WO2025175293A1 - Systems and method for production of bacterial extracellular vesicles - Google Patents

Abstract

Provided are modified bacteria that are modified such that they contain a polynucleotide that includes an inducible promoter that is operably linked to a sequence encoding an endolysin or an autolysin. When expression of the promoter is induced, the modified bacteria exhibit increased production of bacterial extracellular vesicles (BEVs). The BEVs can be used alone or can contain therapeutic or prophylactic cargo for use in treating a variety of conditions.

Description

SYSTEMS AND METHOD FOR PRODUCTION OF BACTERIAL EXTRACELLULAR VESICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S provisional patent application no. 63/554,933, filed February 16, 2024, the entire disclosure of which is incorporated herein by reference.

FIELD

The subject matter of the present disclosure relates generally to systems and methods for the production of bacterial extracellular vesicles.

BACKGROUND

Bacterial extracellular vesicles (BEVs) are nanosized membrane vesicles with extraordinary therapeutic potential in wide-ranging applications. However, they are produced in extremely low yields which has slowed industrial development. Several techniques exist to artificially increase BEV yields, but all do so at the expense of BEV bioactivity or lack operator control. For example, prior techniques include using carcinogenic DNA-damaging agents such as mitomycin C. However, such carcinogenic agents are incompatible with biomanufacturing of therapeutics. Accordingly, there is a need for improved BEV manufacturing techniques. The present disclosure is pertinent to this need.

SUMMARY

The disclosure provides systems and methods for increased production of bacterial extracellular vesicles. The systems and methods allow for low-cost high yield production of bacterial extracellular vesicles. Examples of the present disclosure include a genetic circuit wherein expression of a lysis-inducing protein is coupled to an exogenous signal that is manually added to the culture. The present disclosure provide on-demand, tunable, rapid generation of ~20-fold increased yields of BEVs that have equivalent therapeutic bioactivity to wild type BEVs. It is believed that the performance of the systems and methods described herein are transformative for BEV biomanufacturing and not possible previously available techniques. The disclosure provides a platform for biomanufacturing BEVs for numerous applications including vaccines, and treatment of cancer and inflammatory diseases (e.g., inflammatory bowel disease, depression, anxiety, neurodegenerative disease, liver disease, asthma, atopic dermatitis, psoriasis, and others that are further described below). Additional used of the BEVs include antimicrobial therapies, BEV-based biomaterials, RNA delivery vehicles, or adjuvants for vaccines or immunotherapy.

BRIEF DESCRIPTOIN OF FIGURES

FIG. 1. High yields of BE Vs are rapidly generated upon Al- 1 induction of LysE expression. E. coli Nissle 1917 was inoculated in LB media at OD 0.1 and incubated at 37C at 250 rpm agitation; once cells reached early log-phase (OD 0.4), high yield BEV production was induced by addition of ALl at two concentrations (1 nM or 10 nM). Then, 4 hours later, BEVs were isolated by a standard BEV isolation protocol involving differential centrifugation followed by tangential flow filtration. A) OD600 of control (no ALl) and ALl cultures immediately prior to addition of ALl (+0 hours), and 4- and 12-hours post- AL 1 induction. B-C) BEV yields determined by nanoparticle tracking analysis expressed as relative increases vs control (A) or total BEV yields (C). D) BEV yields expressed as total protein content in BEV samples determined by bicinchoninic acid assay.

FIG. 2. High yield BEVs retain recombinant protein cargo and antiinflammatory efficacy. Lysis was induced as in Figure 1. A) Western blot analysis of the concentration of ClyA-GFP in BEV samples from conventional culture without lysis (0 nM ALl) or lysis induced by low (1 nM) and high (10 nM) doses of ALL B) Levels of TNF-a secreted from RAW264.7 mouse macrophages following pretreatment with wild type (No lysis) or lysed BEVs (1.6E8 particles/mL each) and inflammatory responses stimulated with 10 ng/ml LPS. Statistical significance was determined by ordinary one-way ANOVA.

