteins into the genome of a cell includes: transfecting cells with a recombinant vector carrying exogenous sequences encoding WNT ligand proteins and / or R-spondin ligand proteins. Preferably, the recombinant vector includes a viral vector or a non-viral vector; Preferably, the recombinant vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retroviral vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector; Preferably, the recombinant vector includes at least one of pLVX, pCDH, pQCXIP, pLNCX, pLenti, pRRLSIN, and 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 includes the step of separating the outer vesicles.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the extracellular vesicles and pharmaceutically acceptable carriers described above in this disclosure.

12. The extracellular vesicles of any one of claims 1 to 5, the cells of claim 6 or 7, or the pharmaceutical composition of claim 10 are used for one or more of the following purposes: (1) Promote the differentiation of stem cells, progenitor cells or pluripotent stem cells, preferably into liver cells, skin cells, hair follicle cells, lung cells, intestinal cells, nerve cells and endometrial cells; (2) To prevent, treat or improve diseases or conditions related to WNT signal transduction; (3) Delivery of drugs; (4) Promotes organizational differentiation; (5) Promotes tissue regeneration; (7) Culture of organoids; (8) Prevent or reverse aging; (9) Regulate the weight of the elderly population and prevent spontaneous tumors and fatty liver; (10) Promote liver regeneration after acute injury, reduce the fibrotic area of ​​the liver, improve the granularity and texture of the liver surface, or improve liver function; Preferably, the diseases or conditions associated with WNT signaling include liver diseases, particularly liver injury, liver fibrosis, acute liver failure, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure, cirrhosis, liver fibrosis, portal hypertension, chronic liver dysfunction, 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 to promote liver regeneration after injury; and / or The organoids include those derived from humans or non-human mammals, and the non-human mammals preferably include mice, rats, guinea pigs, sheep, pigs, cattle, dogs, cats, rabbits, alpacas, and horses. Preferably, the organoids include intestinal organoids, heart organoids, stomach organoids, retinal organoids, mammary organoids, prostate organoids, pancreatic organoids, endometrial organoids, nerve organoids, lung organoids, and liver organoids.

13. The use according to claim 12, characterized in that, Engineered exovesicles, including WNT3 or its functional variants or fragments and R-spondin1 or its functional variants or fragments, are intended for one or more of the following purposes: (1) promoting the differentiation of stem cells, progenitor cells or pluripotent stem cells into hepatocytes; (2) preventing or reversing aging, including weight regulation, prevention of spontaneous tumors and fatty liver; (3) promoting liver regeneration, reducing fibrotic areas of the liver, improving the granularity and texture of the liver surface, or improving liver function in cases of acute liver injury; (4) preventing or treating chronic liver fibrosis, and / or Engineered exovesicles, including WNT3A or a functional variant or fragment thereof and R-spondin1 or a functional variant or fragment thereof, are intended for one or more of the following purposes: (1) promoting the differentiation of stem cells, progenitor cells or pluripotent stem cells into hepatocytes; (2) preventing or reversing aging, including weight regulation, prevention of spontaneous tumors and fatty liver; (3) promoting liver regeneration, reducing fibrotic areas of the liver, improving the granularity and texture of the liver surface, or improving liver function in cases of acute liver injury; (4) preventing or treating chronic liver fibrosis, and / or Engineered exovesicles, including WNT5A or its functional variants or fragments and R-spondin2 or its functional variants or fragments, are used to maintain or promote the growth of alveolar organoids, and / or Engineered extravesicles, including WNT7A or its functional variants or fragments and R-spondin3 or its functional variants or fragments, are used for one or more of the following purposes: (1) maintaining or promoting the growth, proliferation, number and / or size of endometrial organoids; (2) promoting regeneration after endometrial injury; and (3) promoting the recovery of endometrial thickness and / or glandular number after endometrial injury.

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