Method for oral delivery of protease by using T0SS secretion system through recombinant probiotics and application thereof

Abstract

The present invention relates to a recombinant Escherichia coli comprising at least one therapeutically active ingredient, preferably a therapeutic enzyme, in its outer membrane vesicles (OMV). The invention also relates to the use of this recombinant EcN to penetrate the intact intestinal epithelial barrier through OMV to deliver said therapeutically active ingredient, preferably a therapeutic enzyme, into the circulation. The OMV containing the therapeutic enzyme can stably catalyze various detoxification reactions in circulation, and is particularly used for clinically treating metabolic disorders (such as hyperuricemia) and the like.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering. More specifically, the present invention relates to an engineered bacterial TOSS system and its use in oral systemic delivery of therapeutically active ingredients (such as proteases) to treat diseases. Background Art

[0002] Among various drug delivery methods, oral administration has advantages in terms of non-invasiveness, patient compliance, and convenience. However, due to their easy degradation, fragile tertiary structure, and poor gastrointestinal epithelial permeability, few therapeutic proteins have been approved for oral administration in clinical practice. There is an urgent need to develop oral protein delivery systems that should overcome the multiple physiological barriers of the gastrointestinal tract, including irritating acids and proteases in the stomach, the sticky mucus layer, and the epithelial cell barrier of the tight junctions of the intestine. To overcome these absorption barriers, nanoparticle (NP)-based drug delivery systems have been developed for oral protein delivery, but NP systems face complex manufacturing procedures, inefficient cargo loading, and delivery. Therefore, the development of convenient and efficient oral protein delivery systems is of great significance for the clinical use of therapeutic proteins.

[0003] Genetically engineered commensal bacteria are promising living medicines, however, the therapeutic molecules they produce are often restricted around the area of ​​their intestinal colonization. The phase 3 clinical failure of the engineered probiotic SYNB1934 in the treatment of phenylketonuria may be attributed to its limited efficacy in reducing toxic blood metabolites by degrading metabolites only in the intestine (Adolfsen, KJ et al. Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering. Nat. Commun. 12, 6215 (2021); Puurunen, MK et al. Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1 / 2 study. Nat. Metab. 3, 1125-1132 (2021); Vockley, J. et al. Efficacy and safety of a synthetic biotic for treatment of phenylketonuria: a phase 2 clinical trial. Nat. Metab. 5, 1685-1690 (2023)). How to deliver a stable therapeutic active ingredient (e.g., protein) in the digestive tract and further pass through the intestinal barrier into the circulation is a major challenge in achieving systemic delivery of oral commensal bacteria.

[0004] Outer membrane vesicles (OMV) are considered to be bacterial type zero secretion systems (TOSS), which mediate microbe-host or microbe-microbe interactions in the intestine (Guerrero-Mandujano, A., Hernández-Cortez, C., Ibarra, JA & Castro-Escarpulli, G. The outer membrane vesicles: secretion system type zero. Traffic 18, 425-432 (2017)). OMV is formed by budding of the outer membrane and then separated from the cell body, and the contents of the OMV of Gram-negative bacteria are separated from the cytoplasm by the inner membrane. Escherichia coli Nissle 1917 (EcN) is a classic Gram-negative probiotic chassis used in synthetic biology and has been approved by the FDA as GRAS (generally recognized as safe) (Sonnenborn, U. & Schulze, J. The non-pathogenic Escherichia coli strain Nissle 1917-features of a versatile probiotic. Microbial. Ecology in Health and Disease 21, 122-158 (2009)). Summary of the invention

[0005] The present invention provides a technology for oral delivery of proteins via outer membrane vesicles (OMVs) using an engineered bacterial type zero secretion system (TOSS) Figure 1 ).

[0006] The present invention is based on the following unexpected discovery: OMVs produced in situ by Escherichia coli such as Nissle 1917 (EcN) in the intestine can penetrate the complete intestinal epithelial barrier and enter the circulation, and the penetration process involves endocytosis and actin-dependent endocytosis. The engineered EcN is loaded with various enzymes into the OMVs, and the secreted OMVs of the encapsulated enzymes can stably catalyze various detoxification reactions in the presence of digestive juices and enter the circulation. This protein secretion system based on TOSS is compatible with various protein payloads, and different protein cargoes can be encapsulated into the OMVs, making the protein-loaded OMVs promising to become biocatalysts for various detoxification reactions. Using hyperuricemia mice and uricase OMVs as examples, we have demonstrated that the therapeutic effect of the engineered EcN equipped with this improved TOSS is superior to the direct secretion protein system. The OMVs of the encapsulated enzymes can also effectively detoxify human serum samples, highlighting the potential of the system for clinical treatment of metabolic disorders.

[0007] Therefore, according to one aspect of the present invention, a recombinant Escherichia coli such as Nissle 1917 (EcN) is provided, which comprises at least one (endogenous or exogenous) therapeutically active ingredient in its outer membrane vesicles (OMV), wherein the therapeutically active ingredient is selected from therapeutic peptides, therapeutic proteins, antigens, therapeutic nucleic acids and small molecule compounds, and preferably the (endogenous or exogenous) therapeutically active ingredient is a therapeutic peptide or therapeutic protein encapsulated in the OMV.

[0008] In one embodiment, the therapeutically active ingredient is a therapeutic enzyme, preferably a therapeutic enzyme for metabolic diseases (preferably in the circulatory system), genetic diseases, cardiovascular diseases, gastrointestinal diseases, inflammatory diseases or cancer, more preferably wherein:

[0009] 1) The metabolic disease is selected from abnormal glucose metabolism, abnormal lipid metabolism, abnormal calcium-phosphorus metabolism, abnormal bone metabolism and abnormal purine metabolism. Most preferably, the metabolic disease is hyperuricemia and / or the therapeutic enzyme is uricase, the metabolic disease is peroxisomal disease and / or the therapeutic enzyme is catalase, or the metabolic disease is hyperlactatemia and / or the therapeutic enzyme is lactate oxidase, or the metabolic disease is hyperalcoholism and / or the therapeutic enzymes are ethanol oxidase and acetaldehyde oxidase;

[0010] 2) the hereditary disease is selected from phenylketonuria, mucopolysaccharidosis, Pompe disease, Gaucher disease, Fabry disease, severe combined immunodeficiency due to adenosine deaminase deficiency, hypophosphatasia and lysosomal acid lipase deficiency, preferably the therapeutic enzyme is selected from phenylalanine deaminase, α-L-iduronidase, iduronidase sulfate, α-glucosidase, alglucosidase, β-galactosidase, adenosine deaminase, alkaline phosphatase and lysosomal acid lipase, most preferably, the metabolic disease is phenylketonuria and / or the therapeutic enzyme is phenylalanine deaminase;

[0011] 3) the cardiovascular disease is selected from coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism, and the therapeutic enzyme is preferably selected from streptokinase, urokinase, tissue-type plasminogen activator (t-PA), snake venom defibrase, hirudin, lumbrokinase, nattokinase and desmoteplase;

[0012] 4) The gastrointestinal disease is selected from dyspepsia, pancreatic secretion deficiency and gallbladder fibrosis, and the therapeutic enzyme is preferably selected from protease, amylase, lipase, cellulase and pancreatin;

[0013] 5) the inflammatory disease is selected from AIDS, endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, food poisoning, skin infection (such as folliculitis, abscess and mastitis, etc.) and burns, and the therapeutic enzyme is preferably selected from lysozyme (such as lysostaphin), trypsin, chymotrypsin, bromelain, collagenase and chondroitinase; and / or

[0014] 6) The cancer is selected from acute lymphocytic leukemia, lymphosarcoma cell leukemia, granulocytic leukemia, melanoma, lung cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, glioblastoma multiforme and thoracic vertebral cancer. Preferably, the therapeutic enzyme is a metabolic enzyme and a prodrug converting enzyme of amino acids required by the tumor, and is more preferably selected from L-asparaginase, arginine deiminase, glutaminase, glucose oxidase, kynurenine enzyme, carboxypeptidase A and β-glucuronidase.

[0015] In another embodiment, the therapeutic nucleic acid is selected from small interfering nucleic acid (siRNA), micro RNA (miRNA), antisense nucleotide (ASO) messenger RNA (mRNA), and aptamer.

[0016] In another preferred embodiment, the recombinant E. coli, such as Nissle 1917 (EcN), is deficient in the nlpI gene. OMV biosynthesis is increased by genomic deletion of the outer membrane lipoprotein encoding gene nlpI. In a preferred embodiment, the nucleotide sequence of the nlpI gene is shown in SEQ ID No.1.

[0017] In another preferred embodiment, the therapeutic peptide or therapeutic protein is fused with a signal peptide secreted into OMV, preferably the signal peptide is selected from Sec signal peptide, Tat signal peptide and Srp signal peptide. In a preferred embodiment, the amino acid sequences of Sec signal peptide, Tat signal peptide and Srp signal peptide are shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.

[0018] In another preferred embodiment, the peptide or therapeutic protein is selected from uricase, catalase, lactate oxidase, phenylalanine deaminase, alcohol oxidase, acetaldehyde oxidase, or a combination thereof.

[0019] In another aspect of the present invention, provided is an outer membrane vesicle (OMV) derived from or prepared by the recombinant Escherichia coli such as Nissle 1917 (EcN) according to the present invention, wherein the OMV comprises the at least one therapeutically active ingredient, and preferably the OMV is not surface-modified.

[0020] In another aspect according to the present invention, there is provided a method for preparing an OMV according to the present invention, the method comprising the step of expressing a therapeutic peptide or therapeutic protein in the periplasm of Escherichia coli such as Nissle 1917 (EcN).

[0021] In a specific embodiment, a therapeutic peptide or therapeutic protein is expressed in the periplasm of EcN using an expression vector comprising a nucleic acid sequence encoding the therapeutic peptide or therapeutic protein operably linked to a nucleic acid encoding a signal sequence for a periplasmic protein.

[0022] Targeting of therapeutic peptides or therapeutic proteins can be achieved by fusing the signal sequence of proteins naturally found in the periplasm and / or OMVs to the therapeutic peptide or therapeutic protein. Protein translocation through the inner membrane and into the periplasm, for example, can be accompanied by one of the following three pathways: SecB-dependent (SEC), signal recognition particle (SRP) or twin arginine ectopic (TAT).

[0023] In a specific embodiment, the therapeutic peptide or therapeutic protein is selected from uricase, catalase, lactate oxidase, phenylalanine deaminase, ethanol oxidase, acetaldehyde oxidase or a combination thereof, preferably the expression of the therapeutic peptide or therapeutic protein is controlled by an inducible or constitutive promoter (i.e., operably linked to an inducible or constitutive promoter), and more preferably the promoter is selected from tac promoter, fnrs promoter, nar promoter, nar618 promoter and J23117 promoter. In a preferred embodiment, the nucleotide sequences of tac promoter, fnrs promoter, nar promoter, nar618 promoter and J23117 promoter are shown in SEQ ID No.30, SEQ ID No.26, SEQ ID No.27, SEQ ID No.28 and SEQ ID No.29, respectively.

[0024] In one embodiment, the catalase is derived from Escherichia coli.

[0025] In a specific embodiment, the uricase is derived from C. utilis or S. auratus AGR0001.

[0026] In a specific embodiment, the phenylalanine deaminase is derived from P. luminescens or Y. enterocolitica.

[0027] In one embodiment, the lactate oxidase is derived from A. viridans.

[0028] In a specific embodiment, the acetaldehyde oxidase is derived from P. blakesleeanus NRRL1555 or C. reinhardtii or C. cardunculus var. scolymus or L. sativa or S. punctatus DAOMBR117.

[0029] In one embodiment, recombinant E. coli, such as Nissle 1917 (EcN), has the outer membrane lipoprotein encoding gene nlpI deleted from its genome to increase OMV biosynthesis.

[0030] According to another aspect of the present invention, a pharmaceutical composition for oral systemic administration for treating a subject is provided, comprising: 1) recombinant Escherichia coli such as Nissle 1917 (EcN) according to the present invention or the outer membrane vesicle (OMV) according to the present invention; and 2) a pharmaceutically acceptable adjuvant, wherein the pharmaceutical composition, recombinant Escherichia coli such as Nissle 1917 (EcN) or OMV is formulated for and / or (completely / only) relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the subject's circulatory system to achieve systemic administration.