FIG. 3. Additional lysis-inducing proteins can be used in Gram positive bacteria. Lacticaseibacillus paracasei 334 was transformed with the inducible expression plasmid pMSP3535-lyb5 (MSP-lyb5) wherein expression of the phage-derived endolysin Lyb5 can be induced by manual addition of the peptide, nisin. Equal volume cultures of either wild type or MSP-lyb5 transformed L. paracasei were cultured in MRS broth until ODeoo = 0.4, at which point 100 ng/ml nisin was added to both cultures. 4 hours later, final OD600 was recorded (A), and BEVs were isolated using tangential flow filtration and total yields determined by NTA (B).

FIG. 4. BEVs are produced by all tested species with similar physical characteristics. A) Particle size distribution of BEVs derived from different species of bacteria determined by nanoparticle tracking analysis, B) Mode diameter of BEVs from different species of bacteria cultured in either defined media (LB or CDM) or rich media (MRS, LAB only), C) Total BEV yields from flask culture determined by nanoparticle tracking analysis and normalized to volume of starting culture. EcN -E.coli Nissle 1917 (“probiotic”), DH5a - E. coli DH5a, Lc - Lacticaseibacillus casei 334, Lre - Limosilactobacillus reuteri F275, Lrh - Lacticaseibacillus rhamnosus GG, Lp - Lactiplantibacillus plantarum WCFS1

FIG. 5. BE Vs limit inflammatory responses from LPS-stimulated macrophages in vitro. A) Mouse RAW264.7 macrophages were pretreated with BEVs followed by stimulation with LPS (10 ng/ml) and analysis of levels of inflammatory TNF-a secreted in conditioned media. B). BEVs from several probiotic species and E. coli strains show dosedependent suppression of TNF TNF-a secretion. Statistical significance determined with oneway ANOVA and Tukey post hoc test. Groups with different letters are significantly different. EcN - E.coli Nissle 1917 (“probiotic”), DH5a - E. coli DH5a, Lc - Lacticaseibacillus casei 334, Lre - Limosilactobacillus reuteri F275, Lrh - Lacticaseibacillus rhamnosus GG, Lp - Lactiplantibacillus plantarum WCFS1

FIG. 6. Lp BEVs reduce colitis severity in acute DSS colitis. Mice undergoing acute DSS-induced colitis were treated with BEVs derived from different species of bacteria. DSS (2.5% w/v) was administered from Day 0 to Day 7, followed by normal water on Day 8; treatments were administered daily by oral gavage starting on Day 1 through the end of the study. Doses were normalized by particle count determined by nanoparticle tracking analysis (2.5E9 particles/mouse/day). A) Body weight changes of mice during acute DSS colitis relative to Day 0 weight, B) Colon length at study endpoint (Day 8). Statistical significance was determined by one-way ANOVA with Holm Sidak post hoc test. EcN - E.coli Nissle 1917 (“probiotic”), DH5a -E. coli DH5a, Lc - Lacticaseibacillus casei 334, Lre - Limosilactobacillus reuteri F275, Lrh - Lacticaseibacillus rhamnosus GG, Lp - Lactiplantibacillus plantarum WCFS1

FIG. 7. L. plantarum (Lp) and L. reuteri (Lre) BEVs reduce markers of inflammation in the mouse colon. Colon tissue was collected at the end of the DSS treatment study and bulk tissue RNA was isolated using commercially available RNA isolation kit (Zymo), cDNA was generated with mulv reverse transcriptase, and gene expression analyzed by RT-qPCR by the delta Ct method and normalized to GAPDH expression.

FIG. 8. Generation of a genetically-programmed hypervesiculating strain of L. plantarum with inducible activation. A) Plasmid design with expression of recombinant endolysin regulated by an inducible promoter system (pSIP403), B) The plasmid was transformed into L. plantarum, and then the engineered L. plantarum strain was cultured in 96 well plates in MRS media at 37 degrees C and growth was monitored by OD600 readings. The inducer peptide (sppIP) was added at the doses indicated to activate endolysin expression; after activation, the expected reduction in bacterial cell number occurs confirming appropriate induction of the gene circuit.

