Engineered extracellular vesicles loaded with wnt proteins and uses thereof

Abstract

An engineered extracellular vesicle loaded with Wnt protein and application thereof, an extracellular vesicle loaded with Wnt protein is isolated from cells, and the extracellular vesicle can be applied in the preparation of a drug for tissue repair and regeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to an engineered extracellular vesicle loaded with Wnt protein and application thereof. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by persistent airflow limitation, and has high morbidity and mortality. According to the statistics of the World Health Organization, COPD ranks the third in the major causes of death and the fifth in the world disease economic burden. Long-term inflammatory damage caused by chronic bronchitis and emphysema is the most common cause of COPD. Normal lungs have endogenous regenerative potential. When lung tissue is damaged, quiescent alveolar stem cells can rapidly proliferate and differentiate into type I alveolar epithelial cells to replace and supplement damaged and dead cells, maintain the integrity of the alveolar barrier structure, and thus improve lung function after injury. However, the endogenous regeneration mechanism of the lung of a COPD patient is severely impaired, and it is difficult to spontaneously initiate the repair and regeneration process.

[0003] Further research has found that the repair and regeneration mechanism disorder mediated by the Wnt signaling pathway in alveolar epithelial cells plays a key role in the occurrence and development of COPD. In recent years, the treatment of COPD by activating the Wnt signaling pathway has become a hot spot in this field. However, the strong toxic side effects of small molecule drugs such as CHIR99021 and LiCl that activate the Wnt signaling pathway and the hydrophobic characteristics of Wnt protein make it difficult to develop drugs, which limits clinical translation. Although some researchers are currently trying to develop Wnt receptor agonists, their clinical effects still need to be further verified. Therefore, a new method that can promote the repair and regeneration of tissues, especially damaged lungs, needs to be developed to provide more possibilities for clinical translation. SUMMARY

[0004] In view of this, one of the main purposes of the present application is to provide an engineered extracellular vesicle loaded with Wnt protein and application thereof to transport biologically active Wnt protein to tissue cells and promote the repair and regeneration of tissue cells by activating the Wnt signaling pathway.

[0005] In order to achieve the above-mentioned purpose, as one aspect of the present application, an engineered extracellular vesicle loaded with Wnt protein is provided.

[0006] As another aspect of the present application, the use of V kinds of engineered extracellular vesicles as described above in the preparation of a drug for the repair and regeneration of tissues is provided.

[0007] As a further aspect of the present application, there is provided use of an engineered extracellular vesicle as described above in the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.

[0008] Based on the above technical solution, the engineered extracellular vesicle loaded with Wnt protein and the use thereof of the present application have one or part of the following beneficial effects:

[0009] The engineered extracellular vesicle loaded with Wnt protein provided by the present application has biological activity, can mediate endogenous regeneration mechanism through activation of Wnt signaling pathway, thereby playing a therapeutic role on damaged tissues such as damaged lungs, and has good biocompatibility, which is of great significance for clinical transformation and application research. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a transmission electron microscope image of the engineered extracellular vesicle loaded with Wnt3a protein in Example 1 of the present application.

[0011] Figure 2 is the nanoparticle analysis result of the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0012] Figure 3 is the Western blot identification result of the marker protein in the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0013] Figure 4 is the Western blot identification result of Wnt3a in the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0014] Figure 5 is the TOPFLASH reporter test result of HEK293T cells in Example 1 of the present application.

[0015] Figure 6 is the Annexin-V analysis result in the in vitro repair function test of the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0016] Figure 7 is the immunostaining analysis result in the in vitro repair function test of the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0017] Figure 8 is the mouse model test result of the engineered extracellular vesicle loaded with Wnt3a in Example 1 of the present application.

[0018] Figure 9are the in vivo safety results of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present application. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to specific examples and with reference to the accompanying drawings.

[0020] The present application provides an engineered extracellular vesicle loaded with Wnt protein and its application. In the process of realizing the present application, it is found that by co-transfecting two constructed plasmids in model cells, extracellular vesicles loaded with Wnt3a protein can be successfully isolated. Through the functional verification test of in vitro model and mouse model, it is verified that the extracellular vesicles can achieve the effect of lung tissue cell proliferation by activating Wnt signaling pathway, indicating that the extracellular vesicles loaded with Wnt protein can promote the tissue repair and regeneration based on the activation of Wnt signaling pathway, and has application potential in the treatment of related diseases, especially in the treatment of chronic obstructive pulmonary disease.