[0031] According to another aspect of the present invention, a pharmaceutical composition for systemic administration to treat the intestinal mucosa of a subject is provided, comprising: 1) recombinant Escherichia coli such as Nissle 1917 (EcN) according to the present invention or the outer membrane vesicle (OMV) according to the present invention; and 2) a pharmaceutically acceptable adjuvant, wherein the pharmaceutical composition, recombinant Escherichia coli such as Nissle 1917 (EcN) or OMV is formulated for and / or (completely / only) relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the circulatory system of the subject to achieve systemic administration.

[0032] According to another aspect of the present invention, there is provided the use of the recombinant Escherichia coli such as Nissle 1917 (EcN) or outer membrane vesicles (OMV) according to the present invention in the preparation of a drug for oral systemic administration or intestinal mucosal systemic administration (to treat a subject), wherein the drug, recombinant Escherichia coli such as Nissle 1917 (EcN) or OMV is formulated for and / or (completely / only) relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the circulatory system (of the subject) to achieve systemic administration.

[0033] According to another aspect of the present invention, there is provided the use of the recombinant Escherichia coli such as Nissle 1917 (EcN) or outer membrane vesicles (OMV) according to the present invention in the preparation of a drug for oral systemic administration or intestinal mucosal systemic administration (to treat a subject), wherein the drug, recombinant Escherichia coli such as Nissle 1917 (EcN) or OMV is formulated for and / or (completely / only) relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the circulatory system (of the subject) to achieve systemic administration.

[0034] In a preferred embodiment, the therapeutically active ingredient is uricase and / or the subject suffers from hyperuricemia, or the therapeutically active ingredient is catalase and / or the subject suffers from a peroxisomal disease, or the therapeutically active ingredient is lactate oxidase and / or the subject suffers from hyperlactatemia, or the therapeutically active ingredient is phenylalanine deaminase and / or the subject suffers from phenylketonuria, or the therapeutically active ingredient is ethanol oxidase and / or acetaldehyde oxidase and / or the subject suffers from hyperalcoholemia.

[0035] In a preferred embodiment, the therapeutic enzyme is derived from a microorganism, a plant or an animal, such as a mammal, more preferably a microorganism.

[0036] In a preferred embodiment, the amino acid sequence of the uricase is shown as SEQ ID No.5 or SEQ ID No.31.

[0037] In a preferred embodiment, the amino acid sequence of the catalase is shown as SEQ ID No.6.

[0038] In a preferred embodiment, the amino acid sequence of the lactate oxidase is shown as SEQ ID No.7.

[0039] In a preferred embodiment, the amino acid sequence of the phenylalanine deaminase is shown as SEQ ID No.8, SEQ ID No.32 or SEQ ID No.33.

[0040] In a preferred embodiment, the amino acid sequence of the acetaldehyde oxidase is shown as SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37, or SEQ ID No.38.

[0041] In a preferred embodiment, the therapeutic enzyme is expressed together with a marker protein or a tag protein fusion. Preferably, the marker protein or tag protein is selected from GFP protein (SEQ ID No.22), RFP protein (SEQ ID No.23), His tag protein (SEQ ID No.24) and HA tag protein (SEQ ID No.25). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0043] Figure 1 Schematic representation of oral delivery of therapeutic proteins by synthetic commensal bacteria, detoxifying blood metabolites via an improved T0SS. EcNs were engineered to increase OMV biogenesis by genomic deletion of the outer membrane lipoprotein encoding gene nlpI, heterologously overexpress the protein of interest (POI) on a stable plasmid in the absence of antibiotic selection (by deleting the essential gene thyA in the genome and compensating it on a plasmid), and endogenously load the POI into OMVs under the guidance of a periplasmic-targeted secretion signal peptide. After oral delivery of our engineered EcNs with an improved T0SS, the protein-loaded OMVs produced in situ by the engineered EcNs resisted proteolysis, penetrated the intact intestinal epithelial barrier, and entered the circulation. Pinocytosis and actin-dependent pathways were involved in this transcellular transport. Engineered EcNs equipped with an improved T0SS can deliver therapeutic proteins to the circulation and detoxify blood metabolites, providing a strategy for oral protein delivery.

[0044] Figure 2 .Yield and morphological analysis of OMVs derived from wild-type EcN and recombinant EcNΔnlpI. a. Protein quantification of OMVs derived from wild-type EcN and recombinant EcNΔnlpI was performed by BCA method (n=3). Data are mean ± SD. b. Transmission electron microscopy images of OMVs derived from EcN and EcNΔnlpI, showing similar spherical morphology. Scale bar = 200 nm. c. Zeta potential of OMVs derived from EcN and EcNΔnlpI. (n=3). Data are mean ± SD. d. Nanoparticle tracking analysis (NTA) determined the size distribution of OMVs from wild-type EcN and recombinant EcNΔnlpI.

[0045] Figure 3.The engineered EcN based on the T0SS secretion system can secrete protein-encapsulated OMVs and package multiple different "cargoes" in a single OMV. a. Western blot analysis of GFP abundance in OMV samples of EcNΔnlpI-OG (abbreviated as "OG"), EcNΔnlpI-GR (abbreviated as "OGR"), and EcNΔnlpI (abbreviated as "NC") using anti-GFP antibodies. b. After modified T0SS and endogenous loading system, the engineered EcN can secrete protein-encapsulated OMVs with an encapsulation efficiency of 97.9%. c. The OMV encapsulation GFP ratio was detected by nanoflow cytometry, calculated as the ratio of GFP-loaded OMVs to total OMVs derived from EcNΔnlpI-OG, and the OMV membrane was stained with EVMembrane Red. d. Western blot analysis of RFP abundance in OMV samples of EcNΔnlpI-OR (abbreviated as “OR”), EcNΔnlpI-GR (abbreviated as “OGR”), and EcNΔnlpI (abbreviated as “NC”) using anti-RFP antibody. e. Engineered EcN synthetic OMVs can encapsulate multiple protein payloads simultaneously in a single OMV carrier, making the synthetic OMVs a promising cascade biocatalyst. f. Engineered OMVs are able to simultaneously encapsulate GFP and RFP in a single OMV as observed by Polar-SIM super-resolution microscopy. Scale bar = 2 μm.

[0046] Figure 4 .Images of GFP-loaded OMVs and RFP-loaded OMVs observed by Polar-SIM super-resolution microscopy. GFP-loaded OMVs are denoted as “OMV GFP ”, OMV loaded by RFP is represented as “OMV RFP ”. Scale bar = 2 μm.

[0047] Figure 5.The protein secretion system based on TOSS is compatible with various enzymes and can realize various detoxification reactions. A. Western blot analysis of the abundance of StlA (abbreviated as "S") in OMVs secreted by EcNΔnlpI-OS (abbreviated as "OS"), the abundance of Lox (abbreviated as "L") in OMVs secreted by EcNΔnlpI-OL (abbreviated as "OL"), and the abundance of Uox (abbreviated as "U") in OMVs secreted by EcNΔnlpI-OU (abbreviated as "OU") using anti-His antibody. OMVs secreted by EcNΔnlpI are "NC". B. Western blot analysis of the abundance of KatE (abbreviated as "K") in OMVs secreted by EcNΔnlpI-OK (abbreviated as "OK") and EcNΔnlpI (abbreviated as "NC") using anti-HA antibody. CF. OMVs from EcNΔnlpI-OS / EcNΔnlpI-OP1 / EcNΔnlpI-OP2 (C), EcNΔnlpI-OK (D), EcNΔnlpI-OL (E), EcNΔnlpI-OU / EcNΔnlpI-OSaU (F), and EcNΔnlpI-OIsA / EcNΔnlpI-OCrA / EcNΔnlpI-OSpA / EcNΔnlpI-OPbA / EcNΔnlpI-OCvA (G) showed the ability to degrade phenylalanine, H2O2, lactate, UA, and acetaldehyde in vitro, respectively (n=3). OMVs secreted by EcNΔnlpI are “NC”. Data are mean ± SD. H. Enzyme-loaded OMVs can effectively catalyze detoxification reactions. These reactions fall into two groups: decomposition reactions and oxidation reactions that are not coupled to NADH. StlA-loaded OMVs and KatE-loaded OMVs can catalyze decomposition reactions. Oxidase (Uox and Lox) are coupled to endogenous catalase in OMVs and catalysis depends on the H2O2 / O2 cycle instead of NAD + / NADH oxidation reaction.

[0048] Figure 6.Protein payloads in OMVs have advantages in terms of protein stability and catalytic activity in simulated intestinal fluid. a. Schematic diagram of Uox secretion methods involving direct T1SS-based protein secretion systems or improved T0SS-based protein secretion systems. b. Western blot analysis of Uox abundance in EcNΔnlpIΔthyA-SU and EcNΔthyA-SUT cells, supernatants, and OMVs using anti-His antibodies. c. EcNΔnlpIΔthyA-SU and EcNΔthyA-SUT based on the T0SS system showed similar uric acid degradation rates in minimal medium supplemented with 0.5 mM uric acid under microaerobic culture conditions (n=3). d. EcNΔnlpIΔthyA-SU and EcNΔthyA-SUT showed higher uric acid degradation efficiency in simulated intestinal fluid supplemented with 0.5 mM uric acid under microaerobic culture conditions (n=3). e. Comparison of the enzymatic activity of purified protein (T1SS-Uox) or protein-loaded OMVs (OMV-Uox) (which were derived from EcNΔthyA-SUT or EcNΔnlpIΔthyA-SU, respectively), tested in PBS at different time points at 37°C (n=3). f, g The stability of T1SS-Uox and OMV-Uox was evaluated by monitoring the enzyme activity (f) and Uox protein levels (g) in simulated intestinal fluid at 37°C (n=3). Data are mean ± SD. P values ​​were determined by two-way analysis of variance (ANOVA) with Tukey's multiple comparison test (* indicates P < 0.05).

[0049] Figure 7 A stable plasmid system was established in the engineered EcN without antibiotic selection. a. Growth curve of the EcN strain cultured in M9 minimal medium, with OD 600Monitoring (n=3). The growth of thymine-dystrophic EcN strains (EcNΔthyA and EcNΔnlpIΔthyA) in the presence (marked as "+ thymine") or absence of exogenous thymine was detected in M9 minimal medium. In addition, the growth of thymine-dystrophic EcN containing complementation plasmids (EcNΔthyA-SUT and EcNΔnlpIΔthyA-SU) was detected in M9 minimal medium in the absence of exogenous thymine. Data are mean ± SD. b. The plasmid retention rate of EcN strains was detected in M9 minimal medium without antibiotic pressure, and the cell survival rate was calculated by CFU counting on LB agar plates containing kanamycin (n=3). Plasmid retention in non-thymine dystrophic EcN strains (EcN-UT2 and EcNΔnlpI-OU) and thymine dystrophic EcN (EcNΔthyA-SUT and EcNΔnlpIΔthyA-SU) with exogenous thymine (annotated as "+thymine") was significantly reduced over 14 days. Complementation plasmids in thymine dystrophic EcN strains (EcNΔthyA-SUT and EcNΔnlpIΔthyA-SU) remained stable in the absence of exogenous thymine. Data are mean ± SD.

[0050] Figure 8 .Exogenous T1SS-modified EcN can effectively secrete Uox into the surrounding environment and degrade UA. a. The membrane proteins HlyB and HlyD of Escherichia coli J96 were added as heterologous T1SS to the engineered EcN to achieve direct secretion of the protein load. EcN-U1 and EcN-U2 contain synthetic T1SS that promotes Uox secretion under the control of a medium-strength promoter (fnrs promoter) (SEQ ID No.26) and a low-strength promoter (nar promoter) (SEQ ID No.27), respectively. The degradation rates of UA in the culture medium by wild-type EcN, recombinant EcN-ut1, and EcN-ut2 were compared (n=3). Data are mean ± SD. b Western blot analysis of the abundance of Uox in the supernatant of engineered EcN loaded with T1SS was performed using anti-His antibody.