FIG. 9: BEV yields are increased from the hypervesiculating strain of L. plantarum. Following activation of endolysin expression, BEVs were isolated by centrifugation followed by tangential flow filtration and, finally, 0.2 um filtration. BEV yields were determined by nanoparticle tracking analysis and compared between the hypervesiculating L. plantarum strain, wild type L. plantarum, and L. plantarum cell pellet treated with sonication to extract BEVs.

FIG. 10: BEVs from the hypervesiculating L. plantarum strain do not show significant changes in anti-inflammatory potency in vitro vs normal BEVs. Mouse RAW264.7 macrophages were pretreated with BEVs followed by stimulation with LPS (10 ng/ml) and quantification of levels of inflammatory cytokine TNF-a secreted in conditioned media by ELISA. BEVs from a hypervesiculating L. plantarum were compared to normal BEVs and BEVs generated by sonication of L. plantarum cell pellet. Statistical analysis by two-way ANOVA, alpha = 0.05.

FIG. 11: BEVs from hypervesiculating L. plantarum are effective in the acute DSS mouse model of IBD. Mice undergoing acute DSS-induced colitis were treated with BEVs from either hypervesiculating L. plantarum strain (Hypervesiculating BEV) or wild type L. plantarum (Normal BEVs); additionally, mice were treated with live L. plantarum cells (Live cells). DSS (2.5% w/v) was administered from Day 0 to Day 5, followed by normal water on Day 6 and 7; treatments were administered daily by oral gavage starting on Day 1 through the end of the study. Mice were evaluated for weight changes daily throughout the study, disease activity index was evaluated on Day 5 (composite score of weight loss, blood in stool, and stool consistency), and colon length was assessed at the end of the study. Statistical significance was determined by one-way ANOVA with Holm Sidak post hoc test.

DETAILED DESCRIPTION

The subject matter of the present disclosure that follows and the references listed herein and/or attached hereto are hereby incorporated by reference each in their respective entirety, except for any statements contradictory to the express disclosure herein, except for any subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the express language of this disclosure controls. Such incorporation of the references shall not be considered an admission by any applicant(s) that the incorporated references constitute prior art or that the incorporated references are material to the patentability of the present disclosure.

While various examples of the present disclosure are described herein, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the exemplary examples provided herein.

Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another example includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another example. The term “about” in relation to a numerical value encompasses variations of +/-10%, +/- 5%, or +/- 1%.

The disclosure includes all polynucleotide and amino acid sequences that are referred to herein by reference to a database. The polynucleotide and amino acids are incorporated herein by reference as the sequence exist in the database as of the effective filing date of this application.

The disclosure enables on-demand, rapid generation of ~20-fold increased yields of BEVs that, without intending to be constrained by any particular theory, are considered to have equivalent therapeutic and/or prophylactic bioactivity to wild type BEVs, an approach that has not previously been possible. The BEVs are produce by modified bacteria as further described herein.

The modified bacteria are configured such that they include a promoter that is inducible and is operably linked to a sequence encoding an autolysin or an endolysin, the expression of which promotes secretion of the BEVs. “Operably linked” means the promoter, along with transcription factors, drives transcription and expression of the autolysin or the endolysin. In non-limiting examples, a preferred promoter system is pSIP403 for Gram -positive bacteria, such as lactic acid bacteria, and pBAD for Gram-negative bacteria such as E. coli. A preferred endolysin is Lyb5 for Gram positive bacteria and LysE for Gram-negative bacteria.

