Engineered exosome for removing intestinal abnormal protein to treat nervous system diseases

Abstract

The invention discloses an engineered exosome for removing intestinal abnormal protein to treat nervous system diseases, which is prepared by the following steps: carrying out competent ice bath on abnormal protein degrading peptide, plasmids of a fluorescent protein sequence and escherichia coli for a first time, immediately transferring into a 42 DEG C metal bath for a second time, and then immediately transferring into an ice-water mixture for an ice bath for a third time; the preparation method comprises the following steps: adding an antibiotic-free liquid culture medium into a culture medium, carrying out mild shaking culture in a shaking table at 37 DEG C for a fourth time to obtain a bacterial solution, and collecting exosomes from the bacterial solution to obtain the engineered exosomes for removing intestinal abnormal proteins to treat nervous system diseases, by combining the engineered exosome with the core design of colon precise delivery, intelligent colonization support and treatment function programmed coupling, multiple bottlenecks of oral delivery and efficacy regulation of the exosome therapy for central nervous system diseases are systematically solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered exosome for treating nervous system diseases by clearing abnormal intestinal proteins. Background Technology

[0002] Central nervous system diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and some neuroinflammatory diseases, have complex pathogenesis and currently lack effective cures. In recent years, an increasing number of studies have shown that there is a close bidirectional communication network between the gut and the brain, known as the "gut-brain axis."

[0003] Intestinal epithelial cells, as a major component of the intestinal mucosal barrier in the "gut-brain axis," are responsible for nutrient absorption, defense against pathogen invasion, and maintaining intestinal homeostasis. When intestinal epithelial cells are damaged due to factors such as genetic mutations, inflammation, infection, toxin exposure, dysbiosis, or stress, they may produce or release "abnormal proteins." These "abnormal proteins" resulting from intestinal epithelial barrier dysfunction enter the bloodstream and can affect the health of the central nervous system through various pathways. Currently, most clinical treatments for central nervous system diseases can only relieve symptoms and cannot stop disease progression. Therefore, intervening in central nervous system diseases through the intestinal pathway has become an emerging strategy.

[0004] Among these approaches, the use of orally administered live bacteria to colonize the gut and continuously secrete therapeutic engineered exosomes to clear abnormal proteins accumulated in intestinal epithelial cells offers a promising new treatment paradigm for the long-term, minimally invasive management of diseases such as Alzheimer's and Parkinson's. However, the successful implementation of this strategy depends entirely on the safe, precise, and efficient delivery of live engineered bacteria and their generated exosomes to the target sites in the gut and the achievement of stable colonization. Existing oral delivery technologies suffer from a series of fundamental deficiencies in this crucial step, severely hindering the translation and application of this therapy.

[0005] First, regarding gastric acid barrier resistance, the pH-dependent dissolution behavior of existing enteric coatings is poorly matched to the complex gastrointestinal physiological environment. Individual differences in gastric emptying time and duodenal acidity often lead to premature disintegration of the coating in the stomach or proximal duodenum, exposing highly acid-sensitive live bacteria to a strongly acidic environment and causing rapid inactivation. This results in the amount of live bacteria and engineered exosomes secreted to intestinal epithelial cells being delivered far below the threshold for effective colonization. Second, current technologies cannot achieve precise spatiotemporal control of release within the intestine. The colonization efficiency of live bacteria varies significantly in different intestinal segments, with the ideal release site often located in the terminal ileum or colon. Traditional enteric-coated formulations lack intelligent responsiveness to intestinal peristalsis, the microbial microenvironment, and specific biomarkers, often releasing prematurely in non-target intestinal segments. This exposes bacteria to unfavorable oxygen environments or highly competitive native flora, hindering effective colonization. More critically, existing formulations consider the mission complete once live bacteria are delivered, completely lacking sustained support during the crucial early colonization stage. Newly delivered bacteria face multiple pressures, including host immune defense, competition from native flora, and intestinal flushing, making it difficult to effectively release engineered exosomes. Current carrier materials cannot provide a temporary physically sheltered microenvironment after delivery, nor do they integrate prebiotics and other nutrients to support initial bacterial adhesion and proliferation, resulting in unreliable survival rates and colonization stability after "seeding." Furthermore, there is an inherent contradiction between traditional formulation preparation processes and bacterial activity preservation; processes such as heating, cross-linking, or drying can easily damage bacterial activity and genetic stability.