[0051] Fig. 9.Quantification of Uox secreted by T0SS or T1SS. a. Coomassie-stained SDS-PAGE gel of His-Uox-HlyAsp fusion protein purified by His-tag-based affinity purification. In order to obtain sufficient levels of secreted Uox by T1SS, Uox was overexpressed in EcN by HlyA signal peptide (abbreviated as "sp") and His tag (His-Uox-HlyAsp) under the control of IPTG-inducible promoter tac promoter (SEQ ID No.30). Channel 1: cell extract without IPTG addition; Channel 2: IPTG-induced His-Uox-HlyAsp expression; Channel 3: His-Uox-HlyAsp purified after his-tag-based affinity purification. Channel M represents a standard protein marker. b. Western blotting was used to quantify the amount of Uox encapsulated in OMVs (abbreviated as "OMV-Uox") and purified protein His-Uox-HlyAsp (abbreviated as "T1SS-Uox").

[0052] Fig.10 .OMVs from engineered EcN can penetrate the intact intestinal barrier and enter the circulation. A. Representative images of colon sections after injection of Cy5.5-labeled OMVs (abbreviated as "Cy5.5-OMV") or PBS (abbreviated as control) into the colonic lumen of healthy mice. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. B. Representative images of different tissues (colon, liver, and kidney) from healthy mice after oral administration of EcNΔnlpIΔthyA-SK or PBS. OMVs were detected using anti-HA antibody (pink). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

[0053] Fig.11 Representative images of liver and kidney sections of healthy mice after intracolonic injection of Cy5.5-labeled OMVs (referred to as "Cy5.5-OMVs") or PBS (referred to as control). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

[0054] Fig.12 Representative images of different tissues (colon, liver and kidney) of healthy mice orally treated with EcNΔnlpIΔthyA-SU or PBS. OMVs were detected using anti-His antibody (pink). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

[0055] Fig.13Protein profiles of wild-type EcN or engineered EcN OMVs. OMVs were isolated from wild-type EcN and engineered EcN and analyzed by SDS-PAGE. Lane 1: OMVs from EcN; Lane 2: OMVs from EcNΔnlp; Lane 3: OMVs from EcNΔnlpIΔthyA; Lane 4: OMVs from EcNΔnlpIΔthyA-OG. Lane M indicates standard protein markers.

[0056] Fig.14.OMVs from engineered EcNs can penetrate the intestinal epithelial barrier via both pinocytosis and dynamin-mediated transcytosis. a. The mechanism by which EcN OMVs penetrate the intestinal epithelial barrier was investigated using an in vitro intestinal epithelial model of Caco-2 monolayers grown in a Transwell culture system. Various inhibitors targeting different endocytic pathways and RFP-loaded OMVs were sequentially added to the upper chamber. b. To determine whether epithelial transcytosis of OMVs occurs via active or passive mechanisms, RFP-loaded OMVs were incubated in the upper chamber of a Transwell chamber at 37°C or 4°C for 4 h. Fluorescence signals were detected in the lower chamber and further normalized to the 37°C group (n=3). c. The mechanism of OMV transepithelial transport was examined by focusing on the first endocytosis step of transcytotoxicity using various chemical inhibitors targeting different endocytic pathways at different concentrations (low, medium, and high), including a dimethyl sulfoxide (DMSO) group as a negative control. The inhibitors used included chlorpromazine (inhibits clathrin-dependent endocytosis), cytochalasin D (inhibits membrane fusion), wortmannin (inhibits phagocytosis), amiloride (inhibits pinocytosis), or nystatin (inhibits caveolin-mediated endocytosis and lipid raft formation). Low concentration: 0.05μM wortmannin; 0.25μM cytochalasin D; 10μM chlorpromazine; 20μM amiloride; 0.1μM nystatin and 20μM dynasore; medium concentration: 0.1μM wortmannin; 0.5μM cytochalasin D; 20μM chlorpromazine; 100μM amiloride; 0.2μM nystatin and 100μM dynasore; high concentration: 1μM wortmannin; 2μM cytochalasin D; 50μM chloropromazine; 200μM amiloride; 1μM nystatin and 200μM dynasore. The fluorescence signals detected in each lower chamber were normalized with the data of DMSO group as the standard (n=3). d. The permeability of epithelial monolayers after incubation with OMVs and DMSO or inhibitors was determined using Lucifer Yellow (n=3). Data are mean ± SD. P values ​​were determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (* indicates P < 0.05).

[0057] Fig.15.The in vivo safety of the modified T0SS-engineered EcN was evaluated by hematological and blood biochemical analysis. Healthy mice were given normal saline (labeled as "control"), EcN (labeled as "EcN") or EcNΔnlpIΔthyA-SU (labeled as "EcNΔnlpIΔthyA-SU") every two days for one week. ag measured white blood cell (WBC) count (a), red blood cell (RBC) count (b), platelet (PLT) count (c), hemoglobin (HGB) level (d), serum alanine aminotransferase (ALT) level (e), creatinine (CRE) level (f) and urea nitrogen (BUN) level (g). Data are mean ± SEM, n = 3.

[0058] Fig.16 .The in vivo safety of the modified TOSS-engineered EcN was evaluated by histopathological analysis of major organs. Representative histopathological images of heart, liver, spleen, lung, and kidney sections of healthy mice were obtained after administration of saline (denoted as "control group"), EcN, or EcNΔnlpIΔthyA-SU every two days for one week. The sections were stained with hematoxylin and eosin (H&E). Scale bar = 100 mm.

[0059] Fig.17.Engineered EcN equipped with improved T0SS showed excellent therapeutic effects in treating hyperuricemic mice and detoxifying human serum samples. Experimental design of UA- and PO-induced hyperuricemic mouse models and treatment procedures. Mice fed a normal diet and injected intraperitoneally with vehicle were denoted as “Con”. Hyperuricemic mice were induced by UA-supplemented diet (2% UA in the diet) and daily intraperitoneal injection of 250 mg / kg PO for 4 weeks. A. At the beginning of hyperuricemia induction, hyperuricemic mice were administered saline (abbreviated as “UA”), EcN (abbreviated as “UA+E”), EcNΔthyA-SUT (abbreviated as “UA+T”), or EcNΔnlpIΔthyA-SU (abbreviated as “UA+O”) by gavage (n=6). B to E: Serum UA (B), urine UA (C), serum CRE (D), and IL-1β (E) levels. (F) Representative histopathological images of colon sections obtained from five groups of mice. Sections were stained with hematoxylin and eosin (H&E). Black arrows indicate infiltration of immune cells, and red arrows indicate sloughed epithelial cells. Scale bar = 50 μm. G and H: UA (G) and glucose (H) degradation effects of Uox-loaded OMVs extracted from EcNΔnlpIΔthyA-SU on serum samples of patients with hyperuricemia, in response to OMV treatment for 30 min (n = 9). I and J: Lox degradation effects of Lox-loaded OMVs extracted from EcNΔnlpI-OL (n = 7), in response to OMV treatment for 30 min on serum samples of patients with lung cancer (n = 7). Data are shown as mean ± SD. P values ​​were determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (BE) or paired t test (GJ) (* represents P < 0.05, ** represents P < 0.01 and *** P < 0.001). DETAILED DESCRIPTION

[0060] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the technician refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0061] The present invention provides a recombinant Escherichia coli such as Nissle 1917 (EcN), which contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) therapeutically active ingredients in its outer membrane vesicles (OMV). When multiple therapeutically active ingredients are contained, these active ingredients can be the same type or different types of active ingredients.

[0062] In the present invention, the term "therapeutically active ingredient" refers to any substance with pharmacological activity or effect that can be used to treat a disease. Therapeutically active ingredients can include therapeutic peptides, therapeutic proteins, antigens, therapeutic nucleic acids and small molecule compounds. Therapeutic peptides, polypeptides or proteins and antigens that can be loaded into the OMV of Gram-negative bacteria have been described and listed in CN108779482A. Therapeutic nucleic acids (such as siRNA) that can be loaded into the OMV of Escherichia coli have been described in CN114958891B. CN 118497094 A describes small molecule compounds such as radionuclides that can be delivered through bacterial vesicles.

[0063] In a preferred embodiment of the present invention, the therapeutically active ingredient is a therapeutic enzyme, in particular a therapeutic enzyme for metabolic diseases, genetic diseases, cardiovascular diseases, gastrointestinal diseases, inflammatory diseases or cancer.

[0064] In the present invention, the term "therapeutic enzyme" refers to a biocatalyst protein used to treat diseases and improve medical conditions. Enzymes have two outstanding characteristics as drugs: (1) enzymes have high affinity and specificity for substrates; (2) enzymes can convert substrates into desired products with relatively few side effects. These two characteristics distinguish enzymes from all other types of drugs and make enzymes valuable therapeutic tools. Therapeutic enzymes can be widely derived from animals, plants and microorganisms, or obtained through recombinant DNA technology. Therapeutic enzymes have been widely used in the treatment of genetic diseases, cardiovascular diseases, gastrointestinal diseases, cancer and other diseases (Shahriari M, Zahiri M, Abnous K, et al. Enzyme responsive drug delivery systems in cancer treatment [J]. Journal of Controlled Release, 2019, 308: 172-189; Gurung N, Ray S, Bose S, et al. A broader view: microbial enzymes and their relevance in industries, medicine, and beyond [J]. BioMed Research International, 2013, 2013: 329121. DOI: 10.1155 / 2013 / 329121; Baldo BA. Enzymes approved for human therapy: indications, mechanisms and adverse reactions [J]. effects[J].BioDrugs,2015,29(1):31-55.DOI:10.1007 / s40259-015-0116-7;Kurosawa Y,Nirengi S,Homma T,et al.Asingle-dose of oral nattokinase potentiates thrombolysis and anti-coagulationprofiles[J].Scientific Reports,2015,5:11601.DOI:10.1038 / srep11601).

[0065] The application of therapeutic enzymes in cancer involves two main types: enzymes that metabolize amino acids required by tumors and enzymes that convert prodrugs. Metabolic enzymes are used to consume amino acids necessary for tumor cells, thereby inhibiting tumor growth; convertases are used to convert prodrugs into cytotoxic drugs in tumor cells, thereby killing tumor cells.

[0066] For example, L-asparaginase hydrolyzes L-asparagine into L-aspartic acid, thereby preventing L-asparagine from feeding tumor cells. L-asparaginase has a good clinical effect on acute lymphocytic leukemia, lymphosarcoma cell leukemia and granulocytic leukemia, and also has a certain effect on melanoma (Meghavarnam AK, Salah M, Sreepriya M, et al. Growth inhibitory and proapoptotic effects of L-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat) [J]. Fundamental & Clinical Pharmacology, 2017, 31 (3): 292-300).

[0067] Arginine deiminase (ADI) hydrolyzes L-arginine into L-citrulline, thereby preventing L-arginine from feeding tumor cells. Under normal circumstances, arginine can be synthesized in vivo by the cell's own urea cycle enzymes, namely arginine deiminase synthetase and arginine succinate lyase (Somani RR, Chaskar PK. Arginine deiminase enzyme evolvingas a potential antitumor agent[J]. Mini-Reviews in Medicinal Chemistry, 2018, 18(4): 363-368. DOI: 10.2174 / 1389557516666160817102701), but some malignant tumors with metabolic defects, such as melanoma, lung cancer, prostate cancer and hepatocellular carcinoma, often lack these enzymes (Xiong LF, Teng JLL, Botelho MG, et al. Arginine metabolismin bacterial pathogenesis and cancer therapy[J]. International Journal of Molecular Sciences, 2016, 17(3): 363. DOI: 10.3390 / ijms17030363; Wu LN, Li L,Meng SC,et al.Expression of argininosuccinatesynthetase in patients with hepatocellular carcinoma[J].Journal ofGastroenterology and Hepatology,2013,28(2):365-368;Ott PA,Carvajal RD,Pandit-Taskar N,et al.PhaseⅠ / Ⅱstudy of pegylated arginine deiminase(ADI-PEG 20)inpatients with advanced melanoma[J]. Investigational New Drugs, 2013, 31(2):425-434.). Since the growth of the above tumor cells depends on arginine in the environment, arginine deiminase can be used to treat these arginine auxotrophic tumors. There are also related reports that ADI-PEG-20 is used as an adjuvant for radiotherapy of pancreatic cancer, and can also be used to treat glioblastoma multiforme and thoracic cancer (mesothelioma and non-small cell lung cancer) (Beddowes E, Spicer J, Chan PY, et al. Phase 1 dose-escalation study of pegylated argininedeiminase, cisplatin, and pemetrexed in patients with argininosuccinate synthetase 1-deficient thoracic cancers [J]. Journal of Clinical Oncology, 2017, 35 (16): 1778-1785; Singh PK, Deorukhkar AA, Venkatesulu BP, et al. Exploiting arginine auxotrophy with pegylated arginine deiminase (ADI-PEG 20) to sensitize pancreatic cancer to radiotherapy via metabolic dysregulation[J].MolecularCancer Therapeutics,2019,18(12):2381-2393.DOI:10.1158 / 1535-7163.MCT-18-0708; Han RZ,Xu GC,Dong JJ,et al.Arginine deiminase:recent advances in discovery,crystal structure,and protein engineering for improved properties as an anti-tumor drug[J].Applied Microbiology and Biotechnology,2016,100(11):4747-4760.DOI:10.1007 / s00253-016-7490-z; Maletzki C,Rosche Y,Riess C,etal.Deciphering molecular mechanisms of arginine deiminase-based therapy-comparative response analysis in paired human primary and recurrentglioblastomas[J].Chemico-Biological Interactions,2017,278:179-188;SzlosarekPW,Steele JP,Nolan L,et al.Arginine deprivation with pegylated argininedeiminase in patients with argininosuccinate synthetase 1-deficient malignantpleural mesothelioma:a randomized clinical trial[J].JAMAOncol,2017,3(1):58-66;Covini D,Tardito S,Bussolati O,et al.Expanding targets for a metabolictherapy of cancer:L-asparaginase[J].Recent Patents on Anti-Cancer DrugDiscovery,2012,7(1):4-13.DOI:10.2174 / 157489212798358001)。.

