Optimized non-chromosomal dynamic activity systems and uses thereof

Abstract

Disclosed herein is a non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one loss of genetic function in a lyase to increase the stability of the ADAS and at least one loss of genetic function in a protease to improve cargo expression, and having or not having an additional constructed cell wall embedded anchoring structure to optimize cargo display.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing, which has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on November 15, 2023, is named 51296-058WO2_Sequence_Listing_11_15_23 and is 187,929 bytes in size. Technical Field

[0003] Provided herein are non-chromosomal dynamic activity systems and methods for their preparation and use. Background Art

[0004] There is a need for delivery vectors capable of targeting cells and delivering biological agents; compositions containing such delivery vectors; and related methods of delivering the vectors to cells to modulate biological systems including animal, plant, and fungal cells, tissues, and organisms. In particular, there is a need for delivery vectors (e.g., non-chromosomal dynamic activity systems (ADAS)) with improved cytoplasmic and surface cargo expression. Summary of the Invention

[0005] In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function alteration in a lyase and at least one loss-of-function alteration in a protease. In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one lyase genomic deletion and at least one protease deletion. In some embodiments, the loss-of-function alteration in the lyase results in increased stability of the ADAS relative to an unmodified ADAS. In some embodiments, the loss-of-function alteration in the protease results in increased cargo expression in the ADAS relative to an unmodified ADAS. In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function alteration in a lyase and a constructed cell wall-embedded anchoring structure for displaying cargo. In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one lyase genomic deletion for stabilizing the ADAS and a constructed cell wall-embedded anchoring structure for displaying cargo.

[0006] In some embodiments, the parental bacterial cell comprises at least one loss-of-function alteration in a lytic enzyme and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more genetic loss-of-function alterations in a protease. In some embodiments, the parental bacterial cell comprises at least one lytic enzyme genomic deletion and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more protease deletions.

[0007] In some embodiments, the parental bacterial cell has been modified to reduce enzyme activity.

[0008] In some embodiments, the loss-of-function or genomic deletion in the parental bacterial cell reduces enzyme activity and / or lytic activity. In some embodiments, the parental bacterial cell has a loss-of-function or genomic deletion that reduces the activity of an endopeptidase, a cell wall lytic enzyme, and / or an autolysin.

[0009] In some embodiments, the parental cell comprises a genetic loss-of-function alteration or genomic deletion in a gene selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0010] In some embodiments, the parental cell comprises a genetic loss-of-function alteration or deletion that disrupts sporulation in a gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof.

[0011] In some embodiments, the parental cell comprises a genetic loss-of-function or genomic deletion in an enzyme selected from the group consisting of proteases, amylases, lipases, cellulases, and combinations thereof.

[0012] In some embodiments, the parental bacterial cell comprises at least one loss-of-function genetic alteration in a protease. In some embodiments, the parental bacterial cell comprises at least one protease deletion. In some embodiments, the loss-of-function alteration or genomic deletion is in aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or a combination thereof.

[0013] In some embodiments, the parental bacterial cell comprises at least one loss-of-function genetic alteration in an amylase. In some embodiments, the parental bacterial cell comprises at least one amylase deletion. In some embodiments, the loss-of-function alteration or genomic deletion is in amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, or a combination thereof.

[0014] In some embodiments, the parental bacterial cell comprises at least one loss-of-function genetic alteration in a lipase. In some embodiments, the parental bacterial cell comprises at least one lipase deletion. In some embodiments, the loss-of-function alteration or genomic deletion is in lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, or a combination thereof.

[0015] In some embodiments, the parental bacterial cell comprises at least one loss-of-function genetic alteration in a cellulase. In some embodiments, the parental bacterial cell comprises at least one cellulase deletion. In some embodiments, the loss-of-function alteration or genomic deletion is in celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof.

[0016] In some embodiments, the parental bacterial cell comprises at least one loss-of-function alteration in a lyase, protease, amylase, lipase, cellulase, a gene affecting the sporulation mechanism, or a combination thereof.

[0017] In some embodiments, the parental bacterial cell comprises at least one genomic deletion in a region encoding a lyase, protease, amylase, lipase, cellulase, a gene affecting the sporulation mechanism, or a combination thereof.

[0018] In some embodiments, the parental bacterial cell is a Gram-positive bacterial cell.

[0019] In some embodiments, the parental bacterial cell is Bacillus subtilis or a Bacillus species.

[0020] In some embodiments, the parental bacterial cell is a Lactobacillus species.

[0021] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0022] In some embodiments, the parental bacterial cell is from the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azorhizobium, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phormidium, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0023] In some embodiments, the parental cell comprises a gene deletion from the group consisting of SPβ, skin, PBSX, prophage 1, pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. [Molecular Biology and Evolution] 20(12):2076–2090, 2003).

[0024] In some embodiments, the parental bacterial cell contains a loss-of-function alteration in a cell division topology-specific factor. In some embodiments, the parental bacterial cell contains a genomic deletion of a cell division topology-specific factor. In some embodiments, the genomic deletion pertains to the DivIVA, minC, minD, minE, minCD, or minCDE operon.

[0025] In some embodiments, the ADAS further comprises at least one cargo.

[0026] In some embodiments, the cargo is a protein or polypeptide. In some embodiments, the cargo is a small molecule, macromolecule, nucleic acid, polynucleotide, or enzyme.

[0027] In some embodiments, the ADAS or the parental bacterial cell has been modified to increase the level of the cargo in the ADAS.

[0028] In some embodiments, the cargo is an enzyme, DNA modifier, chromatin remodeler, gene editor, nuclear targeting agent, binder, immunogenic agent, toxin, mutant protein, cytokine, superkine, or any combination thereof. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editor is a component of the CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binder is an antibody or antibody fragment. In some embodiments, the binder is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulant. In some embodiments, the immunogenic agent is an immunosuppressant. In some embodiments, the immunogenic agent is a tumor antigen. In some embodiments, the tumor antigen is bound to a stabilizer. In some embodiments, the mutant protein is a mutated protein of a known antigen, enzyme, cytokine, or immunogen. In some embodiments, the toxin is an autophagy-inducing agent (e.g., an activator such as listeriolysin O). In some embodiments, the cargo is a heterologous pore-forming toxin (e.g., listeriolysin O). In some embodiments, the toxin aids in the delivery of the ADAS cargo to the target cell. In some embodiments, multiple ADAS cargos are expressed.

[0029] In some embodiments, the cargo is IL-2 or superkine IL-2. In some embodiments, surface cargo is displayed via an engineered cell wall-embedded anchoring structure.

[0030] In some embodiments, the cell wall-embedded anchor construct comprises anti-parallel coiled coils (APCCs) of different lengths. In some embodiments, the cell wall-embedded anchor construct comprises shortened anti-parallel coiled coils. In some embodiments, the cell wall-embedded anchor construct comprises a linker. In some embodiments, the cell wall-embedded anchor construct comprises a coiled linker. In some embodiments, the cell wall-embedded anchor construct is a fusion construct.

[0031] In some embodiments, the cell wall-embedded anchor construct contains a cell wall binding domain.

[0032] In some embodiments, the ADAS contains a fusion promoter.

[0033] In some embodiments, the ADAS contains a proline-rich sequence encoding the cell wall-embedded anchor structure.

[0034] In some embodiments, the ADAS encodes a self-binding helix for the cell wall-embedded anchor structure.

[0035] In some embodiments, the cell wall-embedded anchor structure is an unstructured and proline-rich construct.

[0036] In some embodiments, the cell wall-embedded anchor structure is a structured construct.

[0037] Other features and advantages of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a bar graph showing the immunofluorescence staining intensity of IL-2 (cargo) on MACH2762, MACH2887, MACH2888, and MACH2889 cells (as defined in Tables 1-3) quantified using microscopy and normalized for cell size. High levels of surface-displayed cargo expression were observed on MACH2889 compared to all other strains. The MACH2762 parental cells contain deletions that block sporulation, lytic activity, and protease activity. The MACH2889 parental cells contain the same deletions as well as an expression cassette for surface IL-2. The MACH2888 parental cells contain the same IL-2 cargo but do not contain the protease deletion. MACH2887 contains the same IL-2 cargo without protease, lyase, or sporulation deletions. As confirmed by the lack of IL-2 signal in all cells except MACH2889, one or more protease deletions are required for the expression and surface display of cargo.

[0039] Figure 2AThe middle figure shows the relationship between the dose (number of ADAS per cell) and the absorbance at 620 nm of the HEK-Blue IL-2 cell culture supernatant exposed to various Bacillus subtilis strains characterized by different IL-2 surface display constructs. This effectively determines the ratio of the number of ADAS per cell to IL-2R activation, thereby screening for the functionality and stability of different IL-2 and IL-2 mutant protein constructs. MACH2762 without a display or cargo and MACH2788 with a display but without cargo were used as control strains. MACH2777, MACH2780, MACH2782, MACH2783, MACH2786, MACH2787, MACH2790, and MACH2791 are described in Table 3.

[0040] Figure 2B is a set of schematic diagrams showing exemplary cargo display structures using different cell wall-embedded anchor constructs, and a table depicting Figure 2A the display structures of the individual strains shown in and the designated cargo. IL-2 is shown as an exemplary cargo.

[0041] Figure 3A is a bar graph showing the systemic measurements of IFNγ in mouse plasma at 2 and 6 hours after intravenous administration of a dose of PBS, ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0042] Figure 3B is a bar graph showing the systemic measurements of IL-2 in mouse plasma at 2 and 6 hours after intravenous administration of a dose of PBS, ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0043] Figure 3C is a bar graph showing the systemic measurements of IL-6 in mouse plasma at 2 and 6 hours after intravenous administration of a dose of PBS, ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0044] Figure 3DIs a bar graph showing the systemic measurement of TNFa in mouse plasma at 2 and 6 hours after administration of an intravenous dose of PBS, ADAS derived from MACH2762 (without cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0045] Figure 3E Is a bar graph showing the dose - response measurement of IFNγ secreted by human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (without cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0046] Figure 3F Is a bar graph showing the dose - response measurement of IL - 2 secreted by human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (without cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0047] Figure 3G Is a bar graph showing the dose - response measurement of IL - 6 secreted by human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (without cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0048] Figure 3H Is a bar graph showing the dose - response measurement of TNFa secreted by human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (without cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytoplasmic cargo), and ADAS derived from MACH2880 (with surface and cytoplasmic cargo).

[0049] Figure 4Is a bar graph showing the level of antigen - specific CD8 T - cell activation 24 hours after administration of the designated ADAS. The activation level is measured as the positive granzyme B (GzmB) in live CD45 +, CD8 + T cells as measured using flow cytometry and serves as a measure of antigen - specific CD8 T - cell activation. Measurements were made in OT - 1 mice engineered to have a CD8 T - cell receptor that recognizes ovalbumin (ova). ADAS was produced by Bacillus subtilis strains that either do not have a cargo (MACH2762), have a surface display of ovalbumin (MACH2829), or have a cytoplasmic expression of ovalbumin and listeriolysin O (MACH2670) via subcutaneous or intravenous administration routes. Strain details are provided in Tables 1 - 3.

[0050] Figure 5 The middle panel depicts the MC38 tumor volume in mice two weeks after tumor implantation and administration of PBS (peritumoral), anti - PD - 1 (intraperitoneal), or ADAS (MACH2854 - peritumoral; or MACH2862 - intravenous) on day 10. Strain details are in Tables 1 - 3. Detailed Description

[0051] I. Definitions

[0052] As used herein, the term "achromosomal dynamic system" or "ADAS" refers to a genome - free non - replicating closed membrane system that comprises at least one membrane and has an internal volume suitable for accommodating cargo (e.g., one or more of the following: nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs)). In some embodiments, the ADAS is a minicell or a modified minicell derived from a parental bacterial cell (e.g., a Gram - negative or Gram - positive bacterial cell). In other aspects, the ADAS is derived from a parental cell by modifying the parental cell to remove the genome and is substantially similar in size to the parental cell. In embodiments, the ADAS is derived from a parental bacterium using any suitable method, such as genetically manipulating the parental cell or exposing it to cultures or conditions that enhance the likelihood of bacterial minicell formation. An exemplary method for preparing ADAS is a method that disrupts the cell - division machinery of the parental cell. In some embodiments, the ADAS includes one or more endogenous or heterologous features on the surface of the parental cell, such as a cell wall, cell - wall modification, flagella, or pili, and / or one or more endogenous or heterologous features in the internal volume of the parental cell, such as nucleic acids, plasmids, proteins, small molecules, transcriptional machinery, or translational machinery. In other embodiments, the ADAS lacks one or more features of the parental cell. In still other embodiments, the ADAS is loaded with or otherwise modified by features not contained in the parental cell.

[0053] As used herein, the term "highly active ADAS" refers to an ADAS having a high working potential, e.g., an ADAS having the ability to perform a large amount of useful work. In some embodiments, the work is metabolic work, including chemical synthesis (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), chemical modification (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), or transport (e.g., import, export, or secretion, e.g., secretion by a bacterial secretion system such as the T3SS) under suitable conditions. In certain embodiments, the highly active ADAS starts with a large amount of energy (e.g., energy in the form of ATP). In other embodiments, the ADAS has the ability to absorb or generate energy / ATP from another source. In some embodiments, highly active ADAS is identified by, e.g., an increase in ATP concentration, an increase in the ability to produce ATP, an increase in the ability to produce proteins, an increase in the rate or amount of protein production, and / or an increase in reactivity to biological signals (e.g., induction of a promoter).

[0054] As used herein, the term "parent bacterial cell" refers to the cell (e.g., a Gram-negative or Gram-positive bacterial cell) from which the ADAS is derived. The parent bacterial cell is typically a live bacterial cell. The term "live bacterial cell" refers to a bacterial cell that contains a genome and is capable of cell division. Preferred parent bacterial cells are derived from any of the following strains: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azorhizobium, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxobacter, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacteria.

[0055] An ADAS composition or formulation that is "substantially free" of parental bacterial cells and / or viable bacterial cells is defined herein as a composition having no more than 500, e.g., 400, 300, 200, 150, 100 or fewer colony forming units (CFU) / mL. In some embodiments, an ADAS composition that is substantially free of parental bacterial cells or viable bacterial cells (including no bacterial cells) comprises less than 50, less than 25, less than 10, less than 5, less than 1, less than 0.1 or less than 0.001 CFU / mL.

[0056] The term "cell division topological specificity factor" refers to a component of the cell division apparatus in a bacterial species that is involved in determining the site of the septum and acts by restricting the location of other components of the cell division apparatus (e.g., restricting the location of one or more Z-ring inhibitory proteins). Exemplary cell division topological specificity factors include MinE, which was first discovered in Escherichia coli and has been identified in a large number of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). MinE acts by restricting the Z-ring inhibitory proteins MinC and MinD to the poles of the cell. A second exemplary cell division topological specificity factor is DivIVA, which was first discovered in Bacillus subtilis (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005).

[0057] The term "Z-ring inhibitory protein" refers to a component of the cell division apparatus in a bacterial species that is involved in determining the site of the septum and acts by inhibiting the formation of a stable FtsZ ring or anchoring such a component to the membrane. In some embodiments, the localization of the Z-ring inhibitory protein is regulated by cell division topological specificity factors (e.g., MinE and DivIVA). Exemplary Z-ring inhibitory proteins include MinC and MinD, which were first discovered in Escherichia coli and have been identified in a large number of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). In Escherichia coli and in other species, MinC, MinD and MinE are present at the same genetic locus, which may be referred to as the "min operon", the MinCDE operon or the min or MinCDE genetic locus.

[0058] As used herein, the term "reduced level or activity of a cellular topology-specific factor" refers to a general reduction of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level or activity of a cellular topology-specific factor (e.g., a protein or a nucleic acid (e.g., a gene or an mRNA)) as detected by standard methods, compared to the level in a reference sample (e.g., an ADAS produced by wild-type cells or cells having a wild-type minCDE operon or a wild-type divIVA gene), a reference cell (e.g., a wild-type cell or a cell having a wild-type minC, minD, minE, divIVA or minCDE gene or operon), a control sample or a control cell. In some embodiments, the reduced level or activity refers to a reduction in the level or activity in a sample, the level or activity being at least about 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x or 0.01x of the level or activity of the cellular topology-specific factor in the reference sample, reference cell, control sample or control cell.

[0059] As used herein, the term "percent identity" refers to the percent (%) sequence identity relative to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways within the capabilities of those of skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST or Megalign software. Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. For example, in some embodiments, the sequence comparison computer program BLAST is used to generate percent sequence identity values. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A with, compared to, or relative to a given nucleic acid or amino acid sequence B (which can alternatively be expressed as the phrase: a given nucleic acid or amino acid sequence A having a certain percent sequence identity with, compared to, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows:

[0060] 100×(fraction X / Y)

[0061] Where X is the number of nucleotides or amino acids that are scored as identical matches by a sequence alignment program (e.g., BLAST) in the alignment of A and B, and where Y is the total number of nucleotides or amino acids in B. In some embodiments, for example, in homologs of the MinE or DivIVA proteins, the sequence identity to the native sequence MinE (or minE) or DivIVA (or divIVA) sequences disclosed herein will have at least about 40%, 50%, 60%, 70%, 80%, 85%, 90% or even 95% or greater amino acid or nucleic acid sequence identity, alternatively, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence or nucleic acid identity.

[0062] As used herein, the phrase "modulating the state of a cell" refers to an observable change in the state of a cell (e.g., a transcriptome, proteome, epigenome, biological effect, or health or disease state), such as an animal, plant, or insect cell, as measured using techniques and methods known in the art for such measurements (e.g., methods for measuring the level or expression of proteins, transcripts, epigenetic markers, or for measuring an increase or decrease in biological pathway activity). In some embodiments, modulating the state of a cell results in a change of at least 1% relative to before administration (e.g., a change of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., a change of up to 100% relative to before administration). In some embodiments, modulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In some embodiments, increasing the state of a cell results in a parameter being increased by at least 1% relative to before administration (e.g., increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., increased by up to 100% relative to before administration). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In some embodiments, decreasing the state of a cell results in a parameter being decreased by at least 1% relative to before administration (e.g., decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., decreased by up to 100% relative to before administration).

[0063] As used herein, the term "endogenous type III secretion system" or "endogenous T3SS" refers to a T3SS that is present on a cell (e.g., a parental cell) or an ADAS derived therefrom and that is naturally encoded by the cell (e.g., encoded by the wild-type version of the cell). The T3SS can be expressed from an endogenous gene of the cell, and / or can be encoded and expressed by a synthetic construct in the cell. The expression or abundance of the endogenous T3SS can be increased, for example, by adding a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by reducing or removing a negative regulator of T3SS expression.

[0064] As used herein, the term "heterologous type III secretion system" or "heterologous T3SS" refers to a T3SS that is present on or derived from a cell (e.g., a parental cell) and is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell). The cell may encode another T3SS or may not encode any T3SS. In some embodiments, the T3SS is expressed from a synthetic construct in the cell.

[0065] As used herein, an "endogenous effector" of a secretion system (e.g., T3SS, T4SS, or T6SS) is a moiety (e.g., a protein or polypeptide) that is naturally encoded by the cell (e.g., encoded by the wild-type version of the cell) from which the secretion system (e.g., T3SS) is derived and that is capable of being secreted by the secretion system. One or more of the secretion system and its endogenous effector(s) may be expressed in the cell in which they naturally occur or may be expressed heterologously, e.g., by a cell that does not naturally encode the endogenous effector or the secretion system.