[0021] Definitions:

[0022] The term "extracellular vesicles" (Extracellular Vesicles, EVs) used herein refers to membrane vesicular structures secreted by cells with a diameter of 30-200nm (exosomes) or <1000nm (microvesicles), which contain rich inclusions (including proteins, lipids, nucleic acids, etc.), and are involved in intercellular signal transmission. In recent years, as an important cell communication carrier, extracellular vesicles have shown great prospects in the fields of diagnosis and detection, immunotherapy, nucleic acid drug, protein, small molecule delivery therapy, etc. through participating in normal physiological and pathological processes. Due to the cell natural generation property of extracellular vesicles, they have good biocompatibility, extremely low immunogenicity and toxicity. At the same time, extracellular vesicles are also natural nanocarriers that can load functional proteins, RNA, chemical drugs, etc. The surface of extracellular vesicles has rich membrane proteins, which determines its excellent characteristics of recognizing target cells. The cell and tissue targeting specificity can also be given to extracellular vesicles by modification of the surface molecules of extracellular vesicles, so as to further deliver the effective molecules loaded by extracellular vesicles to specific pathological tissues and organs. Combined with the above advantages of extracellular vesicles, they have obtained extensive attention in tissue repair and regeneration as potential therapeutic agents. At present, the technology based on extracellular vesicles is still in the early stage of clinical transformation and application research, and faces many challenges but also has the possibility of creating numerous new discoveries and new technologies.

[0023] As used herein, the term "Wnt signaling pathway" refers to a large family of secreted proteins that mediate development and post-developmental physiological functions by regulating cellular processes. Currently, two types of Wnt secreted proteins have been discovered, one of which is the canonical Wnt signaling pathway dependent on β-catenin, including Wnt1, Wnt2, Wnt3 and Wnt3a; the other is the non-canonical Wnt signaling pathway independent of β-catenin, including Wnt4a and the like.

[0024] In particular, according to some embodiments of the present application, an engineered extracellular vesicle loaded with a Wnt protein is provided.

[0025] According to embodiments of the present application, the extracellular vesicle loaded with the Wnt protein can promote the repair and regeneration of tissues by exerting its biological activity in the activation of the Wnt signaling pathway.

[0026] According to embodiments of the present application, the Wnt protein described above is Wnt3a protein, and an extracellular vesicle loaded with Wnt3a protein is provided, but is not limited thereto, and may, for example, be other Wnt proteins such as Wnt4a protein.

[0027] According to embodiments of the present application, taking Wnt3a protein as an example, Wnt3a is the main ligand of the canonical Wnt signaling pathway, and can promote cell proliferation and inhibit cell apoptosis. It has been found through experiments that when it is loaded into an engineered extracellular vesicle, it can still exert its biological activity, thereby achieving the effect of promoting tissue repair and regeneration.

[0028] According to embodiments of the present application, the engineered extracellular vesicle described above is derived from HEK293T cells, that is, HEK293T is used as a model cell and an extracellular vesicle supply platform to obtain an extracellular vesicle carrying Wnt3a efficiently and having biological activity.

[0029] According to embodiments of the present application, the plasmids required in the preparation of the engineered extracellular vesicle described above are designed and constructed in the laboratory, and are Wnt3a-T2A-WLS plasmid with the sequence shown in SEQ ID NO. 1 and GPC6 ΔGPI -C1C2 plasmid with the sequence shown in SEQ ID NO. 2. By designing and constructing plasmids corresponding to Wnt proteins, extracellular vesicles loaded with different Wnt proteins can be achieved to target specific tissue cells to deliver Wnt proteins.

[0030] According to embodiments of the present application, the engineered extracellular vesicle described above is prepared by the following preparation method: Wnt3a-T2A-WLS plasmid and GPC6 ΔGPI-C1C2 plasmids are co-transfected into HEK293T cells; after transfection, the engineered extracellular vesicles are isolated from the culture supernatant of HEK293T cells.

[0031] According to an embodiment of the present application, the preparation method further comprises: before transfection, inoculating HEK293T cells in a 10 cm culture dish, and rinsing the cells with PBS when cell confluence reaches 70%.