The disclosure includes but is not necessarily limited to using the following autolysins and endolysins:

In examples, a suitable promoter that drives expression as discussed above is small molecule inducible promoter, a peptide inducible promoter, a light inducible promoter, or a temperature inducible promoter. The promoter may be de-repressible. In examples, the promoter is any an SppIP responsive promoter. SppIP is pheromone that activates its promoter. In an example, the promoter is an AI-1 responsive promoter. AI-1 is a quorum sensing molecule that binds to and activates bacterial R protein which in turn induces gene expression by binding to a regulatory element in DNA. In other non-limiting examples, the promoter/inducer pairs may be any of T71ac, which is inducible by IPTG; araBAD, which is inducible by arabinose; trp, which is repressible by high levels of tryptophan; lac, which is inducible by ITPG or lactose; Ptac, which is inducible in a manner similar to the lac promoter; and pL, which can be regulated by temperature. In an example, the promoter is pSIP403and the inducer is sppIP.

The sequences of each of these promoters are known in the art, as are the types of bacteria in which they are functional. The disclosure includes the proviso that DNA damaging agents, such as Mitomycin C, can be excluded as the inducer. Induction of the promoter and the entire process of producing the BEVs may be performed in a manner that is not reliant on any bacteriophage, prophage, or helper phage and as such, is independent of phage-related lytic and lysogenic processes.

In examples, the described BEVs can be used alone as therapeutic agents, or a described system may be configured so that the BEVs include a cargo of interest. In examples, a protein of interest is modified to initially locate to a bacterial plasma membrane that is incorporated into the BEVs, which when excreted include the protein of interest. In an example, a cargo protein is fused to Cytolysin A (ClyA). The ClyA protein is known to be able to be fused to any protein of interest and used to facilitate exosome-related secretion of the fusion protein. ClyA is a representative bacterial protein that is normally present on the outer membranes of bacteria. Use of ClyA for related purposes is described in Murase K. Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel). 2022 Jan 21;14(2):78. doi: 10.3390/toxinsl4020078. PMID: 35202106; PMCID: PMC8880466, the description of which is incorporated herein by reference. Use of ClyA fusion proteins as a means to incorporate the protein of interests into the described BEVs is not meant to be limiting. Other proteins that are normally trafficked to the bacterial plasma membrane can be adapted to function in a manner similar to ClyA and used as fusion protein components, wherein the fusion protein comprises the bacterial membrane protein or transmembrane segment thereof, and the cargo of interest. Cargo proteins may also be secreted within the BEVs as components of the bacterial cytosol that the BEVs also contain, which would not require a plasma membrane anchor.

The disclosure includes compositions, including but not limited to pharmaceutical compositions suitable for human and/or veterinary uses, wherein the compositions comprise the described BEVs and/or bacteria that produce the BEVs. Where in this disclosure administration of BEVs is discussed, the disclosure includes use of isolated BEV preparations, and use of the described modified bacteria that produce the BEVs.

Protein cargos that may be included in the described BEVs is not particularly limited. In examples, any cargo in this disclosure may be modified to include, in addition to the optional membrane localization signal, a nuclear localization signal or a signal to localize the cargo to any other organelle when used with eukaryotic cells. In examples, the cargo is any one or a combination of enzymes, receptor ligands, transcriptional factors, growth factors, antibodies or antigen-binding fragments thereof including single-chain antibody fragments and Fabs, peptide or protein immunogens that can be used for stimulating an immune response (i.e., a vaccine), protein-based chemotherapeutic agents, and toxins. In examples, the cargo comprises a hormone, a growth factor, a clotting factor, or a cytokine. In an example, the BEVs comprise insulin, and may therefore be used for diabetes.

The disclosure also includes BEVs that contain polynucleotides. Sequences that can be included in polynucleotides such that they selectively sort to extracellular vesicles such as exosomes are known in the art. In examples, the polynucleotides are RNA polynucleotides. The RNA polynucleotides may themselves be functional, such as for use in RNAi -mediated inhibition of gene expression, or may be a CRISPR guide RNA, or the RNA polynucleotides may encode a protein that is expressed in BEV recipient cells.