[0066] As used herein, an effector that is heterologous relative to a secretion system ("heterologous effector") is a moiety (e.g., a protein or polypeptide) that is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell) from which the secretion system (e.g., T3SS) is derived and that is capable of being secreted by the secretion system of the cell from which the heterologous effector is derived. In some embodiments, the effector is capable of being secreted by a secretion system to which it is heterologous or is modified to be secreted by a secretion system to which it is heterologous. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0067] As used herein, the term "heterologous" means not inherent to a cell or composition in its natural state. In some embodiments, "heterologous" refers to a molecule; e.g., a cargo or payload (e.g., a polypeptide, a nucleic acid such as an RNA encoding a protein or a tRNA, or a small molecule) or a structure (e.g., a plasmid or a gene editing system) that is not naturally present in an ADAS or the parental bacterium (e.g., a Gram-negative or Gram-positive bacterial cell) that gave rise to the ADAS.

[0068] As used herein, the terms "phase-light ADAS" and "phase-light parental cell" refer to an ADAS or parental cell body (e.g., a dead ADAS or dead parental cell) that appears brighter or has a ghosting (indicating rupture and lysis) in an image (e.g., a micrograph taken using an optical microscope) compared to a dark ADAS or parental cell (which are described herein as "intact ADAS" and "intact parental cell", respectively).

[0069] As used herein, "increased stability" of an ADAS refers to an overall enhancement of the integrity of the ADAS. The stability of an ADAS (e.g., the stability of an ADAS of a particular population, strain, or variety) can be measured as the ratio (e.g., percentage) of "intact" ADAS to "light-phase" ADAS in one or more representative images of a plurality of ADAS. For example, an increased stability of an ADAS derived from a modified strain containing at least one lyase deletion can be defined as an increase in the percentage of "intact" ADAS in a representative image of the ADAS derived from the modified strain compared to the ADAS derived from a control strain (e.g., a strain not containing the lyase deletion), where the measurement (e.g., scoring) is made under the same conditions and at the same time point. For example, if the modified ADAS has a stability greater than 40%, then more than 40% of the visible ADAS is intact rather than light-phase. In some embodiments, the stability of an ADAS is measured as the unitless ratio of the half-life of an ADAS derived from an unmodified strain (e.g., a strain not containing the lyase deletion) to the half-life of an ADAS derived from a modified strain (e.g., a strain containing the lyase deletion), where the measurement is made under the same environmental conditions. In some embodiments, at one or more time points (e.g., 0 hours, 10 hours, 20 hours, 23 hours, 24 hours, 48 hours, 72 hours, or more than 72 hours after ADAS enrichment), the fold change in the optical density at wavelength 600 (OD600) of an ADAS derived from a modified strain compared to an ADAS derived from a control strain is another measure of ADAS stability. The stability of an ADAS is improved (e.g., increased) by at least one genomic deletion of a lyase in the parental cell. In certain embodiments, an ADAS having one or more lyase deletions (e.g., derived from a parental cell containing one or more lyase deletions) has a stability greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (as measured by the percentage of intact ADAS) compared to an ADAS not having a lyase deletion (e.g., derived from a parental cell not containing the lyase deletion).

[0070] As used herein, "increased stability" or "optimization" of a cargo in an ADAS refers to an overall increase in the expression, presentation, function, or durability of the cargo of the ADAS as compared to a control or unmodified ADAS. In some embodiments, the stability of the cargo is measured as the unitless ratio of the half-life of the unmodified cargo to the half-life of the modified cargo, where the measurements are made under the same environmental conditions. The modified cargo can be, for example, a cargo expressed by a presentation construct provided herein. In some aspects, the stability of the cargo is measured as the fold change in the level of cargo expression between a modified ADAS (e.g., an ADAS derived from a modified parental cell (e.g., a parental cell that has been modified to contain a presentation construct)) and a control ADAS, where the measurements are made under the same environmental conditions. The stability and / or optimization of cargo expression can be improved by at least one genomic deletion of both a lytic enzyme and a protease in the parental bacterial cell. In some embodiments, more than one lytic enzyme, protease, or other enzyme (e.g., lytic enzyme, endopeptidase, autolysin, protease, lipase, cellulase, amylase, peptidase, etc.) is deleted in the parental cell. In certain embodiments, the stability of the cargo in an ADAS derived from a parental cell having one or more lytic enzyme deletions and one or more protease deletions is 2, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 100, 150, 200, 250, or 300 times the stability of the cargo in a control ADAS (e.g., an ADAS derived from a parental cell that does not contain lytic enzyme deletions and protease deletions). In some embodiments, the stability or optimization of cargo expression is measured by the functionality of the cargo, e.g., by measuring or detecting the level of cytokine release, receptor activation, or antigen-specific cell activation.

[0071] As used herein, a "lytic enzyme" refers to an enzyme (e.g., an endopeptidase, cell wall lytic enzyme, or autolysin) that regulates the rupture of the parental bacterial cell wall and membrane. Genomic deletion of one or more of these enzymes reduces the lytic activity in the parental cell and, in some embodiments, can increase the stability and integrity of the ADAS construct, thereby allowing for the production of a greater number of intact ADASs and / or increasing the survival time period of the intact ADASs. Specific genomic deletions that reduce the lytic activity in the parental cell include, but are not limited to, lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, and combinations thereof.

[0072] As used herein, "sporulation" refers to the process by which certain parental bacterial strains form spores from cells during adverse conditions. Genetic deletions that may disrupt sporulation include, but are not limited to, sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof.

[0073] As used herein, "protease" refers to any enzyme that degrades proteins. A loss-of-function mutation (e.g., deletion) of one or more of these enzymes reduces protease activity in the parental cell. Specific genomic deletions or loss-of-function mutations that the parental cells of the present invention may contain include, but are not limited to, aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or combinations thereof. In some embodiments, in addition to lyases and proteases, the parental cells of the present invention further contain one or more additional enzyme deletions or loss-of-function mutations. Examples of other enzymes that may be deleted or disrupted by loss-of-function mutations include, but are not limited to, amylase, lipase, and cellulase. As used herein, "amylase" is an enzyme that breaks down starch, polysaccharides, and complex carbohydrates into sugars. As used herein, "lipase" is an enzyme that is part of the class of hydrolases known for lipid breakdown and for hydrolyzing glycerol esters. Examples of bacterial genes encoding amylase, lipase, and cellulase include, but are not limited to, amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or combinations thereof.

[0074] As used herein, the terms "engineered cell wall-embedded anchoring structure", "cell wall-anchored construct", and "cell wall-embedded anchoring construct" refer to a cell wall scaffold or other functionally equivalent entity that can provide a fixed construct from which surface cargo can be displayed. Engineered cell wall-embedded anchoring constructs can be expressed in ADAS and / or its parental cells. Examples of potential engineered cell wall-embedded anchoring structures include, but are not limited to, antiparallel coiled coils, cell wall-embedded or outer membrane-anchoring domains with one or more coiled or uncoiled linkers, individual cargo proteins (e.g., mCherry) without linkers or with one or more linkers, and cell wall-embedded or outer membrane-anchoring domains without linkers. In some embodiments, (a) the cargo is linked to the cell wall-embedded anchoring structure using a linker (e.g., a polypeptide linker), or (b) the cargo is directly linked to the cell wall-embedded anchoring structure. In some embodiments, the cargo and one or more components of the cell wall-embedded anchoring structure are contained within the same polypeptide chain.

[0075] As used herein, "display structure" or "cargo display structure" refers to any means by which an ADAS expresses and / or displays cargo (e.g., displayed on the surface or in the cytosol of the ADAS). Several surface display structures that include cargo and one or more components involved in displaying the cargo (e.g., binding domains, linkers, and / or scaffolds) are shown in Figure 2BHowever, these display constructs should not be considered restrictive. Display 0 corresponds to a fusion (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain) of a cargo displayed on a cell wall-embedded binding domain that does not have a linker (e.g., does not have a linker domain that attaches the cargo to the binding domain). Display 1 corresponds to a cargo displayed via a cell wall-embedded binding domain having one linker (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain linked by a linker). Display 2 is a construct (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain linked by one or more antiparallel coiled coils) of a cargo linked to a cell wall-embedded binding domain (anchor) via one or more antiparallel coiled coils. Display 3 is a construct using a structure similar to Display 2 but having a shortened antiparallel coiled coil. Display 4 is a construct using a coiled linker that is anchored to the cell wall via a binding domain (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain linked by a coiled linker). Displays 5 and 6 comprise a cargo displayed via a linker fused to a scaffold protein (e.g., mCherry) (e.g., a fusion polypeptide comprising a cargo, a scaffold protein, a linker, and a cell wall-embedded binding domain). Display 6 further comprises a linker that attaches the cargo to the scaffold protein. In some embodiments, the display construct is cytoplasmic rather than by surface expression, e.g., the cargo is displayed by one or more components (e.g., a binding domain, a linker, and / or a scaffold) in the interior (cytosol) of the ADAS. The display construct can be "unstructured", involving a relatively high proportion of proline in the structure to create a non-linear or bent shape for the construct, or can be "structured", such as coiled, with a specific or more defined shape. The expression of the cargo can also be affected by the absence or genetic loss-of-function mutations of different proteases and lyases, as well as different promoters, integration sites, and cell-binding domains.

[0076] As used herein, "genetic loss-of-function" refers to a significant reduction or complete elimination of a protein (e.g., a significant decrease in the protein function, a complete loss of the protein function, a significant decrease in the protein expression, or a complete loss of the protein expression) caused by a change (e.g., a gene mutation) in the gene encoding the protein. Mutations include, but are not limited to, insertions or deletions of one or more nucleotides, non-silent codon changes, and duplications. Proteins in which genetic loss-of-function can occur include, but are not limited to, enzymes such as lyases, proteases, amylases, lipases, or cellulases. In addition to genetic loss-of-function by mutation, promoter inactivation or chemical inhibition can also affect (e.g., reduce or eliminate) the expression of a protein (e.g., a lyase, a protease, an amylase, a lipase, or a cellulase).

[0077] As used herein, "Treatment" and "treating" refer to the medical management of a subject that is intended to improve, alleviate, stabilize, prevent, or cure a disease, disorder, or condition. This term includes active treatment (treatment intended to improve a disease, disorder, or condition); etiological treatment (treatment directed at the cause of the relevant disease, disorder, or condition); palliative treatment (treatment intended to relieve the symptoms of a disease, disorder, or condition); prophylactic treatment (treatment intended to minimize or partially or completely inhibit the development of the relevant disease, disorder, or condition); and supportive treatment (treatment used to supplement another therapy).

[0078] As used herein, the term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. Cancer can be locally advanced or metastatic, for example, it can be stage I, II, III, or IV cancer.

[0079] II. Composition

[0080] A. ADAS and highly active ADAS

[0081] The present invention is at least partially based on the Applicants' discovery of non-chromosomal dynamic active systems (ADAS), including highly active ADAS, which are capable of providing multiple functions in a wide range of environments. An "ADAS" is a genome-free non-replicating closed membrane system that includes at least one membrane (in some embodiments, two membranes, where the two membranes are non-intersecting) and has an internal volume suitable for accommodating cargo (e.g., nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNP)).

[0082] In some embodiments, the ADAS is a microcell or a modified microcell derived from a parental bacterial cell (e.g., a Gram-negative or Gram-positive bacterial cell). In some aspects, any suitable method, such as genetically manipulating the parental cell or exposing it to a culture or conditions that increase the likelihood of bacterial microcell formation, is used to derive the ADAS from the parental bacterium.

[0083] In some embodiments, the ADAS has a major axis cross-section between about 100 nm and 500 μm (e.g., in certain embodiments, about 100 - 600 nm, such as 100 - 400 nm; or between about 0.5 - 10 μm and 10 - 500 μm). In certain embodiments, the minor axis cross-section of the ADAS is between about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100% of the major axis. In certain embodiments, the ADAS has an internal volume between about 0.001 - 1 μm 3 , 0.3 - 5 μm 3 , 5 - 4000 μm 3 or 4000 - 50×10 7 μm 3 . In some embodiments, the ADAS is substantially similar in size to the parental cell, e.g., the size (e.g., internal volume, major axis cross-section, and / or minor axis cross-section) is about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the size of the parental cell, the size is the same as the size of the parental cell, or the size is about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or 110% of the size of the parental cell.

[0084] In some embodiments, the present invention provides highly active ADAS. "Highly active ADAS" is an ADAS having high working potential, e.g., an ADAS having the ability to perform a large amount of useful work. In some embodiments, work is defined as, for example, metabolic work, including chemical synthesis (e.g., synthesis of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), chemical modification (e.g., modification of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), or transport (e.g., import, export, or secretion) under suitable conditions. In some embodiments, the highly active ADAS starts with a large amount of energy (e.g., energy in the form of adenosine triphosphate (ATP)). In other embodiments, the ADAS has the ability to absorb or generate energy (e.g., ATP) from another source.

[0085] The term "ADAS provided by the present invention" encompasses all embodiments of the ADAS described herein, including the highly active ADAS in specific embodiments, and this group can be referred to as "highly active ADAS provided by the present invention", which is a subgroup of the ADAS provided by the present invention.

[0086] In one aspect, the present invention provides a composition comprising a plurality of highly active extrachromosomal dynamic activity systems (ADAS), wherein the initial ATP concentration of the ADAS is at least 1 mM, and wherein the composition is substantially free of live bacterial cells.

[0087] In another aspect, the present invention provides a composition comprising a plurality of highly active extrachromosomal dynamic activity systems (ADAS), wherein the initial ATP concentration of the ADAS is at least 3 mM, and wherein the composition is substantially free of live bacterial cells.

[0088] In some embodiments, the initial ATP concentration of the highly active ADAS is at least 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, 2.5 mM, 3 nM, 3.5 nM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 50 mM. The ATP concentration can be evaluated in various ways, including the BacTiter-Glo TM assay (Promega) for lysed ADAS in certain embodiments.

[0089] In some embodiments, high activity is additionally or alternatively assessed by the rate or amount of increase in the ATP concentration in the ADAS over time. In some embodiments, after incubation under suitable conditions (e.g., incubation at 37 °C for 12 hours), the ATP concentration of the ADAS increases by at least 50%, at least 60%, at least 75%, at least 100%, at least 150%, at least 200%, or more than 200%. In certain embodiments, the ATP production rate of the highly active ADAS is greater than about: 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2, 3, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 500, 1000, 10000 ATP / sec / nm 2 , for at least about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, or two weeks.

[0090] In other aspects, high activity is assessed by the rate of decrease in the ATP concentration over time. In some embodiments, the ATP concentration in the highly active ADAS decreases more slowly than in the non-highly active ADAS. In some embodiments, e.g., as measured using BacTiter-Glo TMAs measured by Promega, the ATP concentration in the ADAS or ADAS composition 24 hours after preparation decreases by less than about 50% (e.g., less than about: 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%) compared to the initial ATP concentration (e.g., ATP per cell volume).

[0091] In some embodiments, high activity is additionally or alternatively rated by the lifespan index of the ADAS. The lifespan index is calculated as the ratio of the GFP production rate at 24 hours to that at 30 minutes. In some embodiments, the lifespan index of the high activity ADAS is greater than about: 0.13, 0.14, 0.15, 0.16, 0.18, 0.2, 0.25, 0.3, 0.35, 0.45, 0.5, 0.60, 0.70, 0.80, 0.90, 1.0 or greater. In more specific embodiments, the lifespan index is measured in an ADAS containing a functional GFP plasmid with a species-appropriate promoter, where at 30 minutes and 24 hours, the GFP concentration is measured with a plate reader relative to the number of ADAS, the average number of plasmids per ADAS, and the solution volume.

[0092] In some aspects, the ADAS produces proteins, such as heterologous proteins. In some aspects, high activity is assessed by the rate, amount, or duration of protein production or the rate of inducing protein expression (e.g., the ADAS's response to a signal). For example, in some embodiments, the ADAS comprises a plasmid that comprises an inducible promoter and a nucleotide sequence encoding the heterologous protein, wherein contacting the ADAS with an inducer of the inducible promoter under appropriate conditions causes production of the heterologous protein. In some aspects, in an ADAS that has been contacted with the inducer (e.g., a high-activity ADAS), the production of the heterologous protein is increased by at least 1.6-fold relative to an ADAS that has not been contacted with the inducer. For example, in some embodiments, in an ADAS that has been contacted with the inducer (e.g., a high-activity ADAS), the production of the heterologous protein is increased by at least 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more than 10-fold. In some embodiments, the production rate of the heterologous protein in the high-activity ADAS reaches a target level within a specific duration after the ADAS is contacted with the inducer, e.g., within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or more than 3 hours. In some embodiments, the protein (e.g., heterologous protein) is produced at a rate of at least 0.1 femtograms per hour per high-activity ADAS, e.g., at least 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10, 25, 50, 100, 250, 500, 1000, 2000, 3000, or 3500 fg / hour / ADAS. In some embodiments, the high activity of the ADAS is assessed by the duration of protein production. In some embodiments, the high-activity ADAS produces protein (e.g., heterologous protein) for a duration of at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or more than 48 hours.

[0093] B. ADAS and high-activity ADAS derived from a parental bacterium defective in a cell division topoispecificity factor

[0094] In some embodiments, as described herein, the ADAS is derived from a bacterial parental cell.

[0095] In some aspects, the present invention provides an ADAS and / or a composition comprising a plurality of ADASs, wherein the ADAS and / or the composition is derived from a parental bacterium having a reduced level, activity, or expression of a cell division topoispecificity factor.

[0096] In some aspects, the present invention provides a composition comprising a plurality of ADASs, wherein the ADASs do not comprise a cell division topoispecificity factor and wherein the composition is substantially free of live bacterial cells.

[0097] In some aspects, the present invention provides a composition comprising multiple ADASs, the composition being substantially free of live bacterial cells, and produced by a method comprising: (a) preparing, providing or obtaining multiple parental bacteria with a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that allow for the formation of microcells, thereby producing the highly active ADASs; and (c) separating the ADASs from the parental bacteria, thereby producing a composition substantially free of live bacterial cells.

[0098] In some embodiments of the above aspects, the cell division topology-specific factor is a polypeptide having an amino acid sequence with at least 20% identity to an Escherichia coli minE polypeptide (e.g., as encoded by SEQ ID NO:1), such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to the polypeptide encoded by SEQ ID NO:1. In some embodiments, the cell division topology-specific factor comprises the amino acid sequence encoded by SEQ ID NO:1. In some embodiments, the cell division topology-specific factor is a minE polypeptide. Exemplary species having a minE polypeptide are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005.

[0099] In some embodiments, the parental bacterium is Escherichia coli, and the MinE polypeptide is Escherichia coli MinE. In other embodiments, the parental bacterium is Salmonella typhimurium, and the MinE polypeptide is Salmonella typhimurium MinE. In still other embodiments, the parental bacterium is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacterium, and the cell division topology-specific factor is the endogenous MinE or DivIVA of the parental bacterium.

[0100] In some embodiments of the above aspects, the cell division topology-specific factor is a polypeptide having an amino acid sequence with at least 20% identity to a Bacillus subtilis DivIVA polypeptide (e.g., as encoded by SEQ ID NO: 6), such as having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to the polypeptide encoded by SEQ ID NO: 6. In some embodiments, the cell division topology-specific factor comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 6. In some embodiments, the cell division topology-specific factor is a DivIVA polypeptide. Exemplary species having a DivIVA polypeptide are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005. In some embodiments, the parental bacterium is Bacillus subtilis, and the cell division topology-specific factor is Bacillus subtilis DivIVA.