[0006] It is evident that existing treatment systems suffer from a singular design philosophy, focusing solely on "delivery" without systematically integrating with downstream therapeutic functions. They lack the ability to locally regulate the function of colonizing microbiota, cannot intelligently adjust exosome secretion levels or components in response to gut microenvironment signals, and fail to synergistically deliver auxiliary components that enhance exosome brain targeting or regulate the local immune microenvironment. This results in an efficiency bottleneck in the transformation chain from "microbiota colonization" to "effective treatment." Furthermore, they cannot meet the functional requirements of the entire strategy, which relies on the long-term stable colonization of live bacteria and the continuous, controllable secretion of therapeutic exosomes to long-term clear abnormal proteins from intestinal epithelial cells. Therefore, developing a novel oral enteric-coated delivery system capable of overcoming these multiple obstacles is crucial for realizing next-generation central nervous system disease therapies based on live bacteria-engineered exosomes.

[0007] Based on this, the purpose of this invention is to address the shortcomings of the prior art by providing an engineered exosome for clearing abnormal intestinal proteins to treat nervous system diseases. This invention achieves highly efficient oral protection of engineered bacteria, targeted intestinal colonization, and programmed regulation of subsequent therapeutic functions, providing a transformative solution for treating central nervous system diseases through the brain-gut axis pathway. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide an engineered exosome for treating nervous system diseases by clearing abnormal intestinal proteins, and an oral enteric-coated formulation containing the engineered exosome and live bacteria, thereby overcoming the multiple bottlenecks in oral delivery and efficacy regulation of exosome therapy for central nervous system diseases in existing technologies.

[0009] Specifically, the technical problem to be solved by the present invention is to address the shortcomings of the prior art. In the first aspect, the present invention provides an engineered exosome for clearing abnormal intestinal proteins to treat nervous system diseases. The process involves first bathing plasmids containing abnormal protein degradation peptides and fluorescent protein sequences with competent E. coli cells on ice for a first time, then immediately transferring them to a 42°C metal bath for a second time, and then immediately transferring them to an ice-water mixture on ice for a third time. Antibiotic-free liquid culture medium is added, and the mixture is then gently shaken and cultured at 37°C for a fourth time to obtain a bacterial culture. Exosomes are collected from the bacterial culture to obtain the engineered exosome for clearing abnormal intestinal proteins to treat nervous system diseases.

[0010] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the abnormal protein degradation peptides include PROTAC, AUTAC, ATTEC, and LYTAC.

[0011] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the fluorescent protein sequence includes EGFP, mCherry, and mPlum.

[0012] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the first time is 10 to 30 minutes.

[0013] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the second time is 30 to 90 seconds.

[0014] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the third time is 2 to 10 minutes.

[0015] In one embodiment of the engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins according to the present invention, the fourth time is 45 to 60 minutes.

[0016] In a second aspect, the present invention provides an oral enteric-coated formulation loaded with live bacteria-engineered exosomes, the oral enteric-coated formulation comprising the engineered exosomes as described above.

[0017] Thirdly, the present invention provides a method for preparing an oral enteric-coated formulation loaded with live bacteria-engineered exosomes, comprising the following steps: Step 1: Screen positive monoclonal colonies of the bacterial culture using antibiotic plate culture medium, expand the culture of single colonies, and identify positive strains by sequencing and Western blot. Step 2: Expand the culture of the positive strain identified in Step 1 until the OD is 0.6-0.8, dispense the bacterial solution into vials, mix it with an equal volume of enteric-coated ingredients, pre-freeze at -80℃ overnight, and then freeze-dry for 12-24 hours using a vacuum freeze dryer.

[0018] In one embodiment of the method for preparing an oral enteric-coated formulation of live bacteria-engineered exosomes according to the present invention, the enteric-coated formulation comprises chitosan, sodium alginate, gelatin, skim milk powder, sodium ascorbate, and sucrose.