The disclosure includes cells modified to produce the described BEVs, cell culture medium comprising the BEVs, as well as isolated and/or purified BEV populations. The disclosure includes methods of making BEVs. Also included are combinations of distinct BEVs that may exhibit an additive or synergistic prophylactic or therapeutic effect. In nonlimiting examples, such as for use as a vaccine, the BEVs may include more than one cargo, or a mixture of BEVs may be used, thereby imparting multivalency to the BEVs or the composition comprising them. Such configurations can be obtained by, for example, using more than one type of fusion protein that contains more than one type of cargo, a single fusion that contains segments of different polypeptides, or more than one type of modified bacteria wherein the different types of bacteria express different cargos that are incorporated into the BEVs. This configuration can be used not just for vaccines, but for any approach where different cargos may be desired. In examples, more than one cargo are encoded within a bacterial operon, and wherein expression of the operon is inducible. Some or all of the genetic components that are used to produce the EBVs may be provided by a plasmid that is introduced into the bacteria. The plasmid will generally include known plasmid components, such as selectable markers, origins of replication, and the like.

Isolated BEVs may be introduced into cells using any suitable technique method and approach. The cells into which the BEVs are introduced may be in vitro or in vivo. In examples, the BEVs are introduced into an individual, such as a human or non-human animal. In examples, the BEVs are modified to produce a toxin or other agent that has bactericidal or bacteriostatic activity such that bacteria that produce the BEVs may selectively kill nearby bacteria that are not resistant to the toxin or the other agent.

In examples, an individual to which the described BEVs or bacteria that produce the BEVs are administered has an autoimmune disease, or cancer, or any other condition that would benefit prophylactically or therapeutically from the BEV cargo and/or the modified bacteria. In examples, the BEVs are used to treat an individual diagnosed with, suspected of having, or at risk for developing or relapsing, of an autoimmune disease that is any of Addison’s disease, Alopecia areata, Celiac disease, Chagas disease, Congenital heart block, Crohn’s disease, Dermatitis herpetiformis, Dermatomyositis, Endometriosis, Fibromyalgia, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hypogammalglobulinemia, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Lupus, Lyme disease, Meniere’s disease, Multiple sclerosis, Myasthenia gravis, PANDAS, Peripheral neuropathy, Pernicious anemia, Primary biliary cirrhosis, Psoriasis, Psoriatic arthritis, Reactive Arthritis, Rheumatoid arthritis, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Type 1 diabetes, Ulcerative colitis, Vasculitis, or Vitiligo.

In examples, the BEVs are introduced into an individual who has cancer. In examples, the cancer can be any type of cancer. In examples, the cancer is a solid tumor which may be a solid tumor that is at risk for metastasis. In examples, the individual may have a tumor that is at risk of or is undergoing metastasis. The individual may have previously had a metastatic tumor and is at risk for recurrence of a tumor and/or metastasis of it. In examples, the cancer may be any one of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, head and neck cancer, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oliodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, thymoma, Waldenstrom's macroglobulinemia, and heavy chain disease.

In examples, the BEVs are introduced to an individual who has an inflammatory condition, including but not necessarily limited in inflammatory bowel disease (IBD).

In examples, an effective amount of the described exosomes can be administered to an individual. The term “therapeutically effective amount” as used herein refers to an amount of EBVs and/or the and the described bacteria the produce the EBVs sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of BEVs and/or modified bacteria to achieve a desired effect. Additional factors which may be taken into account include the type of disease. Additional factors that can be taken into account include the weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. In certain examples, a therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and/or reduces the severity of the disease. A therapeutically effective amount may also inhibit or prevent the onset of a disease, or a disease relapse. For the modified bacteria, in example, a therapeutically effective amount of described bacteria are administered. In examples, when the modified bacteria are used, a therapeutically effective amount may comprise 1 million to 100 billion colony forming units, inclusive, and include all numbers and ranges of numbers there between. When bacteria are used in vivo, the inducer may be administered systemically or locally. In an example, the bacteria are used on the skin of an individual, such as for an allergic reaction, or a wound, or a burn. In this example the inducer can be applied topically, such as in the form of a spray, cream or ointment.