[0101] In some embodiments, the level of one or more Z-ring inhibitory proteins of the ADAS or parental bacterium in which the level or activity of the cell division topology-specific factor is reduced is also reduced.

[0102] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence with at least 20% identity to an Escherichia coli minC polypeptide (e.g., as encoded by SEQ ID NO: 2), such as having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to the polypeptide encoded by SEQ ID NO: 2. In some embodiments, the Z-ring inhibitory protein comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 2. In some embodiments, the Z-ring inhibitory protein is a minC polypeptide.

[0103] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence with at least 20% identity to an Escherichia coli minD polypeptide (e.g., as encoded by SEQ ID NO: 3), such as having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to the polypeptide encoded by SEQ ID NO: 3. In some embodiments, the Z-ring inhibitory protein comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 3. In some embodiments, the Z-ring inhibitory protein is a minD polypeptide.

[0104] In some embodiments, the levels, activities, or expressions of at least two Z-ring inhibitory proteins of the ADAS or parental bacterium are reduced. In some embodiments, the expressions of the MinC polypeptide and MinD polypeptide of the ADAS or parental bacterium are reduced. In some embodiments, the expressions of the MinC polypeptide, MinD polypeptide, and MinE polypeptide of the ADAS or parental bacterium are reduced, for example, the MinCDE operon is deleted (ΔMinCDE).

[0105] In some embodiments, any suitable method is used to achieve a reduction in the level, activity, or expression of a cell division topology-specific factor or Z-ring inhibitory protein, such as a reduction in the ADAS or in a parental bacterial cell. For example, in some embodiments, the reduction in the level or activity is caused by a loss-of-function mutation (such as a gene deletion). In some embodiments, the loss-of-function mutation is an inducible loss-of-function mutation, and the loss of function is induced by exposing the parental cell to an inducing condition. For example, the inducible loss-of-function mutation is a temperature-sensitive mutation, and the inducing condition is a temperature condition.

[0106] In some embodiments, the parental cell has a MinCDE operon (ΔMinCDE) or a homologous operon deletion.

[0107] C. ADAS with increased stability due to a genetic loss of function of a lyase

[0108] In some embodiments, the parental bacterial cell has one or more genetic loss-of-function alterations that stabilize the ADAS derived from the cell (e.g., result in an increase in stability in the ADAS derived from the cell). In some embodiments, the parental bacterial cell has been modified to reduce enzyme activity. In certain embodiments, the ADAS stability is improved by a genetic deletion of one or more enzymes.

[0109] In some embodiments, the parental cell comprises one or more loss-of-function alterations that reduce or eliminate enzymatic activity and / or lytic activity (e.g., reduce or eliminate the enzymatic activity or lytic activity performed by a protein encoded by a gene comprising a loss-of-function genetic alteration). In some embodiments, the parental cell comprises a genomic deletion that reduces or eliminates enzymatic activity and / or lytic activity (e.g., reduces or eliminates the enzymatic activity or lytic activity performed by a protein encoded by a deleted genomic region). In some embodiments, the parental bacterial cell comprises one or more genomic deletions that reduce the activity of one or more endopeptidases, cell wall lytic enzymes, and / or autolysins. In some embodiments, the parental cell comprises a loss-of-function (e.g., genomic deletion) from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0110] In some embodiments, the parental cell comprises a loss-of-function in sigF. For example, in the case where a wild-type parental cell comprises a sigF sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO: 4 and 5. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5. The loss-of-function alteration of sigF may comprise a deletion of all or a portion of the coding region of the gene.

[0111] In some embodiments, the parental cell comprises a loss-of-function in lytC. For example, in the case where a wild-type parental cell comprises a lytC sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO: 8 and 9. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. The loss-of-function alteration of lytC may comprise a deletion of all or a portion of the coding region of the gene.

[0112] In some embodiments, the parental cell comprises a loss of genetic function in aprE. For example, in the case where the wild-type parental cell comprises an aprE sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO: 10 and 11. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11. The loss-of-function alteration of aprE may comprise a deletion of all or a portion of the coding region of the gene.

[0113] In some embodiments, the parental cell comprises a loss of genetic function in ispA. For example, in the case where the wild-type parental cell comprises an ispA sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO: 12 and 13. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13. The loss-of-function alteration of ispA may comprise a deletion of all or a portion of the coding region of the gene.

[0114] In some embodiments, the parental cell comprises a loss of genetic function in wprA. For example, in the case where the wild-type parental cell comprises an wprA sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO: 14 and 15. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. The loss-of-function alteration of wprA may comprise a deletion of all or a portion of the coding region of the gene.

[0115] In some embodiments, the parental cell comprises a loss of genetic function in nprE. For example, where the wild-type parental cell comprises an nprE sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:16, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:16 and 17. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:17. The loss-of-function alteration of nprE may comprise a deletion of all or a portion of the coding region of the gene.

[0116] In some embodiments, the parental cell comprises a loss of genetic function in Epr. For example, where the wild-type parental cell comprises an Epr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:18, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:18 and 19. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:19. The loss-of-function alteration of Epr may comprise a deletion of all or a portion of the coding region of the gene.

[0117] In some embodiments, the parental cell comprises a loss of genetic function in Vpr. For example, where the wild-type parental cell comprises a Vpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:20, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:20 and 21. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:21. The loss-of-function alteration of Vpr may comprise a deletion of all or a portion of the coding region of the gene.

[0118] In some embodiments, the parental cell comprises a loss of genetic function in Bpr. For example, where the wild-type parental cell comprises a Bpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:22, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:22 and 23. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:23. The loss-of-function alteration of Bpr may comprise a deletion of all or a portion of the coding region of the gene.

[0119] In some embodiments, the parental cell comprises a loss of genetic function in Mpr. For example, where the wild-type parental cell comprises an Mpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:24, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:24 and 25. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:25. The loss-of-function alteration of Mpr may comprise a deletion of all or a portion of the coding region of the gene.

[0120] In some embodiments, the parental cell comprises a loss of genetic function in nprB. For example, where the wild-type parental cell comprises an nprB sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:26, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:26 and 27. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:27. The loss-of-function alteration of nprB may comprise a deletion of all or a portion of the coding region of the gene.

[0121] One of ordinary skill in the art can readily identify alterations in nucleotide sequences encoding proteins (e.g., proteins having enzymatic and / or lytic activity, such as lyases) that result in a loss of protein function.

[0122] In some embodiments, the parental cell contains a loss of genetic function in lytC.

[0123] In some embodiments, the parental cell contains a genomic deletion of lytC.

[0124] In some embodiments, the parental bacterial cell comprises at least one loss of genetic function (e.g., genomic deletion) of a gene encoding a lyase, a gene affecting the sporulation mechanism, or a cell division topological factor, and combinations thereof (e.g., comprising two or more loss-of-function alterations affecting one or more lyases, one or more genes affecting the sporulation mechanism, and / or one or more cell division topological factors).

[0125] In some embodiments, the parental bacterial cell comprises a loss of genetic function (e.g., genomic deletion) of lytC, sigF, and divIVa. In some embodiments, the parental bacterial cell comprises a genomic deletion of lytC, sigF, and divIVa.

[0126] In some embodiments, the parental bacterial cell is a Gram-positive bacterial cell.

[0127] In some embodiments, the parental bacterial cell is Bacillus subtilis or belongs to the genus Bacillus (e.g., is a Bacillus species).

[0128] In some embodiments, the parental bacterial cell belongs to the genus Lactobacillus (e.g., is a Lactobacillus species).

[0129] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0130] In some embodiments, the parental bacterial cell is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Dickeya, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

[0131] In some embodiments, the parental cell comprises a gene deletion from the group consisting of SPβ, skin, PBSX, prophage 1, pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. 20(12):2076-2090, 2003). Additional deletions of genomic portions in the parental bacterial cell can result in a reduction of multiple lysis elements by the removal of prophages and prophage-like segments. Such deletions, like certain lytic enzyme deletions or loss-of-function mutations (e.g., lytC), are not necessary for the establishment of a complete and stable ADAS.

[0132] D. ADAS derived from a parental cell with blocked sporulation

[0133] In some embodiments, the parental cell comprises a gene deletion or loss-of-function alteration (e.g., loss-of-function mutation) that blocks sporulation (e.g., reduces or eliminates sporulation). In some embodiments, the loss-of-function alteration is selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof. Although the genomic deletion for blocking sporulation and the genomic deletion for reducing enzyme activity in the ADAS are not required or dependent on each other, each genomic deletion provides unique benefits for ADAS formation and use. Disrupting sporulation is beneficial for ADAS formation because it maintains the parental cell population and removes spores, which may be difficult to distinguish from the ADAS. Thus, in some aspects, the parental cells provided herein comprise one or more cargo display constructs and one or more alterations (e.g., loss-of-function mutations) that block sporulation.

[0134] E. ADAS containing cargo

[0135] In some embodiments, the ADAS provided by the present invention includes cargo contained within the ADAS. In some embodiments, the cargo is any part that is disposed within the ADAS (e.g., encapsulated by the ADAS) or displayed on (e.g., conjugated to) the surface of the ADAS. In some embodiments, the cargo includes nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs), or combinations of the foregoing. In some aspects, the cargo is delivered by a secretion system (e.g., T3SS). In other aspects, the cargo is not delivered by T3SS. In some aspects, the delivery of the cargo to the cytoplasm of a target eukaryotic cell is enhanced by listeriolysin O, a heterologous pore-forming toxin (e.g., listeriolysin O is carried by the ADAS, conjugated to the cargo, or administered separately). In some embodiments, the cargo is secreted by the ADAS.

[0136] In some embodiments, the nucleic acid is DNA, RNA, or a plasmid. In some embodiments, the nucleic acid (e.g., DNA, RNA (e.g., mRNA, ASO, circular RNA, siRNA, shRNA, tRNA, dsRNA, or combinations thereof), or a plasmid) encodes a protein. In some embodiments, the protein is transcribed and / or translated in the ADAS. In some embodiments, the nucleic acid inhibits the translation of a protein or polypeptide, e.g., is siRNA or an antisense oligonucleotide (ASO).

[0137] In some embodiments, the cargo is an agent that can modulate the microbiome of a target organism (e.g., a human, animal, plant, or fungal microbiome), such as a polysaccharide, an amino acid, an antimicrobial agent (e.g., an anti-infective or antimicrobial peptide, protein, and / or natural product), a short-chain fatty acid, or a combination thereof. In some instances, the agent that can modulate the host microbiome is a probiotic agent.

[0138] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme alters a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered.

[0139] In certain embodiments, the cargo is modified to have improved stability (e.g., increased stability when carried by an ADAS) compared to an unmodified version of the cargo. The "stability" of the cargo can be measured as the unitless ratio of the half-life of the unmodified form of the cargo to the half-life of the modified cargo, where measured under the same environmental conditions. In some embodiments, the environment is experimentally controlled, e.g., simulated body fluid, RNase-free water, cytoplasm, extracellular space, or "ADAS protoplasm" (i.e., the contents of the internal volume of the ADAS, e.g., after lysis). In some applications, it is an agricultural environment, e.g., field soil, river water, or sea water. In other embodiments, the environment is an actual or simulated animal intestine, animal skin, animal reproductive tract, animal respiratory tract, animal bloodstream, or animal extracellular space. In certain embodiments, the ADAS does not significantly degrade the cargo (e.g., (a) degrades the unmodified cargo but not significantly the modified cargo, or (b) does not significantly degrade either the modified or unmodified cargo).

[0140] In some embodiments, the ADAS is modified to improve the stability of the cargo and / or optimize cargo expression. In some embodiments, the ADAS is derived from a parental cell that has been modified to improve the stability of the cargo in the ADAS and / or optimize the cargo expression in the ADAS. "Increased stability" or "optimization" of the cargo in the ADAS refers to an overall increase in the expression, display, function, or durability of the cargo carried by the ADAS when compared to a control or unmodified ADAS. In some embodiments, the stability of the cargo is measured as the unitless ratio of the half-life of the unmodified cargo to the half-life of the modified cargo, where measured under the same environmental conditions. In other embodiments, the stability of the cargo is measured as the fold change in the cargo expression level between a modified ADAS (e.g., an ADAS derived from a modified parental cell (e.g., a parental cell that has been modified to contain a display construct)) and a control ADAS, where measured under the same environmental conditions.

[0141] In some embodiments, the stability and / or optimization of cargo expression is improved (e.g., increased) by at least one genomic deletion or other loss-of-function mutation of both a lytic enzyme and a protease in the parental bacterial cell.

[0142] In some embodiments, more than one lytic enzyme, protease, or other enzyme is deleted or mutated in the parental cell. In some embodiments, additional enzymes other than lytic enzymes and proteases are deleted or other loss-of-function mutations are present in the parental cell.

[0143] In some embodiments, an enzyme having oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, lipase, kinase, phosphatase, protease, nuclease, amylase, cellulase, and / or synthetase activity is deleted or mutated in a parental cell. In some embodiments, the deletion or loss-of-function alteration is in a gene selected from: amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof (e.g., the parental cell comprises a deletion or loss-of-function alteration in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the listed genes).

[0144] In some embodiments, the stability of the cargo in an ADAS derived from a parental cell having (i) one or more deletions or loss-of-function mutations in a lyase and (ii) one or more deletions or other loss-of-function mutations in a protease is 2-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, or more than 300-fold the stability of the cargo in a control ADAS. In some embodiments, the stability or optimization of cargo expression is measured by the functionality of the cargo (e.g., by downstream cytokine concentration, receptor activation, or antigen-specific cell activation). In some embodiments, the functional measure of the cargo from an ADAS derived from a modified parental cell is 1, 2, 3, 4, 5, 10, 50, 100, 500, 1000-fold or higher than the same measure from a control or unmodified ADAS (e.g., an ADAS derived from an unmodified parental cell).

[0145] In some embodiments, the parental cell contains one or more deletions or other loss-of-function mutations in proteases. In some embodiments, the one or more protease deletions or loss-of-function alterations increase the stability of the cargo of the ADAS (e.g., increase the expression, display, function, or durability of the cargo, e.g., optimize cargo expression) relative to the ADAS produced by parental cells that do not contain the deletion or loss-of-function mutation. In some embodiments, the loss-of-function mutation is in a gene selected from: aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or combinations thereof (e.g., the parental cell contains a deletion or loss-of-function alteration in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the listed genes).

[0146] In some embodiments, the parental cell contains at least two, at least three, or at least four protease deletions or other loss-of-function mutations. In some embodiments, the parental bacterial cell contains (i) deletions of sigF, divIVA, and lytC, and (ii) deletion of one, two, three, or all four of the proteases aprE, ispA, wprA, and nprE. In some embodiments, the parental bacterial cell contains (i) genetic loss-of-function mutations in sigF, divIVA, and lytC, and (ii) genetic loss-of-function mutations in one, two, three, or all four of aprE, ispA, wprA, and nprE.

[0147] In some embodiments, the ADAS expresses a cytoplasmic cargo, a surface-displayed cargo, or a combination thereof. In some embodiments, the cargo is an antigen, a cytokine, a mutant protein, a protein, a polypeptide, or any combination thereof.

[0148] In some embodiments, the ADAS includes a cell wall-embedded anchoring structure for the construction of surface-displayed cargo. In some embodiments, the constructed cell wall-embedded anchoring structure is a cell wall scaffold, a membrane-anchoring domain, an outer membrane-anchoring domain, an inner membrane-anchoring domain, a cell wall-anchoring domain, or a functional equivalent thereof. In some embodiments, the constructed cell wall-embedded anchoring structure comprises one or more of the following: an antiparallel coiled coil, a cell wall or outer membrane-anchoring domain having one or more coiled or uncoiled linkers, a separate cargo protein (e.g., mCherry) without a linker or having one or more linkers, a cell wall or outer membrane-anchoring domain without a linker, or a combination thereof. In some embodiments, the cell wall-anchoring construct is a shortened antiparallel coiled coil. In some embodiments, the cell wall-anchoring construct is a linker. In some embodiments, the cell wall-anchoring construct is a coiled linker. In some embodiments, the cell wall-anchoring construct is a fusion construct. In some embodiments, the constructed cell wall-embedded anchoring structure has a COOH terminus embedded in the cell wall. In some embodiments, the COOH terminus is included in a sorting signal composed of the COOH terminus, an LPXTG motif, a hydrophobic domain, a tail of mostly positively charged residues, or any combination thereof.

[0149] In some embodiments, the parental bacterial cell comprises a deletion of nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof. In some embodiments, the parental bacterial cell comprises a loss-of-function mutation in nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

[0150] In some embodiments, the parental bacterial cell comprises a deletion of amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof. In some embodiments, the parental bacterial cell comprises a loss-of-function mutation in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

[0151] In some embodiments, the cargo comprises a protein. In some embodiments, the stability of the protein in the cytoplasm or other environment is greater than about: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 10000000. The protein can be any protein, including growth factors; enzymes; hormones; immunomodulatory proteins; antibiotic proteins, such as antibacterial, antifungal, insecticidal proteins, etc.; targeting agents, such as antibodies or nanobodies, etc. In some embodiments, the protein is a hormone, such as a paracrine, endocrine, autocrine hormone.

[0152] In some embodiments, the cargo comprises a plant hormone, such as abscisic acid, auxin, cytokinin, ethylene, gibberellin or a combination thereof.

[0153] In some embodiments, the cargo is an anti-inflammatory agent or a pro-inflammatory agent, such as a cytokine (e.g., a heterologously expressed anti-inflammatory or pro-inflammatory cytokine or a mutant protein thereof (e.g., IL-10, TGF-β, IL-22, IL-2)) or an antibody (e.g., an antibody or antibody fragment targeting tumor necrosis factor (TNF) (e.g., an anti-TNF antibody); an antibody or antibody fragment targeting IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment targeting IL-23 (e.g., an anti-IL-23 antibody)).

[0154] In certain embodiments, the cargo is an immunomodulator. Immunomodulators include, for example, immunostimulants; checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4); chemotherapeutic agents; immunosuppressants; antigens; superantigens; and small molecules (e.g., cyclosporine A, cyclic dinucleotides (CDNs), or STING agonists (e.g., MK-1454, c-di-AMP cyclase (DacA))). In some embodiments, the immunomodulator is a moiety that induces tolerance in a subject, such as an allergen, an autoantigen (e.g., a disease-associated autoantigen), or a microbe-specific antigen. In some embodiments, the immunomodulator is a vaccine, such as an antigen from a pathogen (e.g., a virus (e.g., a viral envelope protein) or a bacterium). In some embodiments, the antigen is a cancer neoantigen. In some embodiments, the pathogen is a coronavirus, such as SARS-CoV-2. In some embodiments, the cargo is an adjuvant, such as an immunomodulatory molecule or a molecule that alters the compartmentalization, presentation, or profile of one or more costimulatory molecules associated with a vaccine antigen. In some instances, the adjuvant is an activator of an immune pathway upstream of the desired immune response (e.g., an activator of an innate immune pathway upstream of an adaptive immune response). In other instances, the adjuvant enhances the presentation of an antigen on immune cells or immune components (e.g., MHC class I) in a target organism. In some instances, the adjuvant is listeriolysin O (LLO). In some instances, the cargo includes a heterologous pore-forming toxin. In some instances, the toxin induces autophagy. In some instances, the pore-forming toxin is listeriolysin O (LLO). In some embodiments, the toxin facilitates delivery of the ADAS cargo to a target cell. In some embodiments, the ADAS comprises an antigen and one or more adjuvants.