[0032] According to an embodiment of the present application, the transfection is performed by liposome method. For example, the operation of the transfection comprises: co-transfecting the two plasmids into HEK293T cells by using Lipo2000 transfection reagent.

[0033] According to an embodiment of the present application, the operation of isolation comprises: continuing to culture HEK293T cells after transfection, and collecting the culture supernatant of HEK293T cells; and performing multiple centrifugations on the collected culture supernatant to isolate the engineered extracellular vesicles.

[0034] According to some embodiments of the present application, there is also provided a use of the engineered extracellular vesicles in the preparation of a medicament for tissue repair and regeneration, in particular, a medicament for lung tissue repair and regeneration.

[0035] According to an embodiment of the present application, there is also provided a use of the engineered extracellular vesicles in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0036] According to an embodiment of the present application, the engineered extracellular vesicles can activate the endogenous regeneration mechanism mediated by Wnt signaling pathway to promote tissue regeneration, such as lung tissue repair and regeneration.

[0037] The technical solutions of the present application are further described and explained below by means of specific embodiments in conjunction with the accompanying drawings. It should be noted that the following specific embodiments are only illustrative, and the protection scope of the present application is not limited thereto. The drugs or reagents used in the following embodiments are commercially available or obtained by known preparation methods. The methods used in the following embodiments, such as H&E staining and Western blot, are known in the art and can be described by textbooks or related literature, and will not be described here.

[0038] Example 1

[0039] Isolation and identification of Wnt3a-loaded extracellular vesicles.

[0040] 1. Cell transfection and collection of culture supernatant

[0041] The culture system of HEK293T cells is: DMEM basic medium + 10% FBS + 1X penicillin-streptomycin mixture. The cells are digested with 0.25% trypsin, and then inoculated into a 10 em culture dish for adhesion overnight. Wnt3a-T2A-WLS and GPC6 ΔGPI -C1C2 plasmid is transfected into HEK293T cells, and the culture supernatant is collected at 24h, 48h and 72h after transfection, and stored at -80°C for long-term storage.

[0042] 2. Isolation of extracellular vesicles.

[0043] Wnt3a WG EVs are isolated from the collected culture supernatant by ultracentrifugation. The detailed speed and time of ultracentrifugation are as follows: the culture supernatant is centrifuged at 300g for 10 min, the supernatant is collected into a new centrifuge tube, and then centrifuged at 2000g for 10 min, the supernatant is collected into a new centrifuge tube, and then centrifuged at 100000g for 70 min, the supernatant is discarded, and a sufficient amount of sterile PBS is added to the precipitate, and then centrifuged at 100000g for 70 min for extracellular vesicle rinsing. Finally, the above extracellular vesicles are resuspended with PBS and stored at -80°C for long-term storage.

[0044] Comparative Example 1

[0045] Similar operations as in Example 1 are used, except that no transfection of two plasmids is performed to obtain native extracellular vesicles (Native EVs), or only Wnt3a-T2A-WLS is transfected to obtain control extracellular vesicles (Control EVs).

[0046] Performance test and results:

[0047] 1. Identification of extracellular vesicles.

[0048] 1) The isolated extracellular vesicles are detected by transmission electron microscopy, as shown in Figure 1 B of the figure, the extracellular vesicles Wnt3a WG EVs isolated in this example are all double-concave disc-shaped, and the size is about 100 nm; similar to the morphology of the native extracellular vesicles (Native EVs) without modification from HEK293T cells in A, indicating that the EVs loaded with Wnt3a protein do not significantly change its morphological characteristics.

[0049] 2) The particle size distribution and particle concentration of the extracellular vesicles are detected by a nanoparticle analyzer, as shown in Figure 2 Wnt3aWG The particle concentrations of EVs and Naive EVs under different particle size distributions show that the collected extracellular vesicles Wnt3a WG The particle size of EVs is distributed in the range of 150-200 nm.

[0050] 3) Western blot (WB) was used to detect the expression of positive marker proteins (including TSG101 and CD63) and negative marker proteins (Calnexin) in donor cells (TCL) and extracellular vesicles, respectively. For example... Figure 3 As shown, the collected extracellular vesicles all expressed TSG101 and CD63, but not Calnexin, indicating that the extracellular vesicles were successfully prepared with high purity.

[0051] 2. Validation of biological activity

[0052] 1) The levels of Wnt3a, Alix, and β-actin in donor cells and extracellular vesicles were detected using Western blotting (WB) assays, respectively. For example... Figure 4 As shown, unmodified donor cells (Native TCL) and Native EVs do not contain Wnt3a, while Wnt3a protein is expressed in both the modified donor cells and extracellular vesicles. Furthermore, the amount of Wnt3a in vesicles was significantly greater in both plasmids when transfected simultaneously compared to transfecting the Wnt3a-T2A-WLS plasmid alone than in the control group. This indicates that simultaneous transfection of the two plasmids can effectively promote the efficient loading of Wnt3a, thereby enhancing biological activity.

[0053] 2) Cells transfected with TOPFlash luciferase and pRL-TK reporter plasmid were treated with EVs obtained from untransfected plasmids and co-transfected cells with both plasmids, along with the corresponding conditioned medium (CM), for 48 hours. Luciferase activity was then measured. Figure 5 As shown, simply expressing the Wnt3a protein does not guarantee its biological activity in activating the Wnt signaling pathway. Furthermore, simultaneous transfection of Wnt3a with two plasmids... wG EVs can significantly activate the Wnt signaling pathway.

[0054] 3) In vitro repair function test: A549 cells were treated with LPS (50 μg / mL) for 2 days to construct a human alveolar epithelial cell injury model. Then, LiCl (5 mM), rhWnt3a (200 ng / mL), and Native EVs (2 × 10⁻⁶) were added respectively. 9 (particles / mL) and Wnt3a WG EVs (2 × 10⁹ particles / mL) were treated for 2 days. Cells were collected for Annexin-V and immunostaining analysis.Figure 6 , 7 As shown, Wnt3a WG EVs can significantly increase cell proliferation and effectively inhibit apoptosis.

[0055] 4) In vivo repair function verification: A mouse model of emphysema was established by intratracheal instillation of elastase (100 U / kg). On day 7, mice were intravenously injected with Native EVs (2 × 10⁻⁶ U / kg). 9 (particles / dose) and Wnt3a WG EVs (2×10 9 The dose was administered once every two days; daily intraperitoneal injection of LiCl (200 mg / kg) was considered a positive control. Mice were sacrificed after 7 days of treatment for further analysis. As shown in Figure 8, H&E-stained lung tissue sections in Figure A and mean linear intercept (MLI) measurements of alveoli in Figure B indicate that Wnt3a injection... WG The lung tissue of mice treated with EVs was repaired and regenerated; such as Figure 9 The mouse body weight shown indicates the amount of Wnt3a injected. WG The mice that produced EVs were robust and grew rapidly, and did not show any toxic side effects.

[0056] As can be seen from the foregoing, Wnt3a WG Compared to other drugs, EVs can promote cell proliferation by activating the Wnt signaling pathway, thereby achieving the effects of tissue regeneration and repair.

[0057] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered extracellular vesicle loaded with a Wnt protein, wherein the engineered extracellular vesicle is prepared by the method of: Wnt3a-T2A-WLS plasmid of sequence as shown in SEQ ID NO. 1 and GPC6 ΔGPI - C1C2 plasmids were co-transfected into HEK293T cells; isolating the engineered extracellular vesicle from the culture supernatant of the HEK293T cells after transfection.

2. The engineered extracellular vesicle of claim 1, wherein, The Wnt protein is a Wnt3a protein.

3. The engineered extracellular vesicle of claim 2, wherein, The engineered extracellular vesicle is derived from HEK293T cells.

4. The engineered extracellular vesicle of claim 1, wherein, The transfection employs a liposome method.

5. Use of the engineered extracellular vesicle of any one of claims 1 to 4 in the preparation of a medicament for tissue repair and regeneration.

6. Use of the engineered extracellular vesicle of any one of claims 1 to 4 in the preparation of a medicament for treating chronic obstructive pulmonary disease.

7. Use according to claim 5 or 6, characterised in that, The engineered extracellular vesicle is capable of activating endogenous regeneration mechanisms mediated by the Wnt signaling pathway to promote tissue repair and regeneration.

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