Bacteria for use in the described methods can be obtained from any suitable source, such as from the American Type Culture Collection (ATCC), or sourced from an existing, stored culture. In examples the bacteria are Gram-negative or Gram-positive bacteria. In an example the bacteria are lactic acid bacteria. In examples, the lactic acid bacteria are any type of Lactobacillus, Streptococcus, Enterococcus, Leuconostoc, or Pediococcu wherein the bacteria produce lactic acid as a primary metabolic product. Lactic acid bacteria are useful in fermented foods and beverages, as probiotics, and are also suitable for other purposes such use as additives in non-human animal feed. In examples, the bacteria are any of Lacticaseibacillus casei, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, or Lactiplantibacillus plantarum. In non-limiting examples, some of which are illustrated by the Figures, lactic acid bacteria are used to produce BEVs that

In an example, the bacteria are any type of Faecalibacterium, including but not necessarily limited to E prausnitzii, which may be particularly beneficial for use in a gastrointestinal environment. In examples, the bacteria may be an E.coli strain that is suitable for use as a probiotic such as E. Coli Nissle 1917.

In examples the disclosure includes a supplement product, such as a nutraceutical product, or a dietary supplement, or a food ingredient, including but not limited to probiotic formulations or functionals food that contains one or more live modified bacteria as described herein. The supplement product can be provided in the form of, for example, a liquid, capsules, tablets, softgels, powders, freeze-dried compositions, and the like. In examples, the supplement product may be provided as enteric-coated capsules. In examples, administration of the described bacteria may be performed such that the bacteria are introduced into the gastrointestinal system. In examples, the administration can be orally or by enema or colonoscope, or via intubation of the small bowel using for example a large bore catheter equipped with a distal balloon to effect rapid passage down the jejunum.

Those skilled in the art will recognize how to separate BEVs made according to this disclosure. In general BEVs are collected from a cellular supernatant and can be isolated by differential centrifugation according to well-known protocols. BEVs that contain cargo can be separated from those that do not by this approach, if desired. The BEVs may be purified to any described degree of purity. The BEVs may be obtained after the modified bacteria are lysed by operation of the described genetic circuity, but lysis is not necessarily required to produce the BEVs.

The disclosure includes kits for making, screening, and using the modified bacteria and/or the BEVs and for use with any desired purpose.

The Examples of this disclosure are illustrated by the accompanying Figures. These Examples are representative and demonstrate proof-of-principle but are not intended to be limiting.