[0155] In some embodiments, the cargo is an agent for treating or preventing cancer, e.g., an agent that reduces the likelihood of a patient developing cancer or an agent that treats cancer (e.g., an agent that increases the progression-free survival and / or overall survival of an individual with cancer). Cancer or tumor includes, but is not limited to, neoplasm, malignant tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth such that it is considered cancerous. Cancer can be primary or metastatic cancer. Specific cancers that can be treated according to the present invention include, but are not limited to, the cancers listed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2nd ed., J.B. Lippincott Co., Philadelphia). Cancers include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological malignancies, including acute lymphocytic leukemia and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphoma, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; ovarian cancer, including ovarian cancer derived from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcoma, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germ cell tumors, such as seminoma, non-seminoma, teratoma, choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and kidney cancer, including adenocarcinoma and nephroblastoma. Common cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer.

[0156] In some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and microsatellite-high (MSIH) / mismatch repair (MMR)-deficient solid malignancies. In some embodiments, NSCLC lacks an EGFR sensitizing mutation and / or an ALK translocation. In some embodiments, the microsatellite-high (MSIH) / mismatch repair (MMR)-deficient solid malignancies are selected from the group consisting of: colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancers of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissue. In some embodiments, the cancer is colorectal cancer.

[0157] Agents for preventing cancer include, but are not limited to, anti-inflammatory agents, growth inhibitors, cytokines, and tumor antigens. Agents for treating cancer (e.g., solid tumor cancer) include, but are not limited to, anti-inflammatory agents, growth inhibitors, chemotherapeutic agents, immunotherapeutic agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as listeriolysin-o), cytotoxins, inflammasome inhibitory agents, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0158] In some embodiments, the cargo is an anti-cancer therapeutic agent. In some embodiments, the cargo embodies one or more wild-type or engineered antigens (or antibodies against antigens). In some embodiments, the antigen is derived from a tumor, e.g., a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the cargo is antigenic, tumor antigenic, or a protein, comprising p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, Claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A (preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12), MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor form BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, Survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, MC38 cancer epitope, or a combination thereof.In some embodiments, the expressed cargo is antigenic, tumor antigenic, or a protein, including CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, LewisY, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

[0159] In some embodiments, the cargo is an IL-2 peptide, IL-2-Ra, tdTomato, GFP, eGFP, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, epoetin alfa (Epogen), filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or a fragment or subunit thereof. In one embodiment, the cargo is an IL-2 peptide, or a fragment or subunit thereof. In one embodiment, the cargo is an IL-15 peptide, or a fragment or subunit thereof. In some embodiments, the cargo is FOXP3. In some embodiments, the cargo is FOXO1. In some embodiments, the ADAS carries two or more types of cargo.

[0160] In some embodiments, the expressed cargo is antigenic, tumor antigenic, or a protein, including STING activators such as cyclic dinucleotides, cGAMP, c-di-AMP, IRF-3, and variants thereof.

[0161] In some embodiments, the tumor antigen polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof. In one embodiment, the tumor antigen polypeptide comprises a lung cancer antigen.

[0162] In some embodiments, the antigen is an autologous antigen polypeptide or an immunogenic variant or fragment thereof. In some embodiments, the autologous antigen polypeptide comprises an antigen typically expressed on cells and recognized as an autologous antigen by the immune system. In some embodiments, the autologous antigen polypeptide comprises: a multiple sclerosis antigen polypeptide, a rheumatoid arthritis antigen polypeptide, a lupus antigen polypeptide, a celiac disease antigen polypeptide, a Sjogren's syndrome antigen polypeptide, or an ankylosing spondylitis antigen polypeptide, or combinations thereof.

[0163] In some embodiments, the cargo is IL-2. In some embodiments, the IL-2 molecule is encoded by a sequence having at least 20% identity (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:59 or SEQ ID NO:60) to wild-type (WT) IL-2 (e.g., human IL-2) or an IL-2 mutant protein (e.g., as shown in SEQ ID NO:59 or SEQ ID NO:60; see Table 5). In some embodiments, the cargo comprises the amino acid sequence encoded by SEQ ID NO:59 or SEQ ID NO:60. In some embodiments, the cargo is an IL-2 polypeptide.

[0164] In another aspect, the present disclosure provides a method of treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutic composition comprising an ADAS (e.g., an ADAS comprising a cargo, the cargo being an agent for treating or preventing cancer) as discussed in the above aspects of the present invention. Thus, in another aspect, the present disclosure provides an ADAS therapeutic composition comprising a plurality of ADASs, the ADASs comprising a cargo as an agent for treating or preventing cancer. The composition may comprise, for example, a physiologically acceptable carrier.

[0165] In some aspects, the ADAS (e.g., the ADAS therapeutic composition) is administered by oral, intravenous, intradermal, intramuscular, intraperitoneal, peritumoral, intranasal, intraocular, or rectal, and / or subcutaneous administration. In certain embodiments, the ADAS (e.g., the ADAS therapeutic composition) is administered by oral, intravenous, intramuscular, and / or subcutaneous administration. In some embodiments, the ADAS (e.g., the ADAS therapeutic composition) is administered to the subject one, two, three, four, or more times. In some embodiments, the ADAS dose is at least 1×10 5 、1×10 6, 1×10 7 , 1×10 8 , 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2×10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 , for example, administration includes administering to a subject at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2×10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 ADASs.

[0166] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme is an enzyme that exerts catalytic activity in a target cell or organism (e.g., in a human, animal, plant, fungus, or insect). In some embodiments, the catalytic activity is extracellular matrix (ECM) digestion (e.g., the enzyme is hyaluronidase and the catalytic activity is ECM digestion) or toxin removal. In some embodiments, the enzyme is an enzyme replacement therapy, such as phenylalanine hydroxylase. In some embodiments, the enzyme is UDP-glucuronosyltransferase. In some embodiments, the enzyme has hepatic enzyme activity (e.g., porphobilinogen deaminase (PBGD), e.g., human PBGD (hPBGD)). In some embodiments, the enzyme is a protease, an oxidoreductase, or a combination thereof.

[0167] In some embodiments, the enzyme modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered. In some embodiments, the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic di-AMP.

[0168] In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally conjugated to the outer membrane via a linker.

[0169] Alternatively, in some embodiments, the cargo is a nucleic acid encoding any of the enzymes described herein.

[0170] In some embodiments, the cargo is an agent that activates or inhibits the autophagy process (e.g., an activator such as listeriolysin O, or an inhibitor such as IcsB).

[0171] In some embodiments, the cargo is an anti-infective agent, such as an antimicrobial agent, such as an anti-infective or antimicrobial peptide, protein, and / or natural product.

[0172] In some embodiments, the cargo is a protein that modulates the host transcriptional response, such as a transcription factor; a protein that promotes host cell growth, such as a growth factor; or a protein that inhibits protein function, such as a nanobody. In some embodiments, the transcription factor is a human transcription factor.

[0173] For an ADAS comprising a cargo, in some embodiments, the cargo is RNA, such as circular RNA, mRNA, siRNA, shRNA, ASO, tRNA, dsRNA, or a combination thereof. In certain embodiments, the stability of the RNA in, for example, the ADAS protoplasm is greater than about: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 10000000. In certain embodiments, the RNA cargo can be stabilized, for example, with an additional step-loop structure, such as a tRNA scaffold. For example, non-human tRNALys3 and Escherichia coli tRNAMet (Nat. Methods [Nature Methods], Ponchon 2007). Both have been well characterized and recombinantly expressed. However, various other types can also be used, such as aptamers, lncRNA, ribozymes, etc. The RNA can also be stabilized when the ADAS is obtained from a parental strain that is ineffective (or hypomorphic alleles) against one or more ribonucleases.

[0174] In some specific embodiments, the RNA is an mRNA encoding a protein. In more specific embodiments, the mRNA encoding a protein encodes an enzyme (e.g., an enzyme conferring hepatic enzyme activity, such as human PBGD (hPBGD) mRNA), or an antigen (e.g., an antigen that elicits an immune response (such as eliciting an effective and persistent neutralizing antibody titer), such as an mRNA encoding CMV glycoprotein gB and / or pentameric complex (PC)). In certain specific embodiments, the RNA is a small non-coding RNA, such as shRNA, ASO, tRNA, dsRNA, or a combination thereof.

[0175] In some embodiments, the ADAS provided by the present invention includes a cargo comprising at least one component of a gene editing system. Components of a "gene editing system" include (or encode) proteins (or nucleic acids encoding such proteins) that can modify a target DNA sequence, such as a genomic DNA sequence, either by inserting or deleting the target sequence, or by altering the methylation state of the target sequence, together with a suitable associated nucleic acid and a nucleic acid related to the function of such proteins (e.g., guide RNA). Exemplary gene editing systems include gene editing systems based on Cas systems such as Cas9, Cpf1 or other RNA targeting systems and their associated RNAs (e.g., CRISPR guide RNAs that are sequence complementary), as well as zinc finger nucleases and TAL effectors conjugated to nucleases.

[0176] Other embodiments of the ADAS provided by the present invention include DNA as a cargo, including plasmids, optionally, wherein the DNA contains a protein coding sequence. In some embodiments, exemplary DNA cargos include plasmids encoding a target RNA sequence (see examples above), e.g., the target RNA sequence may be flanked on each side by tRNA insertion sequences. The present invention encompasses various DNA cargos, including: ADAS production (e.g., driving FTZ overexpression, exonucleases that degrade the genome); long-lived plasmids (ATP synthase expression, rhodopsin expression); cargos expressing stable non-coding RNAs, tRNAs, lncRNAs; expressing secreted system tag proteins, NleE2 effector domains, and localization tags; secreted systems T3 / 4SS, T5SS, T6SS; logic circuits that conditionally express secreted systems; and combinations thereof. In some embodiments, the logic circuits include inducible expression or repression cassettes, such as the IPTG-inducible Plac promoter and the hrpR portion of an AND gate, and, for example, the heat-inducible promoter pL (from bacteriophage λ, which is normally repressed by a heat-labile protein) and the hrpS portion of an AND gate. To engineer an OR gate, the system described by Rosado et al., PLoS Genetics [Public Library of Science Genetics, USA], 2018 can be used. Briefly, cis-repressive mRNAs encoding RFP can be used under a constitutive promoter. Then repression can be removed in the presence of the RAJ11 sRNA. Then plasmids containing the IPTG-inducible promoter PLac and the heat-inducible promoter pL, both of which induce the expression of the RAJ11 sRNA, can be used. Then the output is RFP expression, which is observed in response to either input. These systems can be adjusted for various sensor-type functions.

[0177] In some embodiments, the ADAS provided by the present invention includes transporters in the membrane. In some embodiments, the transporters are specific for glucose, sodium, potassium, metal ions, anionic solutes, cationic solutes, or water.

[0178] In some embodiments, the membrane of the ADAS provided by the present invention comprises an enzyme. In certain embodiments, the enzyme is a protease, a redox enzyme, or a combination thereof. In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally conjugated to the outer membrane via a linker.

[0179] F. ADAS display structure

[0180] In some embodiments, the cargo carried by the ADAS is present inside the ADAS. In some embodiments, the cargo is expressed in the cytosol (e.g., expressed in the cytosol of the parental cell that generates the ADAS and / or inside the ADAS).

[0181] In some embodiments, the cargo carried by the ADAS is present on the surface of the ADAS. In some embodiments, the cargo is expressed on the surface of the ADAS. In some embodiments, the cargo is expressed on the surface of the parental cell from which the ADAS is derived.

[0182] In some embodiments, the cargo carried by the ADAS is secreted by the ADAS.

[0183] In some embodiments, a constructed cell wall-embedded anchoring structure is used to display surface-expressed cargo. The constructed cell wall-embedded anchoring structure (also referred to as a cell wall anchoring construct or a cell wall-embedded anchoring construct) is a construct that provides a fixed construct from which to display surface cargo (e.g., a cell wall scaffold or other functional equivalent). As a general principle, the constructed cell wall-embedded anchoring structure comprises at least one element associated with (e.g., embedded in) the cell wall of the ADAS, wherein the element can be associated with (e.g., conjugated to) the cargo such that the cargo is displayed outside the ADAS.

[0184] The cell wall-embedded anchoring structure can be a component of the display structure. The display structure refers to any means by which the ADAS expresses and / or displays cargo (e.g., displayed on the surface of the ADAS).

[0185] The display structure includes at least one cargo. Other exemplary components of the display structure include, but are not limited to, cell wall-embedded anchoring structures (e.g., binding domains) and scaffold proteins. In some embodiments, the components of the display structure are connected by one or more linkers. In other embodiments, one or more components of the display structure are directly connected (e.g., connected without using a linker).

[0186] For example, in some embodiments, the constructed cell wall-embedded anchoring structure comprises a cell wall-binding domain and one or more antiparallel coiled coils (e.g., wherein the one or more antiparallel coiled coils are linked to the cargo); the cell wall-embedded anchoring structure comprises a cell wall-embedded binding domain having one or more coiled or uncoiled linkers (e.g., wherein the one or more linkers are linked to the cargo); the cell wall-embedded anchoring structure comprises a separate cargo protein or scaffold protein (e.g., mCherry) that is fused to the cell wall-binding domain without a linker or with one or more linkers (e.g., wherein the separate cargo protein or scaffold protein is directly or via one or more linkers linked to the cargo); or the constructed cell wall-embedded anchoring structure comprises a cell wall-embedded binding domain without a linker (e.g., wherein the binding domain is directly linked to the cargo).

[0187] In some embodiments, the display structure comprises one or more polypeptides, and one or more elements of the display structure (e.g., cargo, cell wall-embedded anchoring structure, scaffold protein, and / or linker) are included in the same polypeptide chain. In some embodiments, all elements of the display structure are included in the same polypeptide chain. Exemplary display structures are provided in Figure 2B and the examples, but these examples should not be considered limiting.

[0188] In some embodiments, the display structure is encoded by a nucleotide sequence. In some embodiments, the nucleotide sequence is included in the parental cell from which the ADAS is derived. Thus, in one aspect, the present disclosure features a nucleotide sequence that encodes the components of any one of the display structures provided herein. In another aspect, the present disclosure features a parental cell that comprises a nucleotide sequence encoding the components of any one of the display structures provided herein.

[0189] In some embodiments, the display structure is encoded by a nucleotide sequence that includes a promoter. In some embodiments, the promoter is Pveg. In some embodiments, the Pveg promoter is encoded by a sequence having at least 20% identity to SEQ ID NO:55 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:55). In some embodiments, the Pveg promoter comprises the nucleotide sequence of SEQ ID NO:55. In some embodiments, the promoter is PaprE. In some embodiments, the PaprE promoter is encoded by a sequence having at least 20% identity to SEQ ID NO:56 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:56).

[0190] In some embodiments, the display construct is encoded by a nucleotide sequence comprising a fusion promoter. In some embodiments, the promoter is PrrnI-Pveg. In some embodiments, the PrrnI-Pveg promoter is encoded by a sequence having at least 20% identity to SEQ ID NO:54 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:54). In some embodiments, the PrrnI-Pveg promoter comprises the nucleotide sequence of SEQ ID NO:54. In some embodiments, the fusion promoter is PrrnI-Pveg.

[0191] In some embodiments, the display construct is encoded by a nucleotide sequence encoding a ribosome binding site (RBS). In some embodiments, the binding site is aprE-RBS. In some embodiments, the aprE-RBS is encoded by a sequence having at least 20% identity to SEQ ID NO:57 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:57). In some embodiments, the aprE-RBS comprises the nucleotide sequence of SEQ ID NO:57. In some embodiments, the RBS is aprE-RBS.

[0192] In some embodiments, the display construct is encoded by a nucleotide sequence encoding a secretion signal. In some embodiments, the secretion signal is aprE-SS. In some embodiments, the aprE-SS is encoded by a peptide sequence having at least 20% identity to SEQ ID NO:58 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:58). In some embodiments, the aprE-SS comprises the nucleotide sequence of SEQ ID NO:58. In some embodiments, the secretion signal is the aprE-SS polypeptide.

[0193] In some embodiments, the display construct is encoded by a nucleotide sequence that is segmented by a cargo sequence (e.g., the cargo sequence and one or more elements of the display construct (e.g., a cell wall-embedded anchor structure, a scaffold protein, and / or a linker) are encoded by a single nucleotide sequence, and the cargo sequence appears within or between nucleotide sequences encoding other elements of the display construct). Thus, in some embodiments, the display construct is a polypeptide in which the amino acid sequence corresponding to non-cargo components is segmented by the amino acid sequence corresponding to the cargo. In some embodiments, the display construct sequence is segmented by the cargo sequence at the sites marked with "^" in Table 5. Those skilled in the art will be able to identify other suitable cargo sequence insertion sites (e.g., sites that do not disrupt the function of other elements of the display construct).

[0194] In some embodiments, the display construct comprises a non-covalent cell-binding domain (e.g., comprising a non-covalent cell-binding domain as an alternative or supplement to the cell wall-embedded anchor structure). In some embodiments, the non-covalent cell-binding domain is a LysM domain. In some embodiments, the LysM domain is encoded by a sequence having at least 20% identity to SEQ ID NO:68 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:68). In some embodiments, the LysM domain comprises the amino acid sequence encoded by SEQ ID NO:68. In some embodiments, the non-covalent domain is a LysM domain polypeptide.

[0195] In some embodiments, the display construct comprises a covalent cell-binding domain (e.g., comprising a covalent cell-binding domain as an alternative or supplement to the cell wall-embedded anchor structure). In some embodiments, the covalent cell-binding domain is a CWAD domain. In some embodiments, the CWAD domain is encoded by a sequence having at least 20% identity to SEQ ID NO:69 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:69). In some embodiments, the CWAD domain comprises the amino acid sequence encoded by SEQ ID NO:69. In some embodiments, the covalent domain is a CWAD domain polypeptide.

[0196] In some embodiments, the display construct is encoded by a nucleotide sequence comprising a terminator sequence. In some embodiments, the terminator is TmreBH. In some embodiments, the terminator is encoded by a sequence having at least 20% identity to SEQ ID NO:70 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:70). In some embodiments, the terminator comprises the nucleotide sequence of SEQ ID NO:70. In some embodiments, the terminator is TmreBH.

[0197] In some embodiments, the parental bacterial cell from which the ADAS is derived comprises at least one genomic deletion of a protease, and the nucleotide sequence encoding the display construct is integrated into the deleted protease locus. In some embodiments, the parental bacterial cell from which the ADAS is derived comprises at least one genomic deletion of an amylase, and the nucleotide sequence encoding the display construct is integrated into the deleted amylase locus. In some embodiments, the integration site is amyE (e.g., amyE has been deleted from the genome of the parental cell). In some embodiments, the deleted sequence of the amyE locus has at least 20% identity to SEQ ID NO:50 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:50). In some embodiments, the parental bacterial cell from which the ADAS is derived comprises at least one genomic deletion of a phosphodiesterase, and the nucleotide sequence encoding the display construct is integrated into the deleted phosphodiesterase locus. In some embodiments, the integration site is pdeH. In some embodiments, the deleted sequence of the pdeH locus has at least 20% identity to SEQ ID NO:494 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:49).