[0068] Glutaminase hydrolyzes L-glutamine into L-glutamic acid, thereby preventing L-glutamine from feeding tumor cells. Glutaminase isolated from Pseudomonas 7A can treat lymphomas resistant to asparaginase when used in combination with asparaginase, and also has a certain effect on various leukemias (Li Q, Wang YT, Wu WT. Research on the application of enzymes as therapeutic drugs [A] / / China Bio-Pharmaceutical Innovation Research Forum [C]. Deyang: Chinese Pharmaceutical Association, 2012: 51-62, Li Qian, Wang Youtong, Wu Wutong, Research on the application of enzymes as therapeutic drugs [A] / / China Bio-Pharmaceutical Innovation Research Forum [C]. Deyang: Chinese Pharmaceutical Association, 2012: 51-62 http: / / d.wanfangdata.com.cn / conference / 7981619).

[0069] Glucose oxidase oxidizes glucose into gluconic acid and H2O2, thereby killing tumor cells (Fu LH, Qi C, Lin J, et al. Catalytic chemistry of glucose oxidase in cancer diagnosis and treatment[J]. Chemical Society Reviews, 2018, 47(17): 6454-6472. DOI: 10.1039 / c7cs00891k).

[0070] Kynurenine hydrolyzes kynurenine into L-alanine and anthranilic acid, thereby stimulating the human immune system to attack tumor cells. Studies have shown that PEG-kynurenine treatment in vivo can remove kynurenine, and combined with immune checkpoint inhibitors and tumor vaccines, it has shown good therapeutic effects on melanoma, breast cancer and colon cancer (Triplett TA, Garrison KC, Marshall N, et al. Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme [J]. Nature Biotechnology, 2018, 36 (8): 758-764).

[0071] Carboxypeptidase A hydrolyzes methotrexate-phenylalanine prodrug into methotrexate and L-phenylalanine, restoring the cytotoxicity of methotrexate. Modified carboxypeptidase A can activate the activity of prodrug methotrexate-phenylalanine for the treatment of colon cancer (Deckert PM, Bornmann WG, Ritter G, et al. Specific tumour localisation of ahuA33 antibody-carboxypeptidase A conjugate and activation of methotrexate-phenylalanine [J]. International Journal of Oncology, 2004, 24 (5): 1289-1295. DOI: 10.3892 / ijo.24.5.1289). Human β-glucuronidase is also an attractive enzyme that can activate prodrug glucuronides (Chen KC, Wu CH, Chang CY, et al. Directed evolution of alysosomalenzyme with enhanced activity at neutral pH by mammalian cell-surface display[J]. Chemistry&Biology, 2008, 15(12): 1277-1286. DOI: 10.1016 / j.chembiol.2008.10.008).

[0072] Therapeutic enzymes can also be used to treat metabolic diseases in the subject (in the circulatory system). The metabolic disease is selected from abnormal glucose metabolism, abnormal lipid metabolism, abnormal calcium-phosphorus metabolism, abnormal bone metabolism and abnormal purine metabolism, most preferably, the metabolic disease is hyperuricemia and the therapeutic enzyme is uricase, the metabolic disease is peroxisomal disease and the therapeutic enzyme is catalase, or the metabolic disease is hyperlactatemia and the therapeutic enzyme is lactate oxidase.

[0073] Therapeutic enzymes can also be used to treat genetic diseases in the body (in the circulatory system) of a subject.

[0074] Phenylketonuria (PKU) is a common hereditary disease characterized by the accumulation of phenylalanine in the body. The reason is that the activity of phenylalanine hydroxylase is low or inactive, and it cannot catalyze the conversion of phenylalanine into tyrosine, resulting in the accumulation of phenylalanine and its keto acid. Phenylalanine deaminase (PAL) can catalyze the conversion of phenylalanine into non-toxic and harmless trans-cinnamic acid and ammonia. Trans-cinnamic acid is further metabolized and finally excreted in the urine in the form of hippuric acid. Since the reaction catalyzed by PAL produces non-toxic compounds, PAL is considered a drug for the treatment of PKU (Wang L, Gamez A, Sarkissian CN, et al. Structure-based chemical modification strategy for enzyme replacement treatment of phenylketonuria [J]. Molecular Genetics and Metabolism, 2005, 86 (1 / 2): 134-140).

[0075] Mucopolysaccharidosis (MPSS) is an autosomal recessive genetic disease caused by mutations in the α-L-iduronidase gene. α-L-iduronidase and iduron sulfate esterase have been approved for the treatment of MPSS. CM, Giugliani R. Evaluation of galsulfase for the treatment of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) [J]. Expert Opinion on Orphan Drugs, 2014, 2(4): 407-417).

[0076] Pompe disease is a rare disease caused by a defect or dysfunction of the lysosomal acid alpha-glucosidase (GAA) gene. The FDA approved alpha-glucosidase for the treatment of Pompe disease in 2010.

[0077] Gaucher disease (GD) is a disease in which the body lacks glucocerebrosidase or glucosylceramidase, resulting in the accumulation of glucocerebroside in cells. Alglucosidase is the first drug used to treat GD.

[0078] Fabry's disease is an X-linked genetic disease caused by a deficiency of β-galactosidase A, which leads to the accumulation of sphingolipid trisaccharides in the body. β-galactosidase (fabrazyme) was approved in 2003 for the treatment of Fabry's disease.

[0079] Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is an extremely rare genetic disease caused by adenosine deaminase (ADA) deficiency. In 2018, the FDA approved recombinant adenosine deaminase for the treatment of ADA-SCID in children and adults (Lv Xunlei, Lin Kuaile, Guo Linlin, et al. Introduction to new drugs approved by the US FDA in 2018 [J]. Chinese Journal of Pharmaceutical Industry, 2019, 50(1): 1-33).

[0080] Hypophosphatasia (HPP) is a rare inherited systemic disease caused by loss-of-function mutations in the alkaline phosphatase gene. Alkaline phosphatase was approved by the FDA in 2015 as a drug to treat HPP.

[0081] Lysosomal acid lipase deficiency (LALD) is a rare autosomal recessive lysosomal storage disease in which gene mutations lead to loss or reduction of lysosomal acid lipase function. In 2015, the FDA approved human lysosomal acid lipase as a drug for the treatment of LALD.

[0082] Therapeutic enzymes can also be used to treat cardiovascular disease in the subject (in the circulatory system). Cardiovascular disease is a class of diseases consisting of important diseases such as coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism, one of the causes of which is the formation of blood clots in blood vessels.

[0083] Streptokinase (SK) has been used to treat peripheral arterial occlusive disease. Urokinase has been used to treat embolic diseases. Tissue-type plasminogen activator (t-PA) was approved by the US FDA and approved for the treatment of myocardial infarction in 1987. Snake venom defibrase, which directly acts on fibrin or fibrinogen, has a good therapeutic effect on the treatment of cerebral thrombosis and myocardial infarction. Hirudin is an anticoagulant and antithrombotic agent that acts on thrombin. It is a specific inhibitor of thrombin and can effectively inhibit the formation of thrombus (Li Qian, Wang Youtong, Wu Wutong, Research on the application of enzymes as therapeutic drugs [A] / / Proceedings of the China Biopharmaceutical Innovation Research Forum [C], Deyang: Chinese Pharmaceutical Society, 2012: 51-62). Lumbrokinases (LKs) are a group of proteases isolated from different species of earthworms. They can reduce blood viscosity, increase blood oxygen saturation, and improve blood circulation. They are an effective therapeutic agent for cerebral infarction (Wang XM, Fan SC, Chen Y, et al. Earthworm protease in anti-thrombosis and anti-fibrosis[J]. Biochimica et Biophysica Acta(BBA)-General Subjects, 2019, 1863(2): 379-383. DOI: 10.1016 / j.bbagen.2018.11.006). Nattokinase (NK) is a protease produced by Bacillus subtilis natto with strong thrombolytic function (Duan CR, Feng YF, Zhou H, et al. Optimization offermentation condition of man-made bee-bread by response surface methodology [A] / / Zhang TC, Nakajima M. Advances in Applied Biotechnology [M]. Berlin, Heidelberg: Springer, 2015: 353-363 https: / / link.springer.com / chapter / 10.1007%2F978-3-662-46318-5_38). Desmoteplase is a fibrin-specific and non-neurotoxic thrombolytic drug.It was isolated from the saliva of the vampire bat Desmodus rotundus in 1974 and can be used to reduce the formation of blood clots (Mican J, Toul M, Bednar D, et al. Structural biology and protein engineering of thrombolytics [J]. Computational and Structural Biotechnology Journal, 2019, 17: 917-938. DOI: 10.1016 / j.csbj.2019.06.023).

[0084] Therapeutic enzymes can also be used to treat gastrointestinal diseases in the subject's body (in the circulatory system). The use of enzymes as digestive accelerators is the earliest application of therapeutic enzymes. This type of enzyme acts to hydrolyze and digest components in food, such as proteins, carbohydrates, and lipids. It is often used as a digestive aid to promote digestion. Most of them are compound preparations containing proteases, amylases, lipases, and cellulases. Pancreatic enzyme preparations are compound enzyme preparations containing lipases, proteases, and amylases, which are used to treat malabsorption and insufficient pancreatic secretion, especially in patients with cystic fibrosis.

[0085] Therapeutic enzymes can also be used to treat inflammatory diseases in the subject's body (in the circulatory system). Lysozyme can selectively degrade viral RNA and has anti-HIV activity (Lee-Huang S, Huang PL, Sun YT, et al. Lysozyme and RNases as anti-HIV components in β-core preparations of human chorionic gonadotropin [J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96 (6): 2678-2681). Lysostaphin can be used to treat endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, food poisoning and various skin infections (such as folliculitis, abscesses and mastitis, etc.) caused by Staphylococcus aureus infection. In clinical practice, trypsin, chymotrypsin, bromelain, etc. are often used to treat inflammation and edema to remove necrotic tissue, increase tissue permeability, inhibit edema, promote the discharge of tissue fluid near the lesion and inhibit the formation of granulation. In addition, collagenase can be used to clear burns and sores, and is effective in removing scabs; chondroitinase can be used to treat spinal injuries and can effectively promote the repair of spinal cord injuries.

[0086] In addition to delivering therapeutic proteases, the recombinant Escherichia coli of the present invention, such as Nissle 1917 (EcN), can also be used for oral systemic delivery of therapeutic nucleic acids, such as small interfering nucleic acids (siRNA), microRNA (miRNA), antisense nucleotides (ASOs), messenger RNA (mRNA) or nucleic acid aptamers (Aptamer).