[0019] Compared with the prior art, the positive effects of the present invention are: 1. First, existing oral live bacteria-engineered exosome formulations generally face challenges such as gastric acid inactivation and ineffective release in non-target intestinal segments. One of the core advantages of this invention lies in its unique "pH-enzyme-time triple response" gel delivery system, which does not rely on a single pH trigger. First, the use of outer enteric-coating components such as chitosan, sodium alginate, and hydroxypropyl methylcellulose ensures the integrity of the formulation in the stomach and upper small intestine. Second, the use of colon-specific enzyme-sensitive polysaccharide polymers such as calcium pectin and chitosan / azo polymer complexes enables the in-situ release of active ingredients in the colon. Finally, after the live bacteria arrive in the colon, the live bacteria gel network undergoes specific dissociation under the action of intestinal enzymes such as cellulase, pectinase, and xylanase, and the release of exosomes is further controlled by an inducer. This mechanism ensures that more than 90% of the active bacteria are delivered intact to the colon and that effective exosomes are accurately released upon arrival. Compared with traditional enteric coating technology, this invention not only achieves precise control of the delivery site, but its gel sustained-release characteristics also simulate the process of "microbial seeding", providing a key time window for live bacteria to competitively occupy ecological niches.

[0020] 2. Secondly, addressing the shortcomings of existing technologies that treat the delivery carrier and live bacteria function as independent modules, the greatest breakthrough of this invention lies in transforming the enteric-coated formulation from a passive "container" into an active "initiation platform" and "signal regulator." After the gel matrix is ​​released into the colon, the three-dimensional network formed by its hydration not only provides physical protection for the engineered bacteria, but is also composed of pre-loaded specific prebiotics (such as fructooligosaccharides), adhesion-enhancing peptides (such as adhesin analogs), and pH-sensitive nutrient microspheres. This design actively creates a local microenvironment conducive to the initial survival, adhesion, and proliferation of live bacteria, leaping from "delivery" to "cultivation," ensuring the continuous production of engineered exosomes that have a clearing effect on abnormal intestinal epithelial proteins.

[0021] 3. Finally, this invention has significant advantages in terms of large-scale production and product stability. Utilizing low-temperature in-situ ionic crosslinking and gentle emulsification encapsulation technology, it completely avoids damage to the viability of engineered bacteria caused by high temperatures, organic solvents, or severe shearing, achieving highly efficient encapsulation with a high viable bacterial load. Simultaneously, by precisely controlling the glass transition temperature and water activity of the gel matrix, combined with a vacuum freeze-drying process, solid formulations that are stable for long-term storage at room temperature can be prepared. These formulations can rapidly hydrate and restore their function upon use, solving the problems of storage stability and reliance on cold chain transportation for live bacterial formulations.

[0022] In summary, the oral enteric-coated formulation of live bacteria-engineered exosomes disclosed in this invention systematically solves multiple bottlenecks in oral delivery and efficacy regulation of exosome therapy for central nervous system diseases through its core design of precise colonic delivery, intelligent colonization support, and programmed coupling of therapeutic functions. This invention not only provides a novel, orally administered, and long-lasting "active exosome factory" treatment model for neurodegenerative diseases but also offers an innovative technological platform for other disease treatment strategies based on exosome intervention along the "gut-brain axis," possessing significant clinical translational potential and commercial value. Attached Figure Description

[0023] Figure 1 The image shows the detection results of the positive strains and engineered exosomes successfully detected by WB in this invention.

[0024] Figure 2 This is a graph showing the results of detecting the residence time of live bacterial preparations in the intestine using mouse in vivo imaging in this invention.

[0025] Figure 3 This is a diagram showing the results of Western blot analysis in this invention demonstrating that the oral enteric-coated formulation can clear abnormal proteins in the intestinal epithelium of a mouse model of central nervous system disease. Detailed Implementation

[0026] Hereinafter, exemplary embodiments according to this application will be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments of this application. It should be understood that this application is not limited to the exemplary embodiments described herein.