[0198] In some embodiments in which the display construct comprises a linker, the linker is an unstructured linker. In some embodiments, the linker is proline-rich (e.g., more than 25%, 27%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% proline). In some embodiments in which the display construct comprises a linker, the linker is a structured linker (e.g., a coiled linker, one or more antiparallel coiled coils, etc.).

[0199] In some embodiments, the display structure (e.g., one or more linkers included in the display structure) lacks protease sites, thereby reducing proteolytic cleavage of the cargo from the ADAS. In other embodiments, the display structure (e.g., one or more linkers included in the display structure) contains protease sites to enable release of the cargo from the ADAS surface. In some embodiments, the protease sites are specific for eukaryotes (e.g., plants, animals, mice, humans, etc., such as the organism to which the ADAS is to be delivered). In some embodiments, the protease sites are specific for human tissues or target sites (e.g., lungs, blood, gastrointestinal tract, etc., such as the tissue or target site to which the ADAS is to be delivered).

[0200] In some embodiments, display 0 is used as the cargo display structure. In some embodiments, display 1 is used as the cargo display structure. In some embodiments, display 2 is used as the cargo display structure. In some embodiments, display 3 is used as the cargo display structure. In some embodiments, display 4 is used as the cargo display structure. In some embodiments, display 6 is used as the cargo display structure.

[0201] Display 0

[0202] In some aspects, the display structure is a construct as shown in display 0 (see Figure 2B ). Thus, in some aspects, the present invention provides a display structure polypeptide according to display 0; a nucleotide sequence encoding the display structure polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0203] Briefly, display 0 is a fusion (e.g., a fusion polypeptide) comprising a cargo displayed on a cell wall-embedded binding domain that does not have a linker (e.g., a linker domain that attaches the cargo to the binding domain).

[0204] In some embodiments, the cell wall-embedded anchor construct is a fusion construct. In some embodiments, display 0 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity) to SEQ ID NO:61. In some embodiments, display 0 comprises the amino acid sequence encoded by SEQ ID NO:61. In some embodiments, the display structure is a fusion polypeptide according to display 0.

[0205] Thus, in some embodiments, the display construct is a polypeptide comprising a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or a variant or derivative thereof) and a cargo.

[0206] Display 1

[0207] In some aspects, the display construct is a construct as shown in Display 1 (see Figure 2B ). Thus, in some aspects, the present invention provides a display construct polypeptide according to Display 1; a nucleotide sequence encoding the display construct polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0208] In short, Display 1 is a fusion (e.g., a fusion polypeptide) of a cargo displayed on a cell wall-embedded binding domain having a linker (e.g., a linker domain that links the cargo to the binding domain) (e.g., a fusion polypeptide comprising a cargo, a linker, and a cell wall-embedded binding domain).

[0209] In some embodiments, Display 1 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:61 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:61), and further comprises a linker. In some embodiments, Display 1 comprises the amino acid sequence encoded by SEQ ID NO:61, and further comprises a linker. The linker can be, for example, any linker provided herein (e.g., can be a polypeptide linker, e.g., a structured or unstructured polypeptide linker).

[0210] In some embodiments, Display 1 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:62 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:62). In some embodiments, Display 1 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:62.

[0211] In some embodiments, the display construct is a Display 1 linker polypeptide.

[0212] Thus, in some embodiments, the display construct is a polypeptide comprising a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or a variant or derivative thereof), a linker, and a cargo. In some embodiments, the cell wall-embedded binding domain and the linker are encoded by SEQ ID NO:62 or a variant or derivative thereof.

[0213] Display 2

[0214] In some aspects, the display construct is a construct as shown in Display 2 (see Figure 2B ). Thus, in some aspects, the present invention provides a display construct polypeptide according to Display 2; a nucleotide sequence encoding the display construct polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0215] In short, Display 2 is a construct comprising a cargo linked via one or more antiparallel coiled coils to a cell wall-embedded binding domain (anchor) (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain linked by one or more antiparallel coiled coils).

[0216] In some embodiments, the display construct comprises an antiparallel coiled coil (APCC).

[0217] In some embodiments, Display 2 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:61 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:61), and further comprises one or more antiparallel coiled coils. In some embodiments, Display 1 comprises the amino acid sequence encoded by SEQ ID NO:61, and further comprises one or more antiparallel coiled coils.

[0218] In some embodiments, display 2 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity with SEQ ID NO:63 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:63). In some embodiments, display 2 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:63. As written herein, SEQ ID NO:63 includes an "X" marker indicating the position where the nucleotide sequence encoding the cargo will be included. Thus, those skilled in the art will understand that comparison of a given sequence with SEQ ID NO:63 can be made by excluding the cargo sequence from the analysis (i.e., by comparing only the regions encoding the cell wall-embedded binding domain and one or more anti-parallel coiled coils).

[0219] In some embodiments, the display structure is the display 2 linker polypeptide.

[0220] In some embodiments, the cell wall-embedded anchor construct is a shortened anti-parallel coiled coil.

[0221] Thus, in some embodiments, the display structure is a polypeptide comprising a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or a variant or derivative thereof), one or more anti-parallel coiled coils, and a cargo. In some embodiments, the cell wall-embedded binding domain and the one or more anti-parallel coiled coils are encoded by SEQ ID NO:63 or a variant or derivative thereof.

[0222] Display 3

[0223] In some aspects, the display structure is a construct as shown in display 3 (see Figure 2B ). Thus, in some aspects, the present invention provides a display structure polypeptide according to display 3; a nucleotide sequence encoding the display structure polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0224] In short, display 3 is a construct comprising a cargo linked via one or more anti-parallel coiled coils to a cell wall-embedded binding domain (anchor) (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain linked by one or more anti-parallel coiled coils), wherein the anti-parallel coiled coils are shorter than the anti-parallel coiled coils of display structure 2. Thus, display 3 is a structure similar to display 2 but with shortened anti-parallel coiled coils.

[0225] In some embodiments, display 3 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:61 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:61), and further comprises one or more shortened antiparallel coiled coils. In some embodiments, display 3 comprises the amino acid sequence encoded by SEQ ID NO:61, and further comprises one or more shortened antiparallel coiled coils.

[0226] In some embodiments, display 3 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:64 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:64). In some embodiments, display 3 comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO:64. As written herein, SEQ ID NO:64 includes an "X" marker indicating the position where the nucleotide sequence encoding the cargo will be included. Thus, one of ordinary skill in the art will understand that comparison of a given sequence to SEQ ID NO:64 can be made by excluding the cargo sequence from the analysis (i.e., by comparing only the region encoding the cell wall-embedded binding domain and one or more shortened antiparallel coiled coils).

[0227] In some embodiments, the linker (e.g., an antiparallel coiled coil) lacks protease sites to reduce proteolytic cleavage of the cargo from the ADAS. In some embodiments, the linker contains protease sites to enable release of the cargo from the ADAS surface. In some embodiments, the protease site is specific for eukaryotes (e.g., plants, animals, mice, humans, etc.). In some embodiments, the protease site is specific for human tissues or target sites (e.g., lungs, blood, gastrointestinal tract, etc.).

[0228] Thus, in some embodiments, the display structure is a polypeptide comprising a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or a variant or derivative thereof), one or more shortened antiparallel coiled coils, and a cargo. In some embodiments, the cell wall-embedded binding domain and the one or more shortened antiparallel coiled coils are encoded by SEQ ID NO:64 or a variant or derivative thereof.

[0229] Display 4

[0230] In some aspects, the display structure is a construct as shown in display 4 (see Figure 2B)。Thus, in some aspects, the present invention provides a display - structured polypeptide according to display 4; a nucleotide sequence encoding the display - structured polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0231] In short, display 4 is a construct (e.g., a cell - wall - embedded anchor construct containing a coiled linker) (e.g., a fusion polypeptide comprising a cargo and a cell - wall - embedded binding domain connected by a coiled linker) that contains a cargo connected via a coiled linker to a cell - wall - embedded binding domain (anchor).

[0232] In some embodiments, display 4 comprises a cell - wall - embedded binding domain encoded by a nucleotide sequence having at least 20% identity (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity) with SEQ ID NO:61, and further comprises a coiled linker. In some embodiments, display 4 comprises the amino acid sequence encoded by SEQ ID NO:61, and further comprises a coiled linker.

[0233] In some embodiments, display 4 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity) with SEQ ID NO:65. In some embodiments, display 4 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:65.

[0234] Thus, in some embodiments, the display structure is a polypeptide comprising a cell - wall - embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or its variant or derivative), a coiled linker, and a cargo. In some embodiments, the cell - wall - embedded binding domain and the coiled linker are encoded by SEQ ID NO:65 or its variant or derivative.

[0235] Display 5

[0236] In some aspects, the display structure is a construct as shown in display 5 (see Figure 2B )

[0237] Thus, in some aspects, the present invention provides a display - structured polypeptide according to display 5; a nucleotide sequence encoding the display - structured polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0238] Briefly, display 5 is a construct that includes a cargo attached via a scaffold protein (e.g., mCherry) to a cell wall-embedded binding domain (anchor), where a linker connects the cell wall-embedded binding domain to the scaffold protein (e.g., a fusion polypeptide that includes the cargo, scaffold protein, linker, and cell wall-embedded binding domain).

[0239] In some embodiments, display 5 includes a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 61 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 61), and further includes a linker and a scaffold protein. In some embodiments, display 5 includes an amino acid sequence encoded by SEQ ID NO: 61, and further includes a linker and a scaffold protein.

[0240] In some embodiments, display 5 includes a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 66 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 66). In some embodiments, display 5 includes a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 66. As written herein, SEQ ID NO: 66 includes an "X" marker indicating the position where the nucleotide sequence encoding the cargo will be included. Thus, one of ordinary skill in the art will understand that comparison of a given sequence to SEQ ID NO: 66 can be made by excluding the cargo sequence from the analysis (i.e., by comparing only the regions encoding the cell wall-embedded binding domain, scaffold protein, and linker).

[0241] Thus, in some embodiments, the display construct is a polypeptide that includes a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO: 61 or a variant or derivative thereof), a linker, a scaffold protein, and a cargo. In some embodiments, the cell wall-embedded binding domain, the linker, and the scaffold protein are encoded by SEQ ID NO: 66 or a variant or derivative thereof.

[0242] Display 6

[0243] In some aspects, the display construct is a construct as shown in display 6 (see Figure 2B ). Thus, in some aspects, the present invention provides a display construct polypeptide according to display 6; a nucleotide sequence encoding the display construct polypeptide; a parental bacterial cell comprising the polypeptide and / or the nucleotide sequence; and an ADAS comprising the polypeptide and / or the nucleotide sequence.

[0244] Briefly, display 6 is a construct that includes a cargo attached via a scaffold protein (e.g., mCherry) to a cell wall-embedded binding domain (anchor), where a first linker attaches the cargo to the scaffold protein, and a second linker attaches the cell wall-embedded binding domain to the scaffold protein (e.g., a fusion polypeptide that includes the cargo, the first linker, the scaffold protein, the second linker, and the cell wall-embedded binding domain).

[0245] In some embodiments, display 6 includes a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:61 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:61), and further includes a first linker, a second linker, and a scaffold protein. In some embodiments, display 6 includes the nucleotide sequence of SEQ ID NO:61, and further includes a first linker, a second linker, and a scaffold protein.

[0246] In some embodiments, display 6 includes a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:67 (e.g., having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:67). In some embodiments, display 6 includes a polypeptide encoded by the nucleotide sequence of SEQ ID NO:67. As written herein, SEQ ID NO:67 includes an "X" marker indicating the position where the nucleotide sequence encoding the cargo will be included. Thus, one of ordinary skill in the art will understand that comparison of a given sequence to SEQ ID NO:67 can be made by excluding the cargo sequence from the analysis (i.e., by comparing only the regions encoding the cell wall-embedded binding domain, the scaffold protein, and the linker).

[0247] Thus, in some embodiments, the display construct is a polypeptide that includes a cell wall-embedded binding domain (e.g., a binding domain encoded by SEQ ID NO:61 or a variant or derivative thereof), a first linker, a scaffold protein, a second linker, and a cargo. In some embodiments, the cell wall-embedded binding domain, the linker, and the scaffold protein are encoded by SEQ ID NO:67 or a variant or derivative thereof.

[0248] In some embodiments, the display construct is cytoplasmic rather than surface-expressed, e.g., the cargo is displayed by one or more components (e.g., the binding domain, the linker, and / or the scaffold) in the interior (cytosol) of the ADAS.

[0249] The display structure can be "unstructured", involving a relatively high proportion of proline in the structure to create a non-linear or bent shape for the construct, or can be "structured", such as coiled, with a specific or more defined shape.

[0250] In some embodiments, the cell wall-embedded anchoring structure contains a cell wall-binding domain.

[0251] In some embodiments, the ADAS contains a fusion promoter.

[0252] In some embodiments, the ADAS contains a proline-rich sequence encoding the cell wall-embedded anchoring structure.

[0253] In some embodiments, the ADAS contains a self-binding helix for the cell wall-embedded anchoring structure.

[0254] In some embodiments, the cell wall-embedded anchoring structure is an unstructured and proline-rich construct.

[0255] In some embodiments, the cell wall-embedded anchoring structure is a structured construct.

[0256] In some embodiments, the constructed cell wall-embedded anchoring structure has an N-terminus embedded in the cell wall. In some embodiments, the anchoring structure contains an inner cell wall-embedded anchoring domain. In some embodiments, the cell wall-embedded anchoring structure contains a protease cleavage site. In some embodiments, the display structure contains a protease cleavage site.

[0257] In some embodiments, at least one element of the display structure (e.g., cargo, cell wall-embedded anchoring structure, scaffold protein, and / or linker) is heterologous to the parental cell from which the ADAS is derived.

[0258] G. ADAS Comprising a Secretion System

[0259] In some embodiments, the ADAS provided by the present invention includes a bacterial secretion system (e.g., an endogenous bacterial secretion system or a heterologous secretion system). A "bacterial secretion system" is a protein or protein complex that can export cargo from the cytoplasm of a bacterial cell (or an ADAS derived therefrom, for example) to: the extracellular space, the periplasmic space of Gram-negative bacteria, or the intracellular space of another cell. In some embodiments, the bacterial secretion system functions by an active (e.g., ATP-dependent or PMF-dependent) process, and in certain embodiments, the bacterial secretion system includes a tube or spike that spans the host cell (or ADAS) and the target cell. In other embodiments, the bacterial secretion system is a transmembrane channel. Exemplary bacterial secretion systems include T3SS and T4SS (and T3 / T4SS as defined below), which are tube-containing structures where cargo passes through the interior of the protein tube, and T6SS, which carries cargo at the tip of the spike. Other exemplary bacterial secretion systems include T1SS, T2SS, T5SS, T7SS, Sec, and Tat, which are transmembrane.

[0260] In some aspects, the present disclosure features a non-chromosomal dynamic active system (ADAS) derived from a parental bacterial cell, the ADAS including a bacterial type 3 secretion system (T3SS) heterologous to the parental bacterial cell.

[0261] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0262] In some embodiments, the parental bacterial cell does not contain an endogenous T3SS.

[0263] In some embodiments, the parental bacterial cell is an Escherichia coli cell. In some embodiments, the Escherichia coli cell is an Escherichia coli Nissle cell.

[0264] In some embodiments, the parental bacterial cell is a probiotic cell.

[0265] In some embodiments, the T3SS is a Salmonella T3SS, a Vibrio T3SS, an Escherichia T3SS, a Yersinia T3SS, a Shigella T3SS, a Pseudomonas T3SS, or a Chlamydia T3SS. In some embodiments, the Salmonella T3SS is a Salmonella enterica T3SS. In some embodiments, the Vibrio T3SS is a Vibrio parahaemolyticus T3SS. In some embodiments, the Escherichia T3SS is an enteropathogenic Escherichia coli (EPEC) T3SS. In some embodiments, the Yersinia T3SS is a Yersinia enterocolitica T3SS. In some embodiments, the Shigella T3SS is a Shigella flexneri T3SS.

[0266] In some embodiments, the parental bacterial cell comprises one or more heterologous nucleotide sequences encoding components of the T3SS. In some embodiments, one or more nucleotide sequences encoding components of the T3SS are carried on a vector. In some embodiments, the parental bacterial cell has been transiently transformed with the vector. In some embodiments, the parental bacterial cell has been stably transformed with the vector. In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS. In some embodiments, the moiety is a transcriptional activator of one or more heterologous nucleotide sequences encoding components of the T3SS.

[0267] In another aspect, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the ADAS comprising a bacterial type III secretion system (T3SS) endogenous to the parental bacterial cell, wherein the parental bacterial cell has been modified to reduce the level of an endogenous protein or polypeptide that can be secreted by the T3SS.

[0268] In some embodiments, the parental bacterial cell has been modified by deletion of a transcriptional activator of an endogenous protein or polypeptide that can be secreted by the T3SS.

[0269] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0270] In some embodiments, the parental bacterial cell is a species of Salmonella, a species of Vibrio, a species of Escherichia, a species of Yersinia, or a species of Shigella. In some embodiments, the species of Salmonella is Salmonella enterica. In some embodiments, the species of Vibrio is Vibrio parahaemolyticus. In some embodiments, the species of Escherichia is enteropathogenic Escherichia coli (EPEC). In some embodiments, the species of Yersinia is Yersinia enterocolitica. In some embodiments, the species of Shigella is Shigella flexneri.

[0271] In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of T3SS in the ADAS. In some embodiments, the moiety is a transcriptional activator of a nucleotide sequence encoding a component of the T3SS.

[0272] In some embodiments, the parental bacterial cell has been modified to reduce the level of a negative regulator of a component of the T3SS. In some embodiments, the chromosomal locus encoding the negative regulator has been deleted from the parental bacterial cell.

[0273] In some embodiments, the parental bacterial cell has been modified to reduce the level of one or more of the following: LPS; metabolically non-essential proteins; toxins not associated with the T3SS; endotoxins; flagella; and pili.

[0274] In some embodiments, the ADAS further comprises at least one cargo, wherein the T3SS is capable of delivering the cargo to a target cell. In some embodiments, the delivery is to the cytoplasm of the target cell.

[0275] In some embodiments, the cargo is a protein or polypeptide.

[0276] In some embodiments, the cargo is endogenously secreted by the T3SS.

[0277] In some embodiments, the ADAS or the parental bacterial cell has been modified to increase the level of the cargo in the ADAS.

[0278] In some embodiments, the cargo is not endogenously secreted by the T3SS.

[0279] In some embodiments, the cargo is endogenously secreted by a T3SS from a species other than the ADAS T3SS species.

[0280] In some embodiments, the cargo is endogenously secreted by a type IV secretion system (T4SS) or a type VI secretion system (T6SS).

[0281] In some embodiments, the cargo has been modified for delivery by the T3SS.

[0282] In some embodiments, the cargo is an enzyme, a DNA modifier, a chromatin remodeler, a gene editor, a nuclear targeting agent, a binder, an immunogenic agent, or a toxin. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editor is a component of the CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binder is an antibody or an antibody fragment. In some embodiments, the binder is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulant. In some embodiments, the immunogenic agent is an immunosuppressant.

[0283] In some embodiments, the cargo has been modified by adding a secretion signal.

[0284] In another aspect, the present disclosure features a method for delivering a cargo to the cytoplasm of a target cell, the method comprising contacting the target cell with an ADAS of any of the above aspects.