Examples

FIG. 1 provides graphs depicting results demonstrating high yields of BEVs are rapidly generated upon AI-1 induction of LysE expression. FIG. 2 provides resulting demonstrate that high yield BEVs retain recombinant protein cargo and anti-inflammatory efficacy. FIG. 3 provides results demonstrating other lysis-inducing proteins can be used in Gram positive bacteria. Collectively, FIGS 1-3 and the figures that follow demonstrate the disclosure enables on-demand, rapid generation of ~20-fold increased yields of BEVs that have equivalent therapeutic bioactivity to wild type BEVs which is expected to be transformative for BEV biomanufacturing and is believed not to be possible with previous techniques. Additionally, the Figures show that the degree of increased BEV yields is tunable by adjusting the inducer molecule concentration, thereby further increasing operator control. In examples, after induction of lysis, a minor population of viable bacteria rapidly regrows, permitting multiple cycles of lysis over a 24-hour period. Embodiments of the are compatible with current BEV protein cargo loading technology. In FIG. 2A, green fluorescent protein (GFP) was loaded into BEVs using an existing ClyA fusion approach, and we observed similar ~20-fold increases in GFP content in the BEV sample. Significantly, the antiinflammatory bioactivity of these probiotic BEVs is completely preserved (FIG, 2B). Therefore, FIGs 1-3 show how the presently provided systems involve a genetic circuit wherein expression of a phage-derived lysis-inducing protein using (LysE is a representative example) is coupled to an exogenous signal that is manually added to the culture. The Examples show the inducer can be safe, well-characterized molecules to induce protein expression. In FIGs 1-3 the signal is the bacterial quorum sensing molecule AI-1. However, as described above, any inducible protein expression system is expected to be compatible with this system. As discussed above, the system allows elimination for any requirement for activation of prophages which can introduce system instability, batch-to-batch variability, and additional safety concerns. The data in FIGs 1-3 was obtained in a probiotic strain of E. coli, E. coli Nissle 1917, which is a human commensal, a natural isolate, and is host of choice for engineering probiotics for use in human. As discussed above, this approach is expected to function other species of both Gram positive and Gram negative bacteria owing to conserved mechanisms of lysis. As an example, FIG. 3 demonstrates inducible expression of a different lysis-inducing protein, Lyb5, in Gram positive probiotic, Lacticaseibacillus paracasei 334. The protein Lyb5 is derived from a bacteriophage but lacks other requisite components for phage assembly or activation. This serves to illustrate the flexibility of the system between Gram negative and Gram-positive species, as well as the flexibility across different lysisinducing proteins. Additional examples are shown in FIGs. 4-11. FIG. 4 shows that BEVs are heterogenous and are co-isolated with impurities. FIG. 5 demonstrates that BEVs can show increased potency in vitro. FIG. 6 demonstrates that /.. plantarum is a suitable bacteria for treating IBD, with data from a mouse IBD model. FIG. 7 shows effects on specific genes in colon tissue. FIG. 8 shows a representative plasmid and dose-dependency. This is considered to represent genetically-programming cell wall remodeling (aka lysis). FIG. 9 shows engineered hypervesiculating L. plantarum produces >22-fold more BEVs relative to unmodified bacteria of the same type. FIG. 10 shows that high yield BEVs show dosedependent anti-inflammatory effects in macrophages in vitro. FIG. 11 shows high yield Lp BEVs are effective in a mouse model of IBD (acute DSS), with beneficial effects on weight loss, gross disease severity, and clinical symptoms.

These examples are intended to illustrate aspects of the disclosure but are not meant to be limiting.

Claims

What is claimed is:

1. Modified bacteria that are modified such that they contain a polynucleotide comprising an inducible promoter that is operably linked to a sequence encoding an endolysin or an autolysin, wherein the endolysin or the autolysin is not encoded by a polynucleotide within the bacteria prior to being modified, and wherein inducing expression of the autolysin or the endolysin promotes production of bacterial extracellular vesicles (BEVs) in an amount that is higher than BEVs produced by bacteria that are not modified to contain a sequence that encodes the endolysin or the autolysin.

2. The modified bacteria of claim 1, wherein the bacteria are Gram-positive bacteria.

3. The modified bacteria of claim 2, wherein the Gram-positive bacteria are lactic acid bacteria.

4. The modified bacteria of claim 3, wherein the lactic acid bacteria are modified to express the endolysin.

5. The modified bacteria of claim 3, wherein the lactic acid bacteria are modified to express the autolysin.

6. The modified bacteria of claim 1, wherein the bacteria are Gram-negative bacteria.

7. The modified bacteria of claim 6, wherein the Gram-negative bacteria are modified to express the autolysin.

8. The modified bacteria of claim 6, wherein the Gram -negative bacteria are modified to express the endolysin.

9. The modified bacteria of any one of claims 1-8, wherein the BEVs comprise a therapeutic or prophylactic cargo.

10. A method comprising inducing expression of an endolysin or an autolysin in modified bacteria of any one of claims 1-8, wherein the bacteria are present in a bacteria culture medium and produce the BEVs, the method further comprising separating the BEVs from the bacteria culture medium.

11. An isolated BEV preparation obtained from the method of claim 10.

12. A method comprising introducing into an individual in need thereof a composition comprising an isolated BEV preparation as in claim 11 to thereby provide a therapeutic or prophylactic effect in the individual.

13. The method of claim 12, wherein the individual is in need a treatment for an inflammatory condition.

14. The method of claim 12, wherein the individual is in need of treatment for an autoimmune condition.

15. The method of claim 12, wherein the individual is in need of treatment for cancer.