[0087] Oral protein delivery provides patients with a convenient and highly compliant route of administration. However, the efficiency of oral delivery is greatly limited by the physiological barriers of the gastrointestinal tract, including irritating acids and proteases in the stomach, the intestinal mucus layer, and the intestinal epithelial barrier. In order to overcome these physiological barriers, NP-based drug delivery systems have made significant progress in improving the efficiency of oral protein delivery through material engineering, surface modification, etc. (Mitchell, MJet al. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20, 101-124 (2021); Zhang, F.et al. Lipid-based intelligent vehicle capabilitized with physical and physiological activation. Research (Wash DC) 2022, 9808429 (2022)). Despite these findings, NP-based drug delivery systems still have complex manufacturing procedures, inefficient cargo loading and delivery. In our work, we have established an oral protein delivery technology that can be achieved by oral administration of genetically engineered EcNs equipped with modified TOSSs. The EcN chassis is tolerant to the digestive tract, and it was found that the in situ secreted OMVs can penetrate the intact intestinal barrier and enter the circulation, so the protein payload is escorted by the engineered EcN and the secreted OMV carrier in sequence to deliver the protein orally efficiently and programmably. The protein payload is encapsulated in the OMVs in vivo through the endogenous protein loading strategy, and its encapsulation ratio is 97.9%, which is much higher than the in vitro exogenous protein loading method of EV and NP. Therefore, our engineered EcN equipped with a modified T0SS provides a self-programmable and convenient oral protein delivery technology.

[0088] We unexpectedly found that EcN OMVs without surface modification can enter the circulation through the intact intestinal barrier, suggesting a physiological role for bacterial outer membrane vesicles in host-microbe interactions outside the intestine. Based on this important finding, EcN was designed to improve OMV synthesis and endogenously load the protease of interest into OMVs. OMV carriers have the advantages of high protein stability, resistance to protein degradation and circulation entry ability. The T0SS-based protein secretion system is compatible with a variety of protein payloads and is able to self-package multiple different protein cargoes in a single OMV carrier for cascade reactions. In addition, EcN OMVs can contain endogenous oxidoreductases, including peroxide reductases, making OMVs ideal biocatalysts for detoxification, including decomposition, cascades, and NADH-independent oxidation reactions. Our engineered EcNs that secrete drugs based on T0SS are excellent in detoxifying blood metabolites, thanks to the high stability of OMV-encapsulated proteins and their longer delivery range, providing an oral protein delivery system based on genetically engineered commensal bacteria for the clinical treatment of various metabolic disorders.

[0089] The present invention is further described in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The chemical substances used in the following reactions are all commercially available products unless otherwise indicated.

[0090] Materials and methods

[0091] Material

[0092] DAPI (BL739B) was obtained from Biosharp Life Science. Cell culture plates, 96-well culture plates, kanamycin (GC301008-5g), IPTG (GC205011-1g), chloramphenicol (GC301018-5g), penicillin-streptomycin (G4003-100ml), DMEM (G4525-500ml), anti-HA-tag antibody (GB151252-100, for Western blotting), anti-GFP antibody (GB15603-100), anti-RFP antibody (GB125053-100), and L-phenylalanine (GC304010-10g) were purchased from ServicebioBiotechnology. Cy5.5-NHS ester (A8103) was purchased from APExBIOTechnology. Anti-HA tag antibody (26183, for immunofluorescence), anti-His tag antibody (MA121315), goat anti-IgG (H+L) secondary antibody conjugated with Alexa Fluor 647 (A32728TR) and fetal bovine serum (FBS; A5669401) were purchased from Fisher Thermo Scientific. Mouse IL-1β ELISA kit (KE10003), goat anti-mouse IgG (H&L)-HRP (PR30012) and goat anti-rabbit IgG (H&L)-HRP (PR30011) for Western blotting were purchased from Protentech. EVMembrane Red Stain (NEPU-638-50T) was purchased from NanoFCM. Sodium carboxymethylcellulose (IS9000), uric acid content detection kit (BC1365), creatinine content detection kit (BC4915) and urea nitrogen content detection kit (BC1535) were all obtained from Solarbio Science & Technology. Cytochalasin D (gc13440), chlorpromazine HCl (gc14216), dynasore (gc10395), nystatin (gc10090), amiloride HCl (gc17853), and wortmannin (gc12338) were purchased from Glpbio Technology. Uric acid (U0018) and potassium oxalate (O0164) were purchased from Tokyo Chemical Industry. Sodium lactate (S108838) was purchased from Aladdin Scientific.

[0093] Animals and cells

[0094] Male Kunming mice (4–5 weeks old) were obtained from Beijing Weitong Lihua (China). All animal experiments were approved by the Institutional Animal Use and Animal Experimentation Ethics Committee of Tsinghua University. Mice were housed under specific pathogen-free (SPF) conditions with a 12-h light / dark cycle and free access to water and diet. Caco-2 cells (ATCC HTB-37) were cultured in DMEM supplemented with 20% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin in an incubator at 37 °C with 5% CO2.

[0095] Bacterial strains and culture conditions

[0096] Table 1 provides a list of bacterial strains used in the present invention. Escherichia coli S17-1 strain (Simon R, Priefer U, Puhler A. 1983. A broad host range mobilization system for in vivo genetic engineering-transposon mutagenesis in Gram-negative bacteria. Bio-Technol. 1: 784-791) is used for plasmid construction, and Escherichia coli WM3064 strain (Saltikov CW, Newman DK. 2003. Genetic identification of a respiratory arsenate reductase. Proc. Natl. Acad. Sci. USA 100: 10983-10988) is used as the donor strain for conjugation. The gene (katE gene) encoding catalase (SEQ ID No. 6) is amplified from Escherichia coli Nissle 1917 genomic DNA (GenBank CP007799.1). Heterologous genes, i.e., uricase (SEQ ID No. 5) encoding gene cu_uox from C. utilis (UniProt Entry P78609) or uricase (SEQ ID No. 31) encoding gene sa_uox from S. auratus AGR0001, hlyBD-hlyA (hemolysin gene) from Escherichia coli J96 (Tzschaschel, BD, Guzmán, CA, Timmis, KN ​​& de Lorenzo, V. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat. Biotechnol. 14, 765-769 (1996)), phenylalanine deaminase (SEQ ID No. 5) encoding gene cu_uox from C. utilis (UniProt Entry P78609) or uricase (SEQ ID No. 31) encoding gene sa_uox from S. auratus AGR0001, hlyBD-hlyA (hemolysin gene) from Escherichia coli J96 (Tzschaschel, BD, Guzmán, CA, Timmis, KN ​​& de Lorenzo, V. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat. Biotechnol. 14, 765-769 (1996)), ID No.8) encoding gene stlA or phenylalanine deaminase SEQ from Y. enterocolitica (SEQ ID No.32) encoding gene ye_pal1 (phenylalanine deaminase (SEQ ID No.33) encoding gene ye_pal2 from A.viridans (UniProt Entry Q44467) encoding gene lox for lactate oxidase (SEQ ID No.7), and gene pb_alod for acetaldehyde oxidase (SEQ ID No.34) from P. blakesleeanus NRRL 1555, gene cr_alod for acetaldehyde oxidase (SEQ ID No.35) from C. reinhardtii, gene cv_alod for acetaldehyde oxidase (SEQ ID No.36) from C. cardunculus var. scolymus, gene ls_alod for acetaldehyde oxidase (SEQ ID No.37) from L. sativa, gene sp_alod for acetaldehyde oxidase (SEQ ID No.38) from S. punctatus DAOM BR117 were synthesized and cloned by GenScript (Nanjing, China) and inserted into pYYD plasmid (Yang, Y. et al. Enhancing bidirectional electron transfer of Shewanella oneidensis by a synthetic flavin pathway. ACS Synth.Biol.4,815-823(2015)). Subsequently, the plasmid was transformed into Escherichia coli WM3064 strain and then conjugated with EcN. .

[0097] Suicide plasmid pRE112 (Edwards, RA, Keller, LH & Schifferli, DM I) was used to knock out nlpI (SEQ ID No. 1) and thyA genes (SEQ ID No. 9) in EcN. Plasmids were constructed using Gibson assembly in E. coli S17-1, then transformed into E. coli WM3064, and then joined with EcN. Single exchange mutants were selected on LB agar medium supplemented with chloramphenicol and verified by colony PCR. The resulting double exchange mutants were selected on LB agar medium supplemented with 10% sucrose and 3mM thymidine (for ΔthyA mutants), and further confirmed by colony PCR and sequencing. EcN and E. coli S17-1 were cultured in LB (10 g peptone, 10 g sodium chloride, 5 g yeast extract per liter) at 37°C. E. coli WM3064 was cultured in LB medium supplemented with 50 μg / mL 2,6-diaminocarboxylic acid at 37°C. Antibiotic selection involved the use of 50 μg / mL kanamycin (for pYYD-derived plasmids) and 25 μg / mL chloramphenicol (for pRE112-derived plasmids). The strains were cryopreserved in 15% glycerol at -80°C. The PCR primers used above are shown in Table 2.

[0098] Table 1. Strain genotypes

[0099]

[0100]

[0101] Note: MBP in genotype sp Represents Sec signal peptide (SEQ ID No. 2); torA sp Indicates Tat signal peptide (SEQ ID No. 3); dsbA sp represents Srp signal peptide (SEQ ID No.4); nar , P fnrs , P nar618 and P J23117 They represent nar promoter (SEQ ID No.27), fnrs promoter (SEQ ID No.26), nar618 promoter (SEQ ID No.28) and J23117 promoter (SEQ ID No.29), respectively.

[0102] Table 2. PCR primer sequences

[0103]

[0104] In vitro uric acid (UA) consumption assay

[0105] To evaluate the ability of bacteria to degrade UA, logarithmically grown bacteria in LB medium were resuspended in modified M9 minimal medium (6.78 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 5 g glucose, and 1 g yeast extract per liter) supplemented with 0.5 mM UA for microaerobic culture to a final concentration of 10 9 The samples were collected at different time intervals and the UA concentration was quantified using a uric acid assay kit.

[0106] Outer membrane vesicle isolation and characterization

[0107] To isolate and purify OMVs, the supernatant of the E. coli culture was filtered through a 0.22 μm syringe after centrifugation at 4 °C to remove large contaminants and bacteria. The resulting solution was concentrated by a 100 kDa cutoff centrifugal filter and further ultracentrifuged at 120,000 × g for 2 h at 4 °C. The OMV pellet was washed with PBS, ultracentrifuged again, filtered through a 0.22 μm syringe, and suspended in PBS. The morphology of OMVs was characterized using a Hitachi transmission electron microscope (TEM) system. The Zeta potential was measured by a Zetasizer Nano ZS (Malvern, UK). The hydrodynamic diameter of OMVs was measured by ZetaVIEW (Particle Metrix, Germany).

[0108] Nanoflow cytometer

[0109] Isolated OMV samples were analyzed using a Flow NanoAnalyzer (nanoFCM) instrument equipped with 488 nm and 638 nm lasers. OMVs extracted from EcNΔnlpI and EcNΔnlpI-OG were labeled with EVMembraneRed Stain for 30 min at room temperature. Using the above isolation method, the fluorescently labeled samples were washed twice with PBS to remove excess dye. The particles were then resuspended in PBS and measured using a calibrated instrument with 50 mW laser power, 10% SS attenuation, and 1 kPa sampling pressure. Data analysis was performed using NF professional 2.0 software. By analyzing the green and red fluorescence signals of PBS control and empty OMVs, the threshold lines for green and red fluorescence were determined to reduce the counts of nanoscale impurities and background signals (<5%).

[0110] Measurement of metabolites

[0111] UA, creatinine, and urea nitrogen were quantified using urea acid, creatinine, and urea nitrogen content test kits, respectively. Other metabolites such as phenylalanine and lactate were measured in the culture medium or serum by high-performance liquid chromatography (HPLC). Phenylalanine quantification was performed using an HPLC system equipped with a UV detector and a ZORBAX SB-C18 column. Gradient elution was performed with a mobile phase consisting of acetonitrile and 1.5% acetic acid. Detection was performed at 260 nm and the column temperature was maintained at 40°C. Lactate quantification was performed using an HPLC system equipped with a RID-10A refractive index detector and an Aminex HPX-87H column. Samples were eluted with 5 mM sulfuric acid at 50°C. Identification and quantification of metabolites were completed by comparing retention times and chromatographic peak areas with established standards.

[0112] Animal experiments

[0113] Hyperuricemia induction in Kunming mice: 2% UA in the diet, daily intraperitoneal injection of 250 mg / kg potassium oxonate (PO) (dissolved in 0.5% CMC-Na solution) for 4 weeks. At the beginning of hyperuricemia induction, four groups of mice were treated with EcN (5×10 9 CFU), EcNΔthyA-SUT (5×10 9 CFU), EcNΔnlpIΔthyA-SU (5×10 9 CFU) or an equal volume of saline was administered by gavage. On the last day of the study, the mice were euthanized by carbon dioxide. Blood samples were collected by cardiac puncture, and liver, kidney, and colon were collected for histological analysis.

[0114] In vivo safety assessment of engineered EcNs equipped with improved T0SS: Three groups of healthy male Kunming mice were treated with EcNs (5×10 9 CFU), EcNΔnlpIΔthyA-SU (5×10 9 CFU) or an equivalent volume of saline was administered orally for one week. The mice were euthanized on the last day of the study. Blood samples were collected by cardiac puncture for routine blood tests and serum biochemistry tests, and major organs (heart, liver, spleen, lung, and kidney) were collected for histological analysis.

[0115] Western blotting and immunofluorescence

[0116] The total protein content in the samples was quantified using Bradford analysis. Purified OMVs or proteins were heated at 95°C for 5 min, followed by SDS-PAGE and then transferred to polyvinyl difluoride (PVDF) membranes. After transfer, they were coated with 5% bovine serum albumin (BSA) for 1 h and incubated with specific primary antibodies overnight at 4°C with gentle shaking. Afterwards, the membranes were washed three times for 10 min with TBST and incubated with goat anti-mouse IgG (H&L)-HRP or goat anti-rabbit IgG (H&L)-HRP for another hour, and the bands were visualized by enhanced chemiluminescence (ECL).

[0117] For immunofluorescence, 7 μm OCT-embedded frozen colon, liver, or kidney tissues were placed on slides. After incubation in 0.5% Triton X-100 for 1 hour, incubation in 10% goat serum for 2 hours, and incubation with anti-HIS antibody overnight at 4°C. Then incubation with appropriate secondary antibodies was performed for 2 hours at room temperature. Images were acquired using the Andor DragonFly confocal imaging system.

[0118] In vitro endocytosis studies

[0119] Caco-2 cells were seeded in the upper chamber of the Transwell and cultured for 3 weeks. The integrity of the epithelial monolayer was assessed by transepithelial electrical resistance (TEER) measurement using an EVOM TEER meter. Only cells with TEER values ​​greater than 300 Ω×cm 2 The model was used for transcellular transport studies. To use this model to study OMV transport, OMVs encapsulated with RFP were placed in the upper chamber of the Transwell (~10 10 particles). The effect of temperature was examined by incubating the model at 4 or 37°C. To evaluate the mechanism of endocytosis, the intestinal epithelium was incubated with commercially available inhibitors at 37°C for 1 hour before OMV addition. After 8 hours, the medium was collected from the underside of the chamber and the fluorescent signal was quantified using a microplate reader. To evaluate the effect of OMVs on epithelial barrier integrity, Lucifer Yellow (LY) was added to the Transwell inserts and the fluorescence intensity of the apical and basolateral fluids was measured after 1 hour using a microplate reader. The percentage of LY exclusion was calculated using the following formula: %LY exclusion percentage = 100x(1-RFU basolateral / RFU apical ).

[0120] Detection of metabolites in clinical serum samples

[0121] To test the efficacy of OMVs in degrading metabolites in human blood, 100 μg of purified OMVs were incubated with 50 μL of human serum samples for 30 min. Then, the concentrations of UA, glucose, or lactate in the samples were measured as described above. The acquisition and processing of patient serum samples were approved by the Clinical Research Ethics Committee of the China-Japan Friendship Hospital, approval number 2023-KY-007. This study was conducted in accordance with the ethical standards outlined in the 2013 Declaration of Helsinki.

[0122] Statistics

[0123] Data are expressed as mean ± SD or mean ± SEM. Statistical differences between the control and experimental groups were analyzed by one-way or two-way ANOVA with Tukey's post hoc test. P < 0.05 was considered significant (*P < 0.05; **P < 0.01; ***P < 0.001). Paired samples were compared using paired t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

[0124] Example 1

[0125] Development of improved TOSS for protein secretion

[0126] In order to construct a protein secretion system based on TOSS in EcN, in genetically engineered EcN (i.e., recombinant Escherichia coli Nissle 1917 (EcN)), specific proteins are encapsulated into OMVs and OMV synthesis is enhanced. First, nlpI, which encodes outer membrane lipoproteins involved in regulating peptidoglycan dynamics, is deleted in EcN to increase the production of OMVs (McBroom, AJ, Johnson, AP, Vemulapalli, S. & Kuehn, MJ Outer membrane vesicle production by Escherichia coliis independent of membrane instability. J. Bacteriol. 188, 5385-5392 (2006)). The OMV production of EcNΔnlpI in M9 medium is 2.83 ± 0.24 times higher than that of wild-type EcN, with conservative OMV morphology, zeta membrane potential and size distribution ( Figure 2In the Gram-negative bacterium EcN, its natural secretion system delivers cytoplasmic proteins with signal peptides to the periplasmic space, including general secretion (Sec), twin-arginine translocation (Tat), and signal recognition particle (Srp)-dependent pathways, and the signal peptide is cleaved from the secretory protein by a signal peptidase located on the inner membrane (Green, ER & Mecsas, J. Bacterial secretion systems: an overview. Microbiol. Spectr. 4 (2016)). Next, we investigated whether, like other Escherichia coli strains (Kesty, NC & Kuehn, MJ Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J. Biol. Chem. 279, 2069-2076 (2004); Bartolini, E. et al. Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydia linfection in vitro. J. Extracell. Vesicles 2 (2013)), heterologous expressed proteins with signal peptides can be directed to the periplasm and further packaged into the OMVs of EcN. The fluorescent protein GFP (SEQ ID No. 22) was directed with a Sec signal peptide (SEQ ID No. 2) at the N-terminus and overexpressed in EcNΔnlpI to obtain the recombinant strain EcNΔnlpI-OG. OMVs of EcNΔnlpI-OG were isolated by ultracentrifugation, and Western blot analysis using anti-GFP antibody and fluorescent images confirmed the presence of GFP in OMV samples, indicating that heterologous overexpressed proteins tagged with periplasmic targeting signal peptides in engineered EcNs can be successfully packaged into OMVs ( Figure 3 , a and Figure 4 ).

[0127] The protein encapsulation ratio in OMVs was investigated by nanoflow cytometry. The ratio of GFP-encapsulated OMVs to total OMVs from EcNΔnlpI-OG was calculated by nanoflow cytometry, using GFP as an example of encapsulated protein and EVMembraneRed to stain the membrane of OMVs ( Figure 3, b). GFP-loaded OMVs from EcNΔnlpI-OG showed a substantial increase in GFP fluorescence signal compared to empty OMVs from EcNΔnlpI ( Figure 3 , c). The GFP encapsulation ratio of OMVs is as high as 97.9%, which is calculated as the ratio of OMVs encapsulating GFP to EVMembrane Red-stained OMVs. Traditional exogenous protein loading methods include electroporation, co-incubation, extrusion, freeze-thaw, ultrasound, and surfactant treatment, and their encapsulation efficiency is about 20%-50% (Chen, C. et al. Single-particle assessment of six different drug-loading strategies for incorporating doxorubicin into small extracellular vesicles. Anal. Bioanal. Chem. 415, 1287-1298 (2023)). Compared with exogenous loading strategies, our endogenous loading system is superior in encapsulation ratio and self-programmed assembly.

[0128] Furthermore, we investigated whether this improved protein endogenous packaging system can co-package multiple different protein payloads into a single OMV. In addition to GFP, RFP (SEQ ID No. 23) fused to the Srp signal peptide was overexpressed in EcNΔnlpI. Recombinant EcN can package RFP within the OMV vector, which was confirmed by Western blot analysis of extracted OMV samples using anti-RFP antibodies and fluorescent images ( Figure 3 , d and Figure 4 ). EcNΔnlpI was then engineered to co-express two heterologous proteins, GFP fused to an N-terminal Sec signal peptide and RFP fused to an N-terminal Srp signal peptide. OMVs of the resulting strain EcNΔnlpI-GR were isolated and analyzed. GFP and RFP were detected in OMV samples by Western blot analysis ( Figure 3 , a and d). OMV fluorescence images from EcNΔnlpI-GR showed that a single OMV emitted both green and red fluorescence signals ( Figure 3 , e and f), showing that a single OMV encapsulates GFP and RFP payloads. These results indicate that the T0SS-based protein secretion system in engineered EcNs can assemble multiple different proteins in a single OMV carrier, making engineered OMVs promising cascade biocatalysts.

[0129] Example 2

[0130] The T0SS-based protein secretion system is compatible with a variety of enzymes and can achieve a variety of detoxification reactions

[0131] A series of enzymes were tested for compatibility with the TOSS-based protein secretion system. The cells were fused with an N-terminal Tat signal peptide (SEQ ID No. 3) and a protein tag (His or HA) (His: HHHHHH (SEQ ID No. 24); HA: YPYDVPDYA (SEQ ID No. 25)) and enzymes, including phenylalanine deaminase StlA (SEQ ID No. 8) (from Photorhabdus luminescens), phenylalanine deaminase yePAL1 (SEQ ID No. 32) (from Yersinia enterocolitica), phenylalanine deaminase yePAL2 (SEQ ID No. 33) (from Yersinia enterocolitica), catalase KatE (SEQ ID No. 6) (from EcN), lactate oxidase Lox (SEQ ID No. 7) (from Aerococcus viridans), uricase cuUox (SEQ ID No. 5) (from Candida utilis), uricase saUox (SEQ ID No. 6) (from EcN), and overexpressed in EcNΔnlpI. No.31) (from Streptomyces auratus AGR0001), acetaldehyde oxidase pbAlod (SEQ ID No.34) (from Phycomyces blakesleeanus NRRL 1555), acetaldehyde oxidase crAlod (SEQ ID No.35) (from Chlamydomonas reinhardtii), acetaldehyde oxidase cvAlod (SEQ ID No.36) (from Cynara cardunculus var. scolymus), acetaldehyde oxidase lsAlod (SEQ ID No.37) (from Lactuca sativa), acetaldehyde oxidase spAlod (SEQ ID No.38) (from Spizellomyces punctatus DAOM BR117). After isolating OMV samples from the engineered EcN strains, the respective anti-tag antibodies ( Figure 5, A and B) Enzymes in OMVs were detected by Western blot. Compared with empty OMVs of EcNΔnlpI, synthetic OMVs with enzymes had the catalytic activity of the corresponding enzymes, i.e., engineered OMVs encapsulating StlA, yePAL1, and yePAL2 could degrade phenylalanine at rates of 0.38 ± 0.04 μM / h / mg, 0.31 ± 0.02 μM / h / mg, and 0.49 ± 0.02 μM / h / mg, respectively, OMVs encapsulating KatE could degrade hydrogen peroxide at a rate of 2.92 ± 0.07 μM / h / mg, OMVs encapsulating Lox could degrade lactate at a rate of 0.36 ± 0.02 μM / h / mg, and OMVs encapsulating cuUox and s OMVs containing aUox could degrade uric acid (UA) at rates of 0.34±0.03μM / h / mg and 0.29±0.01μM / h / mg, respectively. OMVs containing lsAlod, crAlod, spAlod, pbAlod, and cvAlod could degrade acetaldehyde at rates of 15.02±0.93μg / mL / h / mg, 15.55±1.02μg / mL / h / mg, 12.51±0.98μg / mL / h / mg, 13.98±1.13μg / mL / h / mg, and 17.02±1.07μg / mL / h / mg, respectively. Figure 5 , C to G). These results indicate that engineered EcNs based on engineered TOSS can self-assemble and secrete OMVs encapsulating functional enzymes, and this TOSS-based protein secretion system is compatible with a variety of proteins.

[0132] Engineered OMVs containing various enzymes are expected to catalyze various detoxification reactions ( Figure 5 , H). The catalytic reactions of OMVs encapsulated with StlA and encapsulated with KatE showed that the synthetic OMVs were able to catalyze the decomposition reaction. In addition, it was found that empty OMVs from EcNΔnlpI could significantly degrade hydrogen peroxide at a rate of 1.61±0.05μM / h / mg. Proteomic analysis of OMV samples (provided by Biotech Biotechnology Co., Ltd.) revealed the presence of endogenous peroxidase KatG in EcN OMVs (see Table 3 below). These results confirm that OMVs from EcN contain endogenous catalase that can degrade hydrogen peroxide into oxygen and water. It should be noted that both Uox and Lox catalyze oxidation reactions and produce hydrogen peroxide ( Figure 5 , G), whereas negligible amounts of hydrogen peroxide were detected when Uox- or Lox-loaded OMVs were used to catalyze the degradation of UA or lactate (data not shown), indicating that the oxidases (Uox and Lox) are coupled to endogenous catalase in OMVs and that catalysis depends on the H2O2 / O2 cycle rather than NAD + / NADH oxidation reaction ( Figure 5, H). Collectively, these results suggest that our synthesized OMVs based on engineered EcNs with engineered T0SS can catalyze various detoxification reactions, including decomposition and oxidation reactions.

[0133] Table 3. Proteomics identified enzymes related to mucus degradation and endogenous catalase in EcN OMVs

[0134]

[0135]

[0136] Example 3

[0137] Proteins encapsulated in OMVs exhibit enhanced stability in simulated intestinal fluid

[0138] Next, we evaluated the performance of our T0SS-based secretion system for protein payloads in a simulated intestinal environment by comparing protein stability and catalytic activity with those of a common secretion system, using the Uox protein as an example ( Figure 6 , a). First, in order to achieve stable expression of heterologous genes in the engineered EcN in the intestinal environment, a stable plasmid system was established by deleting the strain growth essential gene thyA in the EcNΔnlpI genome and then using a plasmid to complement the thyA gene. The growth of EcNΔnlpIΔthyA depends on exogenous thymidine ( Figure 7 , a), while EcNΔnlpIΔthyA carrying a stable plasmid grew well with EcNΔnlpI in the absence of exogenous thymidine ( Figure 7 , a), indicating that the stable plasmid was successfully maintained in an antibiotic-free microenvironment ( Figure 7 , b). The recombinant EcN strain EcNΔnlpIΔthyA-SU overexpressed Uox tagged with Tat signal peptide and His tag on a stable plasmid and was shown to secrete OMVs encapsulating Uox ( Figure 6, b). A genetically engineered EcN strain EcNΔthyA-SUT with a heterologous type I secretion system (T1SS) (Tzschaschel, BD, Guzmán, CA, Timmis, KN ​​& de Lorenzo, V. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat. Biotechnol. 14, 765-769 (1996)) was established, representing a strain for direct protein secretion strategy. Uox fused to HlyA signal peptide and His tag were observed in the cell culture supernatant, and Western blot analysis was performed using anti-His antibody ( Figure 8 , a and b), demonstrating that the protein direct secretion strain successfully secreted Uox.

[0139] Recombinant EcN strains based on engineered T0SS or T1SS ( Figure 6 , a), namely EcNΔnlpIΔthyA-SU and EcNΔthyA-SUT in M9 medium containing 0.5 mM UA, and showed similar UA degradation rates, indicating that there was no significant difference in protein secretion efficiency between T0SS and T1SS in the engineered EcN strains. However, in simulated intestinal fluid containing trypsin and 0.5 mM UA, EcNΔnlpIΔthyA-SU based on the engineered T0SS showed a higher degradation rate than EcNΔthyA-SUT based on the engineered T1SS ( Figure 6 , c and d) showed much higher UA degradation efficiency, indicating that Uox secreted by T0SS is more tolerant to protein degradation in simulated intestinal fluid than Uox secreted by T1SS. In addition, the protein stability and catalytic activity of Uox secreted by T0SS and T1SS were analyzed and compared in simulated intestinal fluid. OMVs of Uox encapsulated by EcNΔnlpIΔthyA-SU (abbreviated as OMV-Uox) and Uox expressed and purified by fusion with the HlyA secretion signal sequence (abbreviated as T1SS-Uox) were extracted and quantified ( Fig. 9 , a and b). OMV-Uox and T1SS-Uox containing similar amounts of Uox were incubated in PBS or simulated intestinal fluid, and their enzyme activities at different time points were measured ( Figure 6 , d and e). It was observed that OMV-Uox and T1SS-Uox had similar enzyme activities and stabilities in PBS within 2 h ( Figure 6, e). However, the enzyme activity of T1SS-Uox decreased rapidly in simulated intestinal fluid, and maintained 37.39±4.91% of its original enzyme activity after 1 h ( Figure 6 , f). In contrast, the enzyme activity reduction rate of OMV-Uox was much lower than that of T1SS-Uox, and more than 79.81±2.51% of the original activity was retained after 1 h of trypsin incubation ( Figure 6 , f and g). These results indicate that the OMV carriers protect the protein cargo from protease degradation, suggesting that the protein payloads delivered by the engineered TOSS-based protein secretion strategy are more stable and catalytically active than those delivered via the direct secretion pathway commonly found in the digestive tract.

[0140] Example 4

[0141] EcN OMVs containing proteins can penetrate the intestinal barrier and enter the circulation

[0142] We explored whether EcN OMVs can penetrate the intact intestinal barrier. OMVs derived from EcNΔnlpIΔthyA were labeled with Cy5.5 and injected into the colon lumen of healthy mice, followed by colon ligation. After eight hours of colon incubation, fluorescent OMVs were observed in the villi at the base of the colon, as well as in the liver and kidneys of healthy mice incubated with OMVs instead of PBS ( Fig.10 , A and Fig.11 ), indicating that EcN-derived OMVs are able to penetrate the intact intestinal epithelial barrier and enter the circulation. Furthermore, we explored whether protein-encapsulated OMVs produced in situ by the engineered EcN strain in the intestine could cross the intestinal barrier and enter the circulation in healthy mice without intestinal permeability defects. Healthy mice were orally administered with EcNΔnlpIΔthyA-SU or EcNΔnlpIΔthyA-OK, and the in vivo distribution of OMVs encapsulated with Uox or KatE was tracked at different time points by anti-His or anti-HA immunofluorescence staining (pink). Four hours after gavage with EcNΔnlpIΔthyA-SU or EcNΔnlpIΔthyA-OK, OMVs encapsulated with Uox or KatE were detected in the colon of mice using anti-His or anti-HA antibodies (pink), respectively. Fig.10 , B and Fig.12 ). At 12 and 24 h after oral gavage of EcNΔnlpIΔthyA-SU or EcNΔnlpIΔthyA-OK, OMVs were observed in the liver and kidney in addition to the colon ( Fig.10 , B and Fig.12), indicating that the protein-encapsulated OMVs produced in situ by the engineered EcN can penetrate the intestinal barrier and enter the circulation of healthy mice. Almost no OMVs were detected in vivo 36 hours after oral administration of EcNΔnlpIΔthyA-SU or EcNΔnlpIΔthyA-OK ( Fig.10 , B and Fig.12 ), indicating that circulating OMVs from intestinal EcNs can persist for up to 36 hours after administration of engineered EcNs. In contrast, no circulating OMVs were detected in healthy mice treated with the excipient vehicle (phosphate-buffered saline, PBS). Together, these results suggest that engineered EcNs in the intestine produce protein-encapsulated OMVs that are able to enter the circulation.

[0143] It has been reported that some bacterial OMVs contain degradative enzymes that may be responsible for accelerating their passage through mucus pores within the mucus layer to reach intestinal epithelial cells (Schwechheimer, C. & Kuehn, MJ. Uter-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat. Rev. Microbiol. 13, 605-619 (2015)). We compared the proteins of native OMVs of wild-type EcN and GFP-encapsulated OMVs of EcNΔnlpIΔthyA-SG by SDS-PAGE and proteomic analysis of OMV samples. The results showed that the protein distribution of OMVs of wild-type or engineered EcN was similar ( Fig.13 ), there was no significant difference in the protein abundance of enzymes (such as proteases, hydrolases, glycosidases, hexosaminidase and sulfatase) between wild-type or engineered EcN OMVs (Table 3). These results indicate that through the engineered TOSS system of engineered EcN, heterologous and secreted proteins do not interfere with endogenous proteins in OMVs, and therefore, protein-encapsulated OMVs of engineered EcN retain the ability to pass through the mucus layer.

[0144] In addition, we used an in vitro intestinal epithelial model using Caco-2 monolayers grown in a Transwell culture system ( Fig.14 , a), the mechanisms behind the ability of protein-coated OMVs to penetrate the intestinal epithelial cell barrier were investigated. To determine whether OMVs are transported across the intestinal epithelium by an active or passive mechanism, RFP-coated OMVs were incubated with the intestinal epithelium in the upper Transwell chamber at either 37°C or 4°C for 4 h. In the lower Transwell chamber, the fluorescence signal detected when incubated at 37°C was significantly higher than that at 4°C, suggesting that the translocation of OMVs across the intestinal epithelial monolayer under physiological conditions involves an active mechanism ( Fig.14, b). In addition, we explored the mechanism of OMV transepithelial transport by focusing on the first step of transcellular transport, endocytosis, using various chemical inhibitors targeting different endocytic pathways (Mulcahy, LA, Pink, RC & Carter, DR Routes and mechanisms of extracellular vesicle uptake. J. Extracell. Vesicles 3 (2014)). Addition of chlorpromazine (inhibits clathrin-dependent endocytosis), cytochalasin D (inhibits membrane fusion), wortmannin (inhibits phagocytosis), or nystatin (inhibits caveolin-mediated endocytosis and lipid raft formation) did not significantly affect the efficiency of endocytic transport of RFP-loaded OMVs in the intestinal epithelium ( Fig.14 , c). In contrast, dynamin (an inhibitor) and aminolone (an inhibitor of pinocytosis) significantly reduced OMV transcellular transport in a dose-dependent manner compared with the control group treated with solvent (dimethyl sulfoxide, DMSO) ( Fig.14 , c). Furthermore, incubation with endocytosis inhibitors and OMVs did not alter the permeability of the intestinal epithelium to Lucifer Yellow ( Fig.14 , d). These results suggest that both pinocytosis and actin-dependent endocytosis are involved in the transcellular transport of protein-loaded OMVs from engineered EcN. To our knowledge, this is the first report on the transport of EcN OMVs across the intestinal epithelial barrier.

[0145] Example 5

[0146] In vivo safety assessment of engineered ECNs equipped with modified TOSS

[0147] To test whether oral administration of engineered EcN equipped with a modified T0SS is a safe therapeutic approach, healthy mice were orally administered saline, EcN, or EcNΔnlpIΔthyA-SU for one week to evaluate potential toxicity, followed by histological analysis of major organs and hematological and blood biochemical analyses. Compared with mice administered saline, no significant changes were observed in white blood cell (WBC) counts, red blood cell (RBC) counts, platelet (PLT) counts, or hemoglobin (HGB) levels in mice treated with EcN or EcNΔnlpIΔthyA-SU ( Fig.15, a to d). These results indicate that neither EcN nor engineered EcN equipped with a modified T0SS to produce OMVs adversely affects the hematopoietic system. In addition, biochemical analysis of plasma samples showed that treatment with EcN or EcNΔnlpIΔthyA-SU did not increase the levels of alanine aminotransferase (ALT), creatinine (CRE), or blood urea nitrogen (BUN), indicating that our engineered EcN does not induce liver or kidney toxicity in vivo ( Fig.15 , e to g). In addition, H&E staining analysis of major organs (including heart, liver, spleen, lung, and kidney) showed no signs of organ damage in any group, indicating that administration of our engineered EcNs did not produce obvious histopathological abnormalities ( Fig.16 ). Overall, the in vivo safety assessment demonstrated that our engineered EcNs equipped with an improved T0SS are biocompatible and safe and have potential for clinical application.

[0148] Example 6

[0149] Oral administration of Uox by engineered EcN equipped with an improved T0SS exhibits superior therapeutic efficacy in treating hyperuricemia mice

[0150] The efficacy and advantages of our engineered EcN equipped with a modified T0SS in the treatment of metabolic diseases were evaluated, using hyperuricemia as a demonstration compared with engineered EcN equipped with a direct protein secretion apparatus (T1SS). The mouse hyperuricemia model was induced by UA-supplemented diet and daily intraperitoneal injection of potassium oxate (PO) for 28 days, where PO served as a mouse Uox inhibitor (Lu, J. et al. Mouse models for human hyperuricaemia: a critical review. Nat. Rev. Rheumatol. 15, 413-426 (2019)). Healthy mice gavaged with normal saline and intraperitoneally injected with sodium carboxymethylcellulose served as healthy controls. During the 28-day UA- and PO-challenges, hyperuricemic mice were orally administered with saline, wild-type EcN, or recombinant EcN strains equipped with T0SS or T1SS, i.e., EcNΔnlpIΔthyA-SU or EcNΔthyA-SUT ( Fig.17, A). The hyperuricemia mouse model treated with normal saline showed a substantial increase in serum UA concentration to ~599.7 μM, approximately 2.18-fold higher than that of the normal control group (serum UA concentration ~274.7 μM). In hyperuricemia mice dosed with wild-type EcN, EcNΔnlpIΔthyA-SU, or EcNΔthyA-SUT, treatment with wild-type EcN or recombinant EcN equipped with T1SS reduced serum UA to ~514.3 μM or ~464.8 μM, respectively. Notably, our engineered EcN treatment equipped with T0SS exhibited therapeutic performance in reducing circulating UA concentrations, significantly reducing serum UA concentrations from ~599.7 μM to ~279.3 μM, close to the level of healthy controls (serum UA concentration approximately 274.7 μM) ( Fig.17 , B). These results demonstrate that our engineered EcN equipped with T0SS is superior to recombinant EcN equipped with T1SS in alleviating hyperuricemia, suggesting that OMV-encapsulated Uox secreted by T0SS has an outstanding effect in degrading circulating UA due to its excellent protein stability and wide delivery range beyond the intestine.

[0151] We further evaluated the performance of our engineered EcNs equipped with a modified T0SS in improving the symptoms of hyperuricemia. Hyperuricemia is often accompanied by increased urinary UA content (Wu, Y. et al. Limosilactobacillus fermentum JL-3 isolated from "Jiangshui" ameliorates hyperuricemia by degrading uricacid. Gut Microbes 13, 1-18 (2021)), which was also observed in a hyperuricemia mouse model ( Fig.17 , C). Compared with the healthy control group, the urine UA level of hyperuricemic mice administered with saline increased by about 2.25 times ( Fig.17 , C). Urinary UA in hyperuricemic mice treated with EcN, EcNΔthyA-SUT or EcNΔnlpIΔthyA-SU was significantly reduced by approximately 21.59%, 41.40% and 46.70%, respectively ( Fig.17 UA is widely considered to be a damage-associated molecular pattern (DAMP) that can activate the NLRP3 inflammasome and increase the release of inflammatory cytokines, such as IL-1β ( G., TO, Klück, V., Popp, RA & Joosten, LAB (2020). Therefore, serum levels of the inflammatory cytokine IL-1β were examined to assess systemic inflammation, and serum creatinine (CRE) levels were detected to assess renal function and the biosafety of drug treatment. Compared with the healthy control group, serum IL-1β and serum creatinine (CRE) levels were significantly increased in hyperuricemic mice administered normal saline ( Fig.17 , D and E). Oral administration of EcNΔnlpIΔthyA-SU, but not EcNΔthyA-SUT or wild-type EcN, significantly reduced serum CRE and serum IL-1β levels by ∼15.94% and ∼32.91%, respectively, compared with hyperuricemic mice given saline ( Fig.17 , D and E). In addition, histological analysis of colon, liver and kidney tissues was performed by H&E staining ( Fig.17 , F). In hyperuricemic mice treated with saline or wild-type EcN, significant immune cell infiltration was observed in the liver and kidney, whereas the group treated with EcNΔnlpIΔthyA-SU showed a significant reduction in immune cell infiltration ( Fig.17 , F). These results indicate that our engineered EcNs equipped with an improved T0SS are superior in terms of oral Uox and serum UA degradation, thus showing excellent efficacy in alleviating the pathological symptoms of hyperuricemia and good biosafety. Using hyperuricemia as a demonstration, we demonstrated that our engineered EcNs equipped with an improved T0SS are superior in treating metabolic disorders compared to conventional recombinant co-formulations that secrete therapeutic proteins in the intestine to the periphery, likely benefiting from the superior stability of therapeutic proteins encapsulated in OMVs, high endogenous encapsulation rate, and wide systemic delivery range beyond the intestine.

[0152] Example 7

[0153] Engineered OMVs can effectively detoxify clinical samples from patients

[0154] To further investigate the clinical potential of our engineered EcN equipped with T0SS for the treatment of metabolic diseases, the efficacy of protein-encapsulated OMVs extracted from engineered EcNs was evaluated for the treatment of human serum samples. Serum samples from nine patients with hyperuricemia were collected and incubated with Uox-loaded OMVs from EcNΔnlpIΔthyA-SU. After 30 min of incubation, serum UA levels were significantly reduced after OMV treatment, with an average decrease of 42.6% after OMV incubation, from 496.9 μM to 285.3 μM ( Fig.17 , G), while the serum glucose levels of these nine samples were not affected by OMV incubation ( Fig.17 , H). These results indicate that synthetic Lox-loaded OMVs derived from our engineered EcNs are effective and specific for reducing UA degradation in the blood of patients with hyperuricemia. In addition, serum samples from seven lung cancer patients with elevated lactate levels were collected and incubated with Lox-loaded OMVs from EcNΔnlpI-OL. Incubation of Lox-loaded OMVs for 30 min resulted in a decrease in serum lactate levels in all seven human samples, from an average of ∼5.67 mM to ∼5.50 mM ( Fig.17 , I). Similarly, Lox-loaded OMVs significantly reduced serum levels of lactate but did not affect serum levels of glucose ( Fig.17 , J). Overall, we demonstrated that therapeutic proteins delivered by our engineered ECNs based on engineered T0SS can effectively detoxify circulating human metabolites and that our engineered ECN-based protein delivery system has the potential to be used in the clinical treatment of a wide range of metabolic disorders.

[0155] Those skilled in the art should understand that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, without departing from the spirit of the present invention, those skilled in the art can make appropriate modifications or improvements, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

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[0157]

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[0160]

[0161]

[0162]

Claims

1. A recombinant Escherichia coli comprising at least one therapeutically active ingredient in its outer membrane vesicle (OMV), wherein the therapeutically active ingredient is selected from therapeutic peptides, therapeutic proteins, antigens, therapeutic nucleic acids and small molecule compounds, and preferably the therapeutically active ingredient is a therapeutic peptide or therapeutic protein encapsulated in the OMV.

2. The recombinant Escherichia coli Nissle 1917 (EcN) according to claim 1, wherein the therapeutic active ingredient is a therapeutic enzyme, preferably a therapeutic enzyme for metabolic diseases, genetic diseases, cardiovascular diseases, gastrointestinal diseases, inflammatory diseases or cancer, more preferably wherein: 1) The metabolic disease is selected from abnormal glucose metabolism, abnormal lipid metabolism, abnormal calcium-phosphorus metabolism, abnormal bone metabolism and abnormal purine metabolism. Most preferably, the metabolic disease is hyperuricemia and / or the therapeutic enzyme is uricase, the metabolic disease is peroxisomal disease and / or the therapeutic enzyme is catalase, or the metabolic disease is hyperlactatemia and / or the therapeutic enzyme is lactate oxidase, or the metabolic disease is hyperalcoholism and / or the therapeutic enzymes are ethanol oxidase and acetaldehyde oxidase; 2) the hereditary disease is selected from phenylketonuria, mucopolysaccharidosis, Pompe disease, Gaucher disease, Fabry disease, severe combined immunodeficiency due to adenosine deaminase deficiency, hypophosphatasia and lysosomal acid lipase deficiency, preferably the therapeutic enzyme is selected from phenylalanine deaminase, α-L-iduronidase, iduronidase sulfate, α-glucosidase, alglucosidase, β-galactosidase, adenosine deaminase, alkaline phosphatase and lysosomal acid lipase, most preferably, the metabolic disease is phenylketonuria and / or the therapeutic enzyme is phenylalanine deaminase; 3) the cardiovascular disease is selected from coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism, and the therapeutic enzyme is preferably selected from streptokinase, urokinase, tissue-type plasminogen activator (t-PA), snake venom defibrase, hirudin, lumbrokinase, nattokinase and desmoteplase; 4) The gastrointestinal disease is selected from dyspepsia, pancreatic secretion deficiency and gallbladder fibrosis, and the therapeutic enzyme is preferably selected from protease, amylase, lipase, cellulase and pancreatin; 5) the inflammatory disease is selected from AIDS, endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, food poisoning, skin infection (such as folliculitis, abscess and mastitis, etc.) and burns, and the therapeutic enzyme is preferably selected from lysozyme (such as lysostaphin), trypsin, chymotrypsin, bromelain, collagenase and chondroitinase; and / or 6) The cancer is selected from acute lymphocytic leukemia, lymphosarcoma cell leukemia, granulocytic leukemia, melanoma, lung cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, glioblastoma multiforme and thoracic vertebral cancer. Preferably, the therapeutic enzyme is a metabolic enzyme and a prodrug converting enzyme of amino acids required by the tumor, and is more preferably selected from L-asparaginase, arginine deiminase, glutaminase, glucose oxidase, kynurenine enzyme, carboxypeptidase A and β-glucuronidase.

3. The recombinant Escherichia coli according to claim 1, wherein the therapeutic nucleic acid is selected from small interfering nucleic acid (siRNA), micro RNA (miRNA), antisense nucleotide (ASO) messenger RNA (mRNA), and aptamer. The recombinant Escherichia coli according to claim 1, which is Nissle 1917 (EcN) and / or has a nlpI gene deleted.

5. The recombinant Escherichia coli according to any one of claims 1 to 4, wherein the therapeutic peptide or therapeutic protein is fused with a signal peptide secreted into OMV, preferably the signal peptide is selected from the group consisting of a Sec signal peptide, a Tat signal peptide and a Srp signal peptide.

6. The recombinant Escherichia coli according to claim 5, wherein the therapeutic peptide or therapeutic protein is selected from uricase, catalase, lactate oxidase, phenylalanine deaminase, ethanol oxidase, acetaldehyde oxidase or a combination thereof, preferably the expression of the therapeutic peptide or therapeutic protein is controlled by an inducible or constitutive promoter, more preferably the promoter is selected from tac promoter, fnrs promoter, nar promoter, nar618 promoter and J23117 promoter.

7. The outer membrane vesicle (OMV) derived from the recombinant Escherichia coli according to any one of claims 1 to 6, wherein the OMV comprises the at least one therapeutically active ingredient, preferably the OMV is not surface-modified.

8. A pharmaceutical composition for oral systemic administration or intestinal mucosal systemic administration for treating a subject, comprising: 1) a recombinant Escherichia coli according to any one of 1 to 6 or an outer membrane vesicle (OMV) according to claim 7; and 2) a pharmaceutically acceptable adjuvant, wherein the pharmaceutical composition, recombinant Escherichia coli or OMV is formulated for and / or relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the subject's circulatory system.

9. Use of the recombinant Escherichia coli according to any one of claims 1 to 6 or the outer membrane vesicles (OMV) according to claim 7 in the preparation of a drug for oral systemic administration or intestinal mucosal systemic administration to treat a subject, wherein the drug, recombinant Escherichia coli or OMV is formulated for and / or relies on the OMV to penetrate the intestinal epithelial barrier, thereby delivering the therapeutically active ingredient to the subject's circulatory system.

10. The oral pharmaceutical composition according to claim 8 or the use according to claim 9, wherein the therapeutically active ingredient is uricase and / or the subject suffers from hyperuricemia, or the therapeutically active ingredient is catalase and / or the subject suffers from a peroxisomal disease, or the therapeutically active ingredient is lactate oxidase and / or the subject suffers from hyperlactatemia, or the therapeutically active ingredient is phenylalanine deaminase and / or the subject suffers from phenylketonuria, or the therapeutically active ingredient is ethanol oxidase and / or acetaldehyde oxidase and / or the subject suffers from hyperalcoholemia.

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