[0285] In some embodiments, the ADAS comprises a cargo, wherein the cargo comprises a portion that directs the output of the bacterial secretion system, e.g., in some embodiments, the portion is a Pho / D, Tat, or synthetic peptide signal.

[0286] In certain embodiments, the ADAS provided by the present invention is a double-membrane ADAS. In more specific embodiments, the double-membrane ADAS further comprises a bacterial secretion system. In still more specific embodiments, the bacterial secretion system is selected from T3SS, T4SS, T3 / 4SS, or T6SS, optionally, wherein T3SS, T4SS, T3 / 4SS, or T6SS has a weakened or non-functional effector that does not affect the fitness of the target cell.

[0287] In some embodiments, the ADAS provided by the present invention includes a bacterial secretion system.

[0288] In some embodiments, the bacterial secretion system (such as T3SS, T4SS, T3 / T4SS, or T6SS) is capable of exporting the cargo through the outer membrane of the ADAS into the target cell (such as an animal, fungal, bacterial, or plant cell).

[0289] In more specific embodiments, the bacterial secretion system is a T3 / 4SS. "T3 / 4SS" is a secretion system based on T3SS or T4SS, including hybrid systems and unmodified forms, which form a protein tube between the bacterium (or ADAS) and the target cell, thereby connecting the two and delivering one or more effectors. The target cell can be an animal, a plant, a fungus, or a bacterium. In some embodiments, the T3 / 4SS includes effectors, which can be modified effectors. Examples of the T3SS system include the Salmonella SPI-1 system, the Enterohemorrhagic Escherichia coli (EHEC coli) ETT1 system, the Xanthomonas Citri / Campestri T3SS system, and the Pseudomonas syringae T3SS system. Examples of the T4SS system include the Agrobacterium Ti plasmid system, the Helicobacter pylori T4SS. In certain embodiments, the T3 / 4SS has altered effector function, for example, an effector selected from SopD2, SopE, Bop, Map, Tir, EspB, EspF, NleC, NleH2, or NleE2. In more specific embodiments, the altered effector function is for intracellular targeting, such as translocation to the nucleus, Golgi apparatus, mitochondria, actin, microvilli, ZO-1, microtubules, or the cytoplasm. In still more specific embodiments, the altered effector function is nuclear targeting based on NleE2 derived from Escherichia coli. In other specific embodiments, the altered effector function is for filopodia formation, tight junction disruption, microvilli elimination, or SGLT-1 deactivation.

[0290] In other embodiments, the ADAS provided by the present invention comprising a bacterial secretion system comprises a T6SS. In some embodiments, the T6SS targets bacteria in its natural host and contains effectors that kill bacteria. In certain specific embodiments, the T6SS is derived from Pseudomonas putida K1-T6SS, and optionally, wherein the effector comprises the amino acid sequence of Tke2 (accession number AUZ59427.1), or a functional fragment thereof. In other embodiments, the T6SS targets fungi in its natural host and contains effectors that kill fungi, for example, the T6SS is derived from Serratia Marcescens, and the effector comprises the amino acid sequence of: Tfe1 (Genbank: SMDB11_RS05530) or Tfe2 (Genbank: SMDB11_RS05390).

[0291] In other embodiments of the ADAS provided by the present invention that contain a bacterial secretion system, the bacterial secretion system is capable of exporting cargo extracellularly. In some more specific embodiments, the bacterial secretion system is T1SS, T2SS, T5SS, T7SS, Sec or Tat.

[0292] ADAS lacking protease, RNase and / or LPS

[0293] In another aspect, the present invention provides a composition further comprising multiple ADASs (e.g., highly active ADASs), wherein the protease level or activity of the ADASs is reduced relative to the ADASs produced by wild-type parental bacteria. In some aspects, the ADASs are produced by parental bacteria that have been modified to reduce or eliminate the expression of at least one protease. In some embodiments, the parental cells contain one or more protease deletions. In some embodiments, the parental cells contain one or more loss-of-function mutations in protease genes. In some embodiments, the protease deletion or other loss-of-function mutation increases the stability of the cargo of the ADAS (e.g., increases the expression, display, function, or durability of the cargo, e.g., optimizes cargo expression) relative to the ADASs produced by parental cells that do not contain deletions or loss-of-function mutations. In some embodiments, the protease deletion or loss-of-function is a deletion or loss-of-function in one or more of the following: aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW or a combination thereof (e.g., deletion or loss-of-function in two, three, four, five, six, seven, eight, nine, ten or more than ten of the listed proteases).

[0294] In some embodiments, the parental cell comprises deletions of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine proteases. In some embodiments, the parental cell comprises loss-of-function mutations in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine protease genes. In some embodiments, the parental bacterial cell comprises deletions of sigF, divIVA, and lytC, and deletions of one, two, three, or all four of the proteases aprE, ispA, wprA, and nprE. In some embodiments, the parental bacterial cell comprises loss-of-function mutations in sigF, divIVA, and lytC, and in one, two, three, or all four of aprE, ispA, wprA, and nprE or combinations thereof.

[0295] In another aspect, the present invention provides a composition comprising multiple ADASs (e.g., highly active ADASs), wherein the RNase level or activity of the ADASs is reduced relative to the ADASs produced by wild-type parental bacteria. In some aspects, the ADASs are produced by parental bacteria that have been modified to reduce or eliminate the expression of at least one RNase. In some embodiments, the RNase is an endoribonuclease or an exoribonuclease.

[0296] In another aspect, the present invention provides a composition comprising multiple ADASs, wherein the ADASs are modified to have reduced lipopolysaccharide (LPS). In some embodiments, the modification is a lipid A biosynthetic myristoyltransferase (msbB) mutation.

[0297] In certain embodiments, the ADASs provided by the present invention lack one or more metabolically non-essential proteins. "Metabolically non-essential proteins" non-exhaustively include: pili, flagella, undesired secretion systems, transposases, effectors, phage elements or regulatory elements thereof, such as flhC or OmpA. In some embodiments, the ADASs provided by the present invention lack one or more of RNases, proteases, or combinations thereof, and in certain embodiments, lack one or more endoribonucleases (such as RNase A, RNase h, RNase III, RNase L, RNase PhyM) or exoribonucleases (such as RNase R, RNase PH, RNase D); or serine, cysteine, threonine, aspartic acid, glutamic acid, and metalloproteases; or any combination of the foregoing.

[0298] I. ADASs Comprising a Targeting Moiety

[0299] In another embodiment, the present invention provides a composition comprising multiple ADASs, wherein the ADASs comprise targeting moieties. In some embodiments, the targeting moiety is a nanobody, a carbohydrate-binding protein, or a tumor-targeting peptide. In some embodiments, the targeting moiety is an endogenous surface ligand of the parental cell (e.g., a surface ligand genetically acquired by the ADAS). In other embodiments, the targeting moiety is an exogenous ligand (e.g., an exogenous tissue-targeting ligand) added to the ADAS using any of the methods described herein for modifying the ADAS. In some embodiments, the targeting moiety facilitates tissue-related targeting of the ADAS to a tissue type or cell type.

[0300] In certain embodiments, the nanobody is a nanobody against a tumor antigen (such as HER2, PSMA, or VEGF-R). In other embodiments, the carbohydrate-binding protein is a lectin, such as mannose-binding lectin (MBL). In still other embodiments, the tumor-targeting peptide is an RGD motif or a CendR peptide.

[0301] J. ADASs derived from symbiotic or pathogenic parental strains

[0302] In another embodiment, the present invention provides a composition comprising multiple ADASs (e.g., highly active ADASs), wherein the ADASs are derived from parental bacteria that are mammalian pathogens or mammalian symbiotic bacteria. In some cases, the mammalian symbiotic bacteria are species of Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus, or Actinomyces, or the mammalian pathogenic bacteria are Escherichia coli (EHEC), Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori.

[0303] In another embodiment, the present invention provides a composition comprising multiple ADASs (e.g., highly active ADASs), wherein the ADASs are derived from parental bacteria that are plant pathogens or plant symbiotic bacteria. In some cases, the plant symbiotic bacteria are Bacillus subtilis or Pseudomonas putida, or the plant pathogenic bacteria are species of Xanthomonas or Pseudomonas syringae.

[0304] K. ADAS Derived from Auxotrophic Parent Strains

[0305] In another embodiment, the present invention provides a composition comprising multiple ADASs (e.g., highly active ADASs), wherein the ADASs are derived from auxotrophic parent bacteria, i.e., parent bacteria that cannot synthesize organic compounds required for growth. Such bacteria can grow only when organic compounds are provided.

[0306] L. ADAS Comprising Additional Moieties

[0307] In certain embodiments, the ADAS includes a functional ATP synthase, and in some embodiments, includes a membrane-embedded proton pump. The ADAS can be derived from different sources, including: parent bacterial strains (''parent strains'') engineered or induced to produce a genome-free closed membrane system, genomically excised bacteria, bacterial cell preparation extracts (e.g., by mechanical or other means), or total synthesis processes, optionally including fractions of bacterial cell preparations. In some embodiments, the ATP synthase concentration of the highly active ADAS is at least: 1 / 10000 nm 2 、1 / 5000 nm 2 、1 / 3500 nm 2 、1 / 1000 nm 2 。

[0308] The ADAS provided by the present invention can include various additional components, including, for example, photovoltaic pumps, one or more retinol-producing cassettes, metabolic enzymes, targeting agents, cargoes, bacterial secretion systems, and transporters, including combinations of the foregoing, including certain specific embodiments described below. In certain embodiments, the ADAS lacks other elements, such as metabolically non-essential genes and / or certain enzymes, nucleases, or proteases.

[0309] In certain embodiments, the ADAS provided by the present invention comprises an ATP synthase, optionally lacking a regulatory domain, such as lacking the ε domain. The deletion can be achieved in various ways. In certain embodiments, the deletion is effected by inducing a natural ε domain deletion. In certain embodiments, the deletion can be achieved by flanking LoxP sites and inducible Cre expression or CRISPR knockout, or can be induced (on a plasmid in the presence of the tTa tet transactivator in an ATP synthase knockout strain)

[0310] In some embodiments, the ADAS can include a photovoltaic proton pump. In certain embodiments, the photovoltaic proton pump is proteorhodopsin. In more specific embodiments, the proteorhodopsin comprises the amino acid sequence of proteorhodopsin from the uncultured marine bacterial lineage SAR86 (GenBank accession number: AAS73014.1). In other embodiments, the photovoltaic proton pump is bacteriorhodopsin. In certain embodiments, the photovoltaic proton pump is a bacteriorhodopsin, δ-rhodopsin, or halorhodopsin from Halobium salinarum, Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica, or Haloarcula marismortui.

[0311] In some embodiments, the ADAS provided by the present invention further comprises retinal. In certain embodiments, the ADAS provided by the present invention further comprises a retinal synthesis protein (or protein system), or a nucleic acid encoding the same.

[0312] In certain embodiments, the ADAS provided by the present invention further comprises one or more glycolytic pathway proteins. In some embodiments, the glycolytic pathway protein is phosphofructokinase (Pfk-A), for example, phosphofructokinase comprising the amino acid sequence of UniProt accession number P0A796 or a functional fragment thereof. In other embodiments, the glycolytic pathway protein is triosephosphate isomerase (tpi), for example, triosephosphate isomerase comprising the amino acid sequence of UniProt accession number P0A858 or a functional fragment thereof.

[0313] M. ADAS Compositions and Formulations

[0314] The present invention provides a composition or formulation containing the ADAS provided by the present invention, which particularly includes the highly active ADAS formulation provided by the present invention, and optionally wherein the ADAS formulation is substantially free of live cells. These are collectively referred to as "the various compositions provided by the present invention" or "a composition provided by the present invention", etc., and can contain any ADAS provided by the present invention and any combination of the ADAS provided by the present invention.

[0315] For example, in some embodiments, the composition provided by the present invention contains at least about: 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of ADAS containing a bacterial secretion system. In certain embodiments, the bacterial secretion system is one of T3SS, T4SS, T3 / 4SS or T6SS.

[0316] In some embodiments, the composition provided by the present invention contains ADAS containing T3SS, wherein the ADAS has an average T3SS membrane density greater than 1 at about 40000, 35000, 30000, 25000, 19600, 15000, 10000 or 5000 nm 2 In certain specific embodiments, the ADAS is derived from a Salmonella typhimurium or Escherichia coli parental strain.

[0317] Certain embodiments of the composition provided by the present invention contain ADAS containing T3SS, wherein the ADAS has an average T3SS membrane density greater than 1 at about 300000, 250000, 200000, 150000, 100000, 50000, 20000, 10000, 5000 nm 2 In certain specific embodiments, the ADAS is derived from an Agrobacterium tumefacien parental strain.

[0318] On the other hand, the present invention provides a composition of ADAS, wherein at least about: 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the ADAS contains a bacterial secretion system, the bacterial secretion system includes T3, T4, T3 / 4SS, T6SS, and optionally includes one or more of the following: exogenous carbohydrates, phosphate-producing synthase, photoreactive proteins, importins, enzymes, functional cargo, organism-specific effectors, fusion proteins.

[0319] It will be apparent that the compositions and formulations provided by the present invention may contain any ADAS provided by the present invention, such as highly active ADAS or ADAS lacking the minE gene product.

[0320] The compositions provided by the present invention can be prepared in any suitable formulation. For example, the formulation can be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosolized or nebulized administration. In some embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.

[0321] In some embodiments, the ADAS compositions described herein contain less than 100 colony forming units (CFU / mL) of live bacterial cells, for example, less than 50 CFU / mL, less than 20 CFL / mL, less than 10 CFU / mL, less than 1 CFU / mL or less than 0.1 CFU / mL of live bacterial cells.

[0322] In some embodiments, the present invention provides an ADAS composition, wherein the ADAS is lyophilized and reconstituted, and wherein the ATP concentration of the reconstituted ADAS is at least 90% of the ATP concentration of the non-lyophilized ADAS, for example, at least 95%, 98% or at least equal to the ATP concentration of the non-lyophilized ADAS.

[0323] In some embodiments, the present invention provides an ADAS composition, wherein the ADAS is stored, for example, stored at 4°C, and wherein the ATP concentration of the ADAS after storage is at least 90% of the ATP concentration of the non-stored ADAS, for example, at least 95%, 98% or at least equal to the ATP concentration of the non-stored ADAS. In some embodiments, the storage lasts for at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least six months or at least one year.

[0324] In some embodiments, the ADAS is stored in a "quiescent" state or otherwise processed and then rapidly activated.

[0325] In some embodiments, the ADAS composition is formulated for delivery to an animal, for example, formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosolized or nebulized administration.

[0326] In some embodiments, the ADAS composition is formulated for delivery to a plant. In some aspects, the composition includes adjuvants such as surfactants (e.g., nonionic surfactants, surfactant plus nitrogen source, silicone surfactants, or high surfactant oil concentrates), crop oil concentrates, vegetable oil concentrates, modified vegetable oils, nitrogen sources, deposition (drift control) and / or retention aids (with or without ammonium sulfate and / or defoamers), compatibilizers, buffers and / or acidifying agents, water conditioners, alkaline mixtures, sticker-spreaders and / or extenders, adjuvant plus foliar fertilizer, defoamers, foam markers, odorants, or tank cleaners and / or neutralizers. In some embodiments, the adjuvant is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (13th Edition), Purdue University).

[0327] In some embodiments, the ADAS composition is formulated for delivery to an invertebrate (e.g., an arthropod (e.g., an insect or arachnid), nematode, protozoan, or annelid). In some embodiments, the ADAS composition is formulated for delivery to an insect.

[0328] In some embodiments, the composition is formulated as a liquid, solid, aerosol, paste, gel, or gas composition.

[0329] N. ADAS Comprising Enzymes

[0330] In one aspect, the invention features a composition comprising multiple ADASs, wherein the ADAS comprises an enzyme, and wherein the enzyme modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS, and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered. In some embodiments, the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic di-AMP.

[0331] O. ADAS Comprising Cancer Therapeutic Agents

[0332] In one aspect, the invention features a composition comprising multiple ADASs, wherein the ADASs comprise cancer therapeutics. Exemplary cancer therapeutics are described, for example, in Section II(E) above and include, but are not limited to, anti-inflammatory agents, growth inhibitors, chemotherapeutic agents, immunotherapy agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines comprising cancer neoantigens), agents that induce autophagy (e.g., activators such as listeriolysin-o), cytotoxins, inflammasome inhibitory agents, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0333] In some embodiments, the cancer therapeutics are displayed on an anchored structure embedded in the constructed cell wall. In some embodiments, the cancer therapeutics are displayed on a display structure (e.g., as described in Section II(F) above).

[0334] III. Methods of Making ADAS

[0335] A. Preparation of ADAS and Highly Active ADAS

[0336] In some aspects, the production of ADAS features a method for making a composition comprising multiple ADASs that is substantially free of live bacterial cells, the method comprising (a) preparing, providing, or obtaining a plurality of parental bacteria with a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of microcells, thereby producing the highly active ADAS; and (c) separating the highly active ADAS from the parental bacteria, thereby producing a composition that is substantially free of live bacterial cells.

[0337] Parental bacteria include any suitable bacterial species that can produce an ADAS (e.g., species that can be modified using the methods described herein to produce an ADAS). A non-limiting list of suitable genera from which an ADAS can be derived is provided below: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacter, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacteria.

[0338] In some aspects, methods for making any of the ADAS compositions described in Part I herein (e.g., highly active ADAS compositions) are used. For example, methods for preparing highly active ADAS are provided herein; methods for preparing an ADAS that lacks a cell division topological specificity factor and optionally lacks a Z-ring inhibitor protein (e.g., a method for preparing an ADAS from ΔminCDE parental bacteria), and methods for preparing any ADAS mentioned herein, wherein the ADAS contains cargo.

[0339] In some embodiments, the ADAS (highly active ADAS) is prepared from a parental strain that is a plant bacterium, such as a plant symbiotic bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogenic bacterium (e.g., a Xanthomonas species or Pseudomonas syringae), or a bacterium capable of colonizing the plant rhizosphere and / or forming root nodules, such as Rhizobium.

[0340] In some embodiments, the ADAS (highly active ADAS) is prepared from a parental strain that is a symbiont of an invertebrate, such as a symbiont of an arthropod (e.g., an insect or an arachnid), a nematode, a protozoan, or an annelid. In an embodiment, the invertebrate is a pest or pathogen of a plant or an animal.

[0341] In some embodiments, the ADAS (e.g., highly active ADAS) is prepared from a parental strain capable of genetic transformation (e.g., Agrobacterium).

[0342] In some embodiments, the ADAS (e.g., highly active ADAS) is prepared from a parental strain that is a human bacterium, such as a symbiotic human bacterium (e.g., Escherichia coli, Staphylococcus species, Bifidobacterium species, Micrococcus species, Lactobacillus species, or Actinomyces species) or a human pathogenic bacterium (e.g., Escherichia coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica, or Helicobacter pylori) or an extremophile.

[0343] In some embodiments, the ADAS and / or the parental strain is a functionalized derivative of any of the foregoing, for example, including a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial agent, digest plastic, secrete an insecticide, survive in an extreme environment, manufacture nanoparticles, integrate in other organisms, respond to the environment, and produce a reporter signal.