[0027] The following is combined with Figure 1-3 The present invention will be further described with reference to specific embodiments. The terms used in the specification and accompanying drawings are defined as follows: PROTAC: Proteolysis Targeting Chimeras AUTAC: Autophagy-targeting Chimera (AUTAC) ATTEC: Autophagosome-Tethering Compound (ATTEC) LYTAC: Lysosome-Targeting Chimaeras (LYTAC) EGFP: Enhanced Green Fluorescent Protein (EGFP) MCherry: Monomeric red fluorescent protein (a red fluorescent protein derived from mushroom coral; the "m" in mCherry stands for "monmer," indicating that the mCherry fluorescent protein is in monomeric form, MCherry). mPlum: Deep red fluorescent protein The preparation process of this invention is as follows: ① Strains Construction: Plasmids containing abnormal protein degradation peptides such as PROTAC, AUTAC, ATTEC, and LYTAC, as well as fluorescent protein sequences such as EGFP, mCherry, and mPlum, were incubated with competent *E. coli* cells on ice for 10–30 minutes. Immediately afterwards, the cells were transferred to a 42°C metal bath for 30–90 seconds, followed by an immediate transfer to an ice-water mixture for 2–10 minutes. Antibiotic-free liquid culture medium was added, and the cells were then gently incubated at 37°C with shaking for 45–60 minutes to obtain the bacterial culture. Positive single colonies were screened using antibiotic-treated agar plates. Single colonies were expanded and identified by sequencing and Western blotting to determine the expression of positive strains and engineered exosome proteins.

[0028] ② Preparation of enteric-coated formulation: When the identified positive strains are cultured to an OD of 0.6-0.8, the bacterial solution is divided into vials and mixed with chitosan, sodium alginate, gelatin, skim milk powder, sodium ascorbate, sucrose and other components in equal volumes. After pre-freezing at -80℃ overnight, the mixture is freeze-dried in a vacuum freeze dryer for 12-24 hours.

[0029] ③ Gavage and in vivo imaging: The prepared formulation was administered to 6-8 week old C57BL / 6 mice via gavage. The mice were sacrificed and dissected at 0, 2, 4, 8, 12, 24, 48, and 72 hours, and the intestinal fluorescence signal was detected by IVIS spectral imaging system.

[0030] ④ Abnormal protein clearance assay: The prepared formulation was administered to 2-3 month old central nervous system disease model mice by gavage 1-2 times per week until 9-10 months of age. The clearance of abnormal proteins in the intestinal epithelium of the mice was measured by Western blot (WB) experiment.

[0031] The experimental results are as follows: Example 1: Successful detection of positive strains and engineered exosomes by Western blotting (results are shown in...) Figure 1 To confirm the successful loading of the exogenous plasmid into the engineered bacterial strain (E. coli) and the successful expression of the target protein in E. coli and the engineered bacterial exosomes (E. coli-EV), the purified bacterial culture and exosome samples were subjected to protein lysis buffer extraction, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then Western blot analysis. After incubation with a specific primary antibody targeting the target protein, the sample was developed using a horseradish peroxidase (HRP)-labeled secondary antibody and an enhanced chemiluminescence (ECL) reagent. Figure 1 As shown, a positive band corresponding to the expected molecular weight appears, indicating that the target protein is successfully expressed in the strain and its secreted exosomes.

[0032] Example 2: The long-term residence of this formulation in the intestine was demonstrated using a mouse in vivo imaging system (results are shown in...). Figure 2 This invention employs an In Vivo Imaging System (IVIS), a highly sensitive optical imaging instrument for non-invasive, real-time observation of biological processes within living animals. It is primarily used in preclinical research (such as in laboratory animals like mice and rats) to visualize cellular or molecular activity by detecting light signals on the animal's surface. This invention provides real-time, dynamic visualization and monitoring of the retention of formulations in the mouse intestine to verify their long-term residence characteristics in the intestinal lumen. Figure 2As shown in the experiment, mice were administered a strain of engineered bacteria containing the detectable fluorescent protein MPL (mplum) via gavage. The mice were euthanized at 0, 2, 4, 8, 12, 24, 48, and 72 hours post-administration, and their gastrointestinal tissues were harvested. Fluorescence signals from the mouse tissues were acquired using an IVIS imaging system at specific excitation and emission wavelengths. The results showed that the strain maintained a high and stable signal intensity in the intestine for a prolonged period after administration, indicating a significant retention capacity in the intestinal lumen and a sustained local effect.

[0033] Example 3: Western blot analysis demonstrated that this enteric-coated formulation could clear abnormal proteins in the intestinal epithelium of a mouse model of central nervous system disease (see results). Figure 3 To verify the effect of the enteric-coated formulation constructed in this invention on clearing abnormally aggregated proteins in the intestinal epithelial tissue of a central nervous system disease model mouse, we used Western blot (WB) technology for detection and analysis. First, a mouse model with a central nervous system lesion phenotype (A53T-SNCA transgenic mouse model) was selected and randomly divided into wild-type mouse control group (SNCA-), model mouse control group (SNCA+), induced group (SNCA+Inducer+), non-induced group (SNCA+Inducer-), degraded peptide group (SNCA+Peptide+), engineered bacteria group (SNCA+E. coli+), and enteric-coated formulation treatment group (SNCA+E. coliPeptide+). After gavage administration, colon tissue samples were collected from the mice and immediately flash-frozen in liquid nitrogen and stored at –80 °C to prevent protein degradation.

[0034] During tissue protein extraction, the protein was thoroughly homogenized and sonicated using a protein lysis buffer containing protease inhibitors and phosphatase inhibitors. The supernatant, or total protein lysate, was obtained by centrifugation at 10,000g at 4°C. The protein concentration of each sample was precisely quantified using the BCA method to ensure consistent loading. Equal volumes of protein samples were then separated by SDS-PAGE electrophoresis and electrotransferred to a PVDF membrane. After blocking the membrane with 5% BSA, it was incubated with a specific primary antibody against the target protein α-syn, followed by binding with the corresponding HRP-labeled secondary antibody. The final protein signal was visualized by enhanced chemiluminescence (ECL). Figure 3 As shown, the results indicated that, compared with the control group, the band intensity of the target abnormal protein in the intestinal tissue of mice treated with the enteric-coated preparation was significantly reduced, indicating that the preparation can effectively reduce the α-syn protein load in the intestinal epithelium of the pathological model.

[0035] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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.

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined in this invention may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins, characterized in that, Plasmids containing abnormal protein degradation peptides and fluorescent protein sequences were incubated with competent E. coli cells on ice for the first time, then immediately transferred to a 42°C metal bath for the second time, and then immediately transferred to an ice-water mixture for the third time. Antibiotic-free liquid culture medium was added, and the cells were then gently shaken and cultured at 37°C for the fourth time to obtain a bacterial culture. Exosomes were collected from the bacterial culture to obtain the engineered exosomes used to clear abnormal intestinal proteins for the treatment of nervous system diseases.

2. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The abnormal protein degradation peptides include PROTAC, AUTAC, ATTEC, and LYTAC.

3. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The fluorescent protein sequences include EGFP, mCherry, and mPlum.

4. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The first time is 10 to 30 minutes.

5. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The second time is 30 to 90 seconds.

6. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The third time interval is 2 to 10 minutes.

7. The engineered exosomes for treating nervous system diseases by clearing abnormal intestinal proteins as described in claim 1, characterized in that, The fourth time period is 45 to 60 minutes.

8. An oral enteric-coated formulation loaded with live bacteria-engineered exosomes, characterized in that, The oral enteric-coated formulation comprises engineered exosomes as described in any one of claims 1 to 7.

9. The method for preparing the oral enteric-coated formulation of live bacteria-engineered exosomes as described in claim 8, characterized in that, Includes the following steps: Step 1: Screen positive monoclonal colonies of the bacterial culture using antibiotic plate culture medium, expand the culture of single colonies, and identify positive strains by sequencing and Western blot. Step 2: Expand the culture of the positive strain identified in Step 1 until the OD is 0.6-0.8, dispense the bacterial solution into vials, mix it with an equal volume of enteric-coated ingredients, pre-freeze at -80℃ overnight, and then freeze-dry for 12-24 hours using a vacuum freeze dryer.

10. The preparation method according to claim 9, characterized in that, The enteric-coated formulation comprises chitosan, sodium alginate, gelatin, skim milk powder, sodium ascorbate, and sucrose.

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