[0344] In some embodiments, the parental bacterium includes a functionalized derivative of any of the foregoing, for example, including a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial agent, digest plastic, secrete an insecticide, survive in an extreme environment, manufacture nanoparticles, integrate in other organisms, respond to the environment, and produce a reporter signal.

[0345] In some embodiments, the ADAS is derived from a parental strain engineered or induced to overexpress ATP synthase. In some more specific embodiments, the ATP synthase is heterologous to the parental strain. In certain specific embodiments, the parental strain is modified to express a functional F o F1 ATP synthase.

[0346] In certain embodiments, the ADAS provided by the present invention is obtained from a parental strain cultured under conditions selected from the following: an applied voltage (e.g., 37 mV), a non-atmospheric oxygen concentration (e.g., 1%-5% O2, 5%-10% O2, 10%-15% O2, 25%-30% O2), a low pH (about: 4.5, 5.0, 5.5, 6.0, 6.5), or a combination thereof.

[0347] The highly active ADAS as described in any one of the preceding claims, which is prepared from extremophilic microorganisms and includes functional derivatives of any one of the foregoing, for example, includes functional cassettes, such as cassettes that induce bacteria to perform one or more of the following: secrete antimicrobial agents, digest plastics, secrete insecticides, survive in extreme environments, manufacture nanoparticles, integrate into other organisms, respond to the environment, and generate reporting signals.

[0348] Due to the diversity of bacteria, ADAS with modified membranes can be prepared. For example, to improve the biodistribution of ADAS after administration to target cells. In certain embodiments, the membrane is modified to have less immunogenicity or immunostimulatory properties in plants or animals. For example, in certain embodiments, ADAS is obtained from a parental strain, and the immunostimulatory ability of the parental strain is reduced or eliminated by post-production treatment with detergents, enzymes, or PEG functionalization. In certain embodiments, ADAS is prepared from a parental strain, and the membrane is modified by knocking out the LPS synthesis pathway in the parental strain, for example, by knocking out msbB. In other specific embodiments, ADAS is prepared from a parental strain that generates cell wall-deficient particles by exposure to hypertonic conditions.

[0349] In some embodiments, the method includes transforming the parental strain with an inducible DNA enzyme system, such as exoI (NCBI GeneID: 946529) & sbcD (NCBI GeneID: 945049) nucleases, or I-CeuI (e.g., Swissprot: P32761.1) nuclease. In more specific embodiments, the method includes using one, two, three, or four auxotrophic strains and having complementary genes on a plasmid encoding the inducible nuclease.

[0350] In some embodiments, the parental strain is cultured under conditions selected from: applied voltage (e.g., 37 mV), non-atmospheric oxygen concentration (e.g., 1%-5% O2, 5%-10% O2, 10%-15% O2, 25%-30% O2), low pH (4.5-6.5), or a combination thereof.

[0351] In certain embodiments, the parental strain lacks flagella and undesired secretion systems, optionally wherein λred recombination engineering is used to remove the flagella and undesired secretion systems.

[0352] In some embodiments, the flagella control components are excised from the parental strain genome via, for example, inserting a plasmid containing a CRISPR domain targeting flagella control genes (such as flhD and flhC).

[0353] In certain embodiments, the provided method is for preparing highly active ADAS, wherein the ADAS containing a plasmid encoding a rhodopsin gene is cultured in the presence of light. In more specific embodiments, the rhodopsin is proteorhodopsin having the amino acid sequence with GenBank accession number: AAS73014.1 from uncultured bacteria of SAR86 or a functional fragment thereof. In still more specific embodiments, the culture is supplemented with retinal. In other more specific embodiments, the rhodopsin is proteorhodopsin, and the plasmid additionally contains a gene for synthesizing retinal (such a plasmid is the pACYC-RDS plasmid from Kim et al., Microb CellFact [Microbial Cell Factories], 2012).

[0354] In certain specific embodiments, the parental strain contains a nucleic acid sequence encoding a nanobody, and then the nanobody is expressed on the membrane of the ADAS.

[0355] In some embodiments of the method provided by the present invention, the parental strain contains a nucleic acid sequence encoding one or more bacterial secretion system operons. Exemplary plasmids include Salmonella SPI-1 T3SS, Shigella flexneri T3SS, Agrobacterium Ti plasmid, and Pseudomonas putida K1-T6SS system.

[0356] In certain embodiments, the parental strain contains cargo. In some embodiments, the parental strain contains a nucleic acid sequence encoding a gene for a set of synthetic small molecule cargo.

[0357] IV. Purification of ADAS and ADAS Compositions

[0358] In some embodiments of the methods and compositions provided herein, ADAS is purified from a composition (e.g., a culture) containing live bacteria (e.g., parental bacteria). For example, the present invention features a method for making a composition containing multiple ADAS that is substantially free of live bacterial cells, the method comprising (a) preparing, providing, or obtaining a plurality of parental bacteria with a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of microcells, thereby producing the ADAS; and (c) separating the ADAS from the parental bacteria, thereby producing a composition substantially free of live bacterial cells.

[0359] Purification separates the ADAS from live parental bacterial cells, which are larger and contain a genome. Separation of highly active ADAS from parental bacteria can be performed using a variety of methods as described herein. Exemplary methods for the purification described herein include centrifugation, selective growth, and buffer exchange / concentration methods.

[0360] In some aspects, the present disclosure provides ADAS compositions and methods for comparing such compositions, wherein the compositions are substantially free of parental bacterial cells and / or live bacterial cells, e.g., having no more than 500, e.g., 400, 300, 200, 150, or 100 or less than 50, less than 25, less than 10, less than 5, less than 1, less than 0.1 colony forming units (CFU) / mL. In some embodiments, an ADAS composition that is substantially free of parental bacterial cells does not include bacterial cells.

[0361] Auxotrophic parental strains can be used to prepare the ADAS provided by the present invention. As described in more detail below, such manufacturing methods can be used to purify ADAS. For example, in some embodiments, after ADAS production, parental bacterial cells are removed by growing in a medium lacking nutrients (e.g., amino acids) necessary for parental bacterial survival. In some embodiments, the ADAS provided by the present invention is derived from at least 1, 2, 3, 4 or more auxotrophic parental strains of the following: arginine (e.g., argA knockout, such as strains JW2786-1 and NK5992), cysteine (cysE knockout, such as strains JW3582-2 and JM15), glutamine (e.g., glnA knockout, such as strains JW3841-1 and M5004), glycine (e.g., glyA knockout, such as strains JW2535-1 and AT2457), histidine (e.g., hisB knockout, such as strains JW2004-1 and SB3930), isoleucine (e.g., ilvA knockout, such as strains JW3745-2 and AB1255), leucine (e.g., leuB knockout, such as strains JW5807-2 and CV514), lysine (e.g., lysA knockout, such as strains JW2806-1 and KL334), methionine (e.g., metA knockout, such as strains JW3973-1 and DL41), phenylalanine (e.g., pheA knockout, such as strains JW2580-1 and KA197), proline (e.g., proA knockout, such as strains JW0233-2 and NK5525), serine (e.g., serA knockout, such as strains JW2880-1 and JC158), threonine (e.g., thrC knockout, such as strains JW0003-2 and Gif 41), tryptophan (e.g., trpC knockout, such as strains JW1254-2 and CAG18455), tyrosine (e.g., tyrA knockout, such as strains JW2581-1 and N3087), valine / isoleucine / leucine (e.g., ilvd knockout, such as strains JW5605-1 and CAG18431).

[0362] In certain embodiments, the method includes using one, two, three, or four auxotrophic parental strains, optionally wherein the parental strains further comprise a plasmid expressing ftsZ.

[0363] V. Method of Using ADAS

[0364] A. Method of Delivering ADAS

[0365] In one aspect, the present invention features a method for delivering an ADAS (e.g., a highly active ADAS) to a target cell, the method comprising (a) providing a composition comprising a plurality of any of the ADAS provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a). In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0366] In another aspect, the present invention features a method for delivering an ADAS to a target cell, the method comprising: (a) providing a composition comprising a plurality of any of the ADAS provided herein; and (b) contacting the target cell with the composition of step (a).

[0367] In some embodiments, the target cell is, for example, an animal cell, a plant cell, or a fungal cell.

[0368] B. Method of Delivering Cargo

[0369] In another aspect, the present invention features a method for delivering a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of any of the ADAS provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a). In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0370] In another aspect, the present invention features a method for delivering a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of any of the ADAS provided herein; and (b) contacting the target cell with the composition of step (a).

[0371] In another aspect, the present invention features a method for delivering a cargo (e.g., nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of any of the ADASs provided herein, wherein the ADAS is derived from a parental bacterium with reduced levels or activity of a cell division topology-specific factor, the ADAS comprises the cargo, and the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a).

[0372] In some embodiments, the target cells to which the cargo is to be delivered are, for example, animal cells, plant cells, or fungal cells.

[0373] In another aspect, the present disclosure features a method for delivering a therapeutic agent (e.g., a cancer therapeutic agent) to a subject, the method comprising administering to the subject an ADAS derived from a parental bacterial cell, or a composition comprising the ADAS, the parental bacterial cell comprising at least one loss-of-function alteration in a lytic enzyme and at least one loss-of-function alteration in a protease, wherein the ADAS has anti-cancer properties.

[0374] In another aspect, the present disclosure features a method for delivering a therapeutic agent (e.g., a cancer therapeutic agent) to a subject, the method comprising administering to the subject an ADAS derived from a parental bacterial cell, or a composition comprising the ADAS, the parental bacterial cell comprising a cell wall-embedded anchoring structure for displaying a cargo, wherein the ADAS has anti-cancer properties.

[0375] C. Method for regulating cell state

[0376] In one aspect, the present invention features a method for regulating the state of an animal cell,

[0377] the method comprising: (a) providing a composition comprising a plurality of any of the non-chromosomal dynamic activity systems (ADASs) provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal cell with the composition of step (a), thereby regulating the state of the animal cell. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0378] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADASs) provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant cell with the composition of step (a), thereby modulating the state of the plant cell. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0379] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADASs) provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby modulating the state of the insect cell. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0380] In another aspect, the present invention features a method of modulating the state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of any ADASs provided herein, wherein the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topology-specific factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal cell with the composition of step (a), thereby modulating the state of the animal cell.

[0381] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of any ADASs provided herein, wherein the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topology-specific factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant cell with the composition of step (a), thereby modulating the state of the plant cell.

[0382] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of any ADASs provided herein, wherein the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topology-specific factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby modulating the state of the insect cell.

[0383] In one aspect, the present invention features a method of modulating the state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADAS) provided herein; and (b) contacting the animal cell with the composition of step (a), thereby modulating the state of the animal cell.

[0384] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADAS) provided herein; and (b) contacting the plant cell with the composition of step (a), thereby modulating the state of the plant cell.

[0385] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADAS) provided herein; and (b) contacting the insect cell with the composition of step (a), thereby modulating the state of the insect cell.

[0386] In some embodiments, the modulation is any observable change in the state of a cell (e.g., an animal, plant, or insect cell) (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state) as measured using techniques and methods known in the art for such measurements (e.g., methods for measuring the levels or expression of proteins, transcripts, epigenetic markers, or measuring increases or decreases in biological pathway activity). In some embodiments, modulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway).

[0387] D. Methods of Treating Animals, Plants, Insects, or Fungi

[0388] In some aspects, the present invention features a method of treating an animal in need thereof, the method comprising (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADAS) provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal. In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0389] In some embodiments, the present invention features the use of any ADAS provided herein in the manufacture of a medicament for treating an animal, plant, insect, or fungus.

[0390] In some embodiments, the invention features the use of any of the ADASs provided herein in the manufacture of a medicament for treating an animal, wherein the composition is substantially free of live bacterial cells. In some embodiments, the initial ATP concentration of the ADAS is at least 1.25 mM.

[0391] In other aspects, the invention features a method of treating an animal in need thereof, the method comprising: (a) providing a composition comprising a plurality of any of the ADASs provided herein, wherein the ADAS is derived from a parental bacterium having a reduced level or activity of a cell division topoispecificity factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.

[0392] In some aspects, the invention features a method of treating an animal in need thereof, the method comprising (a) providing a composition comprising a plurality of any non-chromosomal dynamic activity systems (ADASs) provided herein; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.

[0393] In some embodiments, the animal in need of treatment has a disease, such as cancer. In some embodiments, the ADAS carries a chemotherapeutic cargo or an immunotherapeutic cargo.

[0394] In some embodiments, the cargo is an anti-cancer therapeutic agent. In some embodiments, the cargo embodies one or more wild-type or engineered antigens (or antibodies against antigens). In some embodiments, the antigen is derived from a tumor, e.g., a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the cargo is antigenic, tumor antigenic, or a protein, including p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, Claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A (preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12), MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor form BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, Survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, MC38 cancer epitope, or a combination thereof.In some embodiments, the expressed cargo is antigenic, tumor antigenic, or a protein, including CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, LewisY, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, or variants thereof.

[0395] In some embodiments, the cargo is an IL-2 peptide, IL-2-Ra, IL-15 peptide, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or fragments or subunits thereof. In one embodiment, the cargo is an IL-2 peptide, or a fragment or subunit thereof. In one embodiment, the cargo is an IL-15 peptide, or a fragment or subunit thereof. In some embodiments, the cargo is FOXP3. In some embodiments, the cargo is FOXO1. In some embodiments, the ADAS carries two or more cargos.

[0396] In some embodiments, the expressed cargo is antigenic, tumor antigenic, or a protein, including STING activators such as cyclic dinucleotides, cGAMP, c-di-AMP, IRF-3, and variants thereof.

[0397] In some embodiments, the cargo (e.g., an anti-cancer therapeutic agent or immunotherapy) is cisplatin, cyclophosphamide, 5-fluorouracil, bleomycin, an alkylating agent, an antimetabolite, an anthracycline, a topoisomerase inhibitor, a mitotic inhibitor, or a corticosteroid.

[0398] In some aspects, the invention features a method of treating a plant in need thereof, the method comprising (a) providing a composition comprising a plurality of any of the non-chromosomal dynamic activity systems (ADASs) provided herein, wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) to thereby treat the plant. In some embodiments, the initial ATP concentration of the ADAS is at least 1.25 mM.

[0399] In other aspects, the invention features a method of treating a plant in need thereof, the method comprising: (a) providing a composition comprising a plurality of any of the ADASs provided herein, wherein the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topological specificity factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) to thereby treat the plant.

[0400] In some aspects, the invention features a method of treating a plant in need thereof, the method comprising (a) providing a composition comprising a plurality of any of the non-chromosomal dynamic activity systems (ADASs) provided herein; and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) to thereby treat the plant.

[0401] In another aspect, the present invention provides methods for modulating a target cell. The target cell can be any cell, including animal cells (e.g., including human and non-human animals, including farm animals or livestock, pests), plant cells (including plant cells from crops or pests), fungal cells or bacterial cells. In some embodiments, the cells are isolated, e.g., in vitro, or in other embodiments, in vivo, within an organism. These methods involve providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention in proximity to the target cell. In some embodiments, the proximity to the target cell is direct, e.g., where the target cell is directly modulated by the ADAS, such as by secreting a certain agent in proximity to the target cell or injecting the agent into the target cell, or is indirect. Indirect modulation of the target cell can be effected by targeting different cells, e.g., by modulating cells adjacent to the target cell, which adjacent cells can be symbiotic with or pathogenic to the target cell. The adjacent cells can be in vitro or in vivo, just like the target cell - i.e., within an organism, where they can be symbiotic or pathogenic. These methods are collectively referred to as "methods of use provided by the present invention" and the like. In a related aspect, the present invention provides the target uses of the ADAS and composition provided by the present invention, which target uses are consistent with the methods of use provided by the present invention.

[0402] For example, in some embodiments, the present invention provides methods for modulating the state of an animal cell by providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention in proximity to the animal cell. In certain embodiments, the ADAS or composition is provided to be in proximity to the animal cell within an animal (such as a mammal, such as a human). In some embodiments, animal cells in a healthy animal are exposed to bacteria. In more specific embodiments, the animal cell is a lung epithelial cell, an immune cell, a skin cell, an oral epithelial cell, an intestinal epithelial cell, a reproductive tract epithelial cell, or a urinary tract cell. In still more specific embodiments, the animal cell is an intestinal epithelial cell, such as an intestinal epithelial cell from a human subject suffering from an inflammatory bowel disease (such as Crohn's disease or colitis). In yet more specific embodiments, the animal cell is an intestinal epithelial cell from a subject suffering from an inflammatory bowel disease, and the ADAS comprises a bacterial secretion system and a cargo comprising an anti-inflammatory agent, such as an antibody or antibody fragment targeting tumor necrosis factor (TNF) (e.g., an anti-TNF antibody); an antibody or antibody fragment targeting IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment targeting IL-23 (e.g., an anti-IL-23 antibody)).

[0403] In other embodiments, animal cells in a diseased state are exposed to bacteria. In certain embodiments, the animal cells are pathogenic, such as tumors. In other embodiments, animal cells in a diseased state, such as a wound, ulcer, tumor, or inflammatory disease, are exposed to bacteria.

[0404] In certain embodiments, the ADAS is derived from an animal symbiotic parental strain. In other embodiments, the ADAS is derived from an animal pathogenic parental strain.

[0405] In certain specific embodiments, the animal cells are contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and a cargo, wherein the cargo is delivered into the animal cells. In some specific embodiments, the animal cells are provided in proximity to an effective amount of an ADAS comprising a cargo and a secretion system, wherein the cargo is secreted extracellularly and contacts the animal cells.

[0406] In some embodiments, the state of the animal cells is regulated by providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention in proximity to bacterial or fungal cells near the animal cells. That is, these methods require indirectly regulating the state of the animal cells. In certain embodiments, the bacterial or fungal cells are pathogenic. In more specific embodiments, the fitness of the pathogenic bacterial or fungal cells is reduced. In certain other embodiments, the bacterial or fungal cells are symbiotic. In more specific embodiments, the fitness of the symbiotic bacterial or fungal cells is increased. In still more specific embodiments, the fitness of the symbiotic bacterial or fungal strain is increased by reducing the fitness of a large number of competing bacterial or fungal cells that may be neutral, symbiotic, or pathogenic.

[0407] In certain specific embodiments, the bacterial or fungal cells near the animal cells are contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and a cargo, wherein the cargo is delivered into the bacterial or fungal cells. In other specific embodiments, the bacterial or fungal cells near the animal cells are provided in proximity to an effective amount of an ADAS that secretes a cargo extracellularly, and the cargo contacts the bacterial or fungal cells.

[0408] In certain embodiments, the ADAS is derived from a parental strain that is a competitor of the bacterial or fungal cells. In other embodiments, the ADAS is derived from a parental strain that is a mutualistic bacterium of the bacterial or fungal cells.

[0409] As will be appreciated, the various methods of use for regulating the state of animal cells provided by the present invention can be readily adapted to corresponding methods for regulating the state of plant, fungal, or bacterial cells. For illustrative purposes, methods for regulating plant or fungal cells will be described more particularly.

[0410] Accordingly, in relevant aspects, the present invention provides a method of modulating the state of a plant or fungal cell by providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention to approach the following: a) a plant or fungal cell, b) an adjacent bacterium or adjacent fungal cell near the plant or fungal cell, or c) an invertebrate (e.g., an arthropod (e.g., an insect or an arachnid), a nematode, a protozoan, or an annelid) cell near the plant or fungal cell.

[0411] In certain embodiments, the ADAS is provided in situ, e.g., within a plant near plant cells such as crop plants, such as row crops including corn, wheat, soybeans, and rice, and vegetable crops including Solanaceae, such as tomatoes and peppers; Cucurbitaceae, such as melons and cucumbers; Brassicaceae, such as cabbages and broccoli; leafy greens, such as kale and lettuce; roots and tubers, such as potatoes and carrots; large-seeded vegetables, such as beans and corn; and mushrooms. In some embodiments, plant or fungal cells in healthy plants or fungi are exposed to bacteria. In other embodiments, plant or fungal cells in a diseased state are exposed to bacteria.

[0412] In certain embodiments, the plant or fungal cell divides, such as a meristematic cell, or is pathogenic, such as a tumor. In some embodiments, plant or fungal cells in a diseased state such as a wound are exposed to bacteria, or wherein the plant or fungal cell is not part of human food.

[0413] In certain embodiments, the ADAS is derived from a symbiotic parental strain. In other embodiments, the ADAS is derived from a plant- or fungal-pathogenic parental strain.

[0414] In some embodiments, the ADAS comprises a T3 / 4SS or T6SS and cargo, and the cargo is delivered into the plant or fungal cell. In other embodiments, the plant or fungal cell is provided to approach an effective amount of an ADAS comprising a bacterial secretion system and cargo, wherein the bacterial secretion system extracellularly secretes the cargo, thereby bringing the plant or fungal cell into contact with the cargo.

[0415] In some embodiments, these methods require providing an effective amount of an ADAS or composition to approach an adjacent bacterium or adjacent fungal cell near the plant or fungal cell. In more specific embodiments, the adjacent bacterium or adjacent fungal cell is pathogenic, optionally, wherein the fitness of the pathogenic adjacent bacterium or adjacent fungal cell is reduced. In other more specific embodiments, the adjacent bacterium or adjacent fungal cell is symbiotic, optionally, wherein the fitness of the symbiotic adjacent bacterium or adjacent fungal cell is increased. In still more specific embodiments, the fitness is increased by reducing competing bacteria or competing fungi that may be neutral, symbiotic, or pathogenic.

[0416] In some embodiments, the adjacent bacterial or adjacent fungal cells are contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and a cargo, wherein the cargo is delivered into the adjacent bacterial or adjacent fungal cells.

[0417] In other embodiments, the adjacent bacterial or adjacent fungal cells are provided in proximity to an effective amount of an ADAS comprising a bacterial secretion system and a cargo, wherein the bacterial secretion system extracellularly secretes the cargo such that the adjacent bacterial or adjacent fungal cells are contacted with the cargo.

[0418] In some embodiments, the ADAS is derived from a parental strain that is a competitor of the adjacent bacterial or adjacent fungal cells. In other embodiments, the ADAS is derived from a parental strain that is a mutualistic bacterium of the adjacent bacterial or adjacent fungal cells.

[0419] In certain embodiments, the method includes providing an effective amount of an ADAS or composition in proximity to invertebrate (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cells near a plant or fungus. In more specific embodiments, the invertebrate is pathogenic. In still more specific embodiments, the fitness of the pathogenic invertebrate cells is reduced. In yet more specific embodiments, the fitness of the pathogenic invertebrate cells is reduced by modulating symbionts in the invertebrate cells. In other specific embodiments, the invertebrate is symbiotic. In more specific embodiments, the fitness of the symbiotic invertebrate cells is increased. In still more specific embodiments, the fitness is increased by reducing competing bacteria or fungi that may be neutral, symbiotic, or pathogenic.

[0420] In yet another aspect, the present invention provides a method for removing one or more undesirable materials from an environment, the method comprising contacting the environment with an effective amount of an ADAS provided by the present invention or a composition provided by the present invention, wherein the ADAS comprises one or more molecules (such as proteins, polymers, nanoparticles, binders, or combinations thereof) that solubilize, chelate, or degrade one or more undesirable materials. "Environment" is defined as a non-cell target such as the ocean, soil, Superfund contaminated sites, skin, ponds, the intestinal lumen, and food in a container.

[0421] In certain embodiments, the undesired material includes heavy metals, such as mercury, and the ADAS comprises one or more molecules that bind heavy metals (such as proteins, polymers, nanoparticles, binders, or combinations thereof), such as MerR for mercury. In some embodiments, the undesired material includes plastics, such as PET, and the ADAS comprises one or more plastic-degrading enzymes, such as PETase. In certain embodiments, the undesired material includes one or more small organic molecules, and the ADAS comprises one or more enzymes capable of metabolizing the one or more small organic molecules.

[0422] E. RNA delivery method

[0423] In another aspect, the present invention provides a composition comprising the bacterium or ADAS provided by the present invention, wherein the bacterium or ADAS comprises a T4SS, an RNA-binding protein cargo, and an RNA cargo bound by the RNA-binding protein and adapted to be delivered to a target cell through the T4SS. In certain embodiments, the RNA-binding protein is Cas9 fused with VirE2 and VirF, the RNA cargo is a guide RNA, and optionally, the T4SS is the Ti system from Agrobacterium. In other embodiments, the RNA-binding protein is p19 from Carnation Italian Ringspot Virus fused with VirE2 or VirF, the RNA cargo is siRNA, and optionally, wherein the T4SS is the Ti system from Agrobacterium.

[0424] In a related aspect, the present invention provides a method for preparing these specific compositions, such methods requiring transfection of a plasmid containing Cas9 fused with VirE2 and VirF and an RNA cargo into Agrobacterium cells.

[0425] In another related aspect, the present invention provides a method for delivering RNA to a plant cell or an animal cell, the method comprising contacting the plant cell or the animal cell with a bacterium or ADAS, wherein the bacterium or ADAS comprises a T4SS, an RNA-binding protein cargo, and an RNA cargo, wherein the RNA is delivered to the plant cell or the animal cell. In more specific embodiments, the RNA-binding protein cargo is also delivered to the plant cell or the animal cell.

[0426] VI. Other embodiments

[0427] Some embodiments of the techniques described herein can be defined according to any one of the following numbered embodiments:

[0428] Example 1. A non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function alteration in a lytic enzyme and at least one loss-of-function alteration in a protease.

[0429] Example 2. The ADAS according to Example 1, wherein the loss-of-function alteration in the protease results in an increased expression of the cargo by the ADAS as compared to an ADAS derived from a parental bacterial cell that does not contain the alteration.

[0430] Example 3. The ADAS according to Example 1, wherein the loss-of-function alteration in the lytic enzyme results in an increased stability of the ADAS as compared to an ADAS derived from a parental bacterial cell that does not contain the alteration.

[0431] Example 4. The ADAS according to Example 1, wherein the loss-of-function alteration is a non-silent codon alteration, deletion, insertion, mutation, or any combination thereof.

[0432] Example 5. The ADAS according to Example 1, wherein the loss-of-function alteration is a deletion.

[0433] Example 6. The ADAS according to Example 1, wherein the lytic enzyme is an endopeptidase, a cell wall lytic enzyme, and / or an autolysin.

[0434] Example 7. The ADAS according to Example 6, wherein the lytic enzyme is selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0435] Example 8. The ADAS according to Example 6, wherein the loss-of-function alteration is in lytC.

[0436] Example 9. The ADAS according to Example 1, wherein the parental bacterial cell further comprises a loss-of-function alteration in a cell division topological specificity factor.

[0437] Example 10. The ADAS according to Example 9, wherein the cell division topological specificity factor is a genetic deletion or loss-of-function alteration of DivIVA, minC, minD, minE, minCD, or the minCDE operon.

[0438] Example 11. The ADAS according to Example 1, wherein the parental bacterial cell is Gram-positive.

[0439] Example 12. The ADAS as described in Example 1, wherein the parental bacterial cell is Gram-negative.

[0440] Example 13. The ADAS as described in Example 1, wherein the parental bacterial cell further comprises a loss-of-function genetic alteration that disrupts sporulation.

[0441] Example 14. The ADAS as described in Example 13, wherein the loss-of-function genetic alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, or a combination thereof.

[0442] Example 15. The ADAS as described in Example 13, wherein the loss-of-function genetic alteration in the sporulation gene is in SigF.

[0443] Example 16. The ADAS as described in Example 1, wherein the parental bacterium is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseovarius, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

[0444] Example 17. The ADAS as described in Example 11, wherein the parental bacterial cell is Bacillus subtilis.

[0445] Example 18. The ADAS as described in Example 1, wherein the loss-of-function genetic alteration in the protease is aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or a combination thereof.

[0446] Example 19. The ADAS as described in Example 1, wherein the parental cell contains loss-of-function genetic alterations in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine proteases.

[0447] Example 20. The ADAS as described in Example 19, wherein the loss-of-function genetic alteration is in sigF, divIVA, lytC, and is a protease deletion of aprE, ispA, wprA, nprB, Mpr, Vpr, Epr, Bpr, and nprE, or a combination thereof.

[0448] Example 21. The ADAS as described in any one of Examples 1-20, wherein the ADAS expresses one or more cargos.

[0449] Example 22. The ADAS as described in Example 21, wherein the ADAS expresses cytoplasmic cargos, surface-displayed cargos, secreted cargos, or a combination thereof.

[0450] Example 23. The ADAS as described in Example 22, wherein the cargo is an antigen, a cytokine, a mutant protein, a protein, a polypeptide, a small molecule, a macromolecule, a nucleic acid, a polynucleotide, an enzyme, or any combination thereof.

[0451] Example 24. A non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell containing a cell wall-embedded anchoring structure for displaying cargos.

[0452] Example 25. The ADAS as described in Example 24, wherein the parental bacterial cell further contains one or more loss-of-function genetic alterations in lytic enzymes.

[0453] Example 26. The ADAS as described in Example 25, wherein the parental bacterial cell further contains one or more loss-of-function genetic alterations in proteases.

[0454] Example 27. The ADAS as described in Example 25, wherein the lytic enzyme is selected from the group consisting of: lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0455] Example 28. The ADAS as described in Example 25, wherein the loss-of-function genetic alteration in the lytic enzyme is in lytC.

[0456] Example 29. The ADAS as described in Example 26, wherein the parental bacterial cell further comprises a loss-of-function alteration in a cell division topology-specific factor.

[0457] Example 30. The ADAS as described in Example 29, wherein the cell division topology-specific factor is DivIVA, minC, minD, minE, minCD, or the minCDE operon.

[0458] Example 31. The ADAS as described in Example 24, wherein the parental bacterial cell is Gram-positive.

[0459] Example 32. The ADAS as described in Example 24, wherein the parental bacterial cell is Gram-negative.

[0460] Example 33. The ADAS as described in Example 24, wherein the parental bacterial cell further comprises a loss-of-function genetic alteration that disrupts sporulation.

[0461] Example 34. The ADAS as described in Example 33, wherein the loss-of-function genetic alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of: sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, or combinations thereof.

[0462] Example 35. The ADAS as described in Example 33, wherein the loss-of-function alteration in the sporulation gene is in SigF.

[0463] Example 36. The ADAS as described in Example 24, wherein the parental bacterial cell is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Dickeya, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodoferax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

[0464] Example 37. The ADAS as described in Example 31, wherein the parental bacterial cell is Bacillus subtilis.

[0465] Example 38. The ADAS as described in any one of Examples 26 - 30, wherein the loss-of-genetic-function alteration in the protease gene is aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW or a combination thereof.

[0466] Example 39. The ADAS as described in Example 26, wherein the parental cell comprises loss-of-genetic-function alterations in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more proteases.

[0467] Example 40. The ADAS as described in Example 39, wherein the parental bacterial cell contains each of sigF, divIVA, and lytC, and a loss-of-function alteration in one or more of the protease genes aprE, ispA, wprA, nprE, epr, vpr, bpr, mpr, nprB, or a combination thereof.

[0468] Example 41. The ADAS as described in any one of Examples 24 - 40, wherein the ADAS expresses one or more of a cytoplasmic cargo, a surface-displayed cargo, a secreted cargo, or any combination thereof.

[0469] Example 42. The ADAS as described in Example 41, wherein the cargo is an antigen, a tumor antigen, a cytokine, a mutant protein, a protein, a polypeptide, a small molecule, a macromolecule, a nucleic acid, a polynucleotide, an enzyme, IL-2, an IL-2 superkine, or any combination thereof.

[0470] Example 43. The ADAS as described in Example 42, wherein the ADAS expresses the cargo via a constructed cell-wall-embedded anchoring structure.

[0471] Example 44. The ADAS as described in Example 43, wherein the constructed cell-wall-embedded anchoring structure is a membrane-anchoring domain, an outer-membrane-anchoring domain, an inner-membrane-anchoring domain, a cell-wall-anchoring domain, or a functional equivalent thereof.

[0472] Example 45. The ADAS as described in Example 43, wherein the constructed cell-wall-embedded anchoring structure is an antiparallel coiled coil.

[0473] Example 46. The ADAS as described in Example 43, wherein the constructed cell-wall-embedded anchoring structure has a COOH terminus embedded in the cell wall.

[0474] Example 47. The ADAS as described in Example 46, wherein the COOH terminus is included in a sorting signal that includes the COOH terminus and an LPXTG motif, a hydrophobic domain, a tail of mostly positively charged residues, or any combination thereof.

[0475] Example 48. The ADAS as described in Example 43, wherein the constructed cell-wall-embedded anchoring structure is a shortened antiparallel coiled coil, a linker, a coiled linker, or a fusion construct.

[0476] Example 49. The ADAS as described in any one of Examples 1 - 48, wherein the parental bacterial cell further contains at least one additional loss-of-function alteration in an enzyme.

[0477] Example 50. The ADAS as described in Example 49, wherein the parental bacterial cell comprises a loss-of-function alteration in one or more amylases.

[0478] Example 51. The ADAS as described in Example 50, wherein the amylase is amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, or a combination thereof.

[0479] Example 52. The ADAS as described in Example 51, wherein the parental bacterial cell comprises a loss-of-function alteration in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

[0480] Example 53. The ADAS as described in Example 49, wherein the parental bacterial cell further comprises a loss-of-function alteration in one or more lipases.

[0481] Example 54. The ADAS as described in Example 53, wherein the lipase is lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, or a combination thereof.

[0482] Example 55. The ADAS as described in Example 49, wherein the parental bacterial cell further comprises a loss of one or more cellulases.

[0483] Example 56. The ADAS as described in Example 55, wherein the cellulase is in celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof.

[0484] Example 57. The ADAS as described in Example 43, wherein the stability of the cargo is 2-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, or more than 300-fold the stability of the cargo in the control ADAS.

[0485] Example 58. The ADAS as described in Example 43, wherein the ADAS is administered at a dose of more than 1, more than 10, more than 100, more than 1000, or more than 2000 ADAS / cell.

[0486] Example 59. The ADAS as described in Example 43, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or more of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.

[0487] Example 60. The ADAS as described in any one of Examples 1-59, wherein the ADAS is delivered by subcutaneous, intraperitoneal, intravenous, intramuscular, oral, nebulized, or aerosolized administration.

[0488] Example 61. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 61.

[0489] Example 62. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 62.

[0490] Example 63. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 63.

[0491] Example 64. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 64.

[0492] Example 65. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 65.

[0493] Example 66. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 66.

[0494] Example 67. The ADAS as described in Example 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 67.

[0495] Example 68. The ADAS as described in any one of Examples 43-67, wherein the cell wall-embedded anchoring structure contains a cell wall-binding domain.

[0496] Example 69. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure contains a fusion promoter.

[0497] Example 70. The ADAS as described in Example 69, wherein the promoter is PrrnI-Pveg.

[0498] Example 71. The ADAS as described in Example 70, wherein the PrrnI-Pveg promoter is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 54.

[0499] Example 72. The ADAS as described in Example 43, wherein the ADAS contains a proline-rich sequence encoding the cell wall-embedded anchoring structure.

[0500] Example 73. The ADAS as described in Example 43, wherein the ADAS contains a self-binding helix for the cell wall-embedded anchoring structure.

[0501] Example 74. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure is an unstructured and proline-rich construct.

[0502] Example 75. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure is a structured construct.

[0503] Example 76. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure contains a promoter.

[0504] Example 77. The ADAS as described in Example 76, wherein the promoter is Pveg and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 55.

[0505] Example 78. The ADAS as described in Example 76, wherein the promoter is PaprE and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 56.

[0506] Example 79. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure sequence contains a ribosome binding site.

[0507] Example 80. The ADAS as described in Example 79, wherein the binding site is aprE-RBS and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 57.

[0508] Example 81. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure sequence contains a secretion signal.

[0509] Example 82. The ADAS as described in Example 81, wherein the secretion signal is aprE-SS and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 58.

[0510] Example 83. The ADAS as described in any one of Examples 43-67, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is interrupted by the nucleotide sequence encoding the cargo.

[0511] Example 84. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure uses a non-covalent cell-binding domain.

[0512] Example 85. The ADAS as described in Example 84, wherein the binding domain is LysM and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 68.

[0513] Example 86. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure uses a covalent cell-binding domain.

[0514] Example 87. The ADAS as described in Example 86, wherein the binding domain is CWAD and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 69.

[0515] Example 88. The ADAS as described in Example 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure includes a terminator sequence.

[0516] Example 89. The ADAS as described in Example 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is integrated into a deleted protease site.

[0517] Example 90. The ADAS as described in Example 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is integrated into a deleted amylase site.

[0518] Example 91. The ADAS as described in Example 43, wherein the cell wall-embedded anchoring structure is integrated into a deleted phosphodiesterase site.

[0519] Example 92. The ADAS as described in Example 72, wherein the proline-rich sequence is a linker having at least 25%, 27%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% proline.

[0520] Example 93. A method of treating or preventing cancer in a subject, the method comprising: administering to the subject one or more doses of the ADAS as described in any one of Examples 1-92 or a composition comprising the ADAS.

[0521] Example 94. The ADAS as described in Example 93, wherein the ADAS has anti-cancer properties.

[0522] Example 95. The method as described in Example 94, wherein the ADAS is administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly, rectally, intraperitoneally, intratumorally, by aerosolization, and / or subcutaneously or any combination thereof.

[0523] Example 96. The method as described in Example 95, wherein the ADAS is at least 1×10 5 、1×10 6 、1×10 7 、1×10 8, 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2×10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 doses of ADAS are administered to the subject.

[0524] Example 97. The method according to Example 94, wherein one, two, three, four, or more than four doses of ADAS are administered to the subject.

[0525] Example 98. The method according to Example 97, wherein one or two doses of the ADAS composition are administered to the subject.

[0526] Example 99. The ADAS according to Example 94, wherein the cargo comprises one or more tumor antigens.

[0527] Example 100. The ADAS according to Example 99, wherein the cargo further comprises a known anti-cancer cytokine.

[0528] Example 101. The ADAS according to Example 100, wherein the cytokine is IL-2 or an IL-2 mutant protein.

[0529] Example 102. The ADAS according to Example 101, wherein the ADAS cargo further comprises a pore-forming toxin.

[0530] Example 103. The ADAS according to Example 43, wherein the ADAS cargo comprises a pore-forming toxin and at least one other cargo component.

[0531] Example 104. The ADAS according to Example 102 or 103, wherein the toxin is listeriolysin O.

[0532] Example 105. The ADAS according to Example 43, wherein the constructed cell wall-embedded anchoring structure has an N-terminus embedded in the cell wall.

[0533] Example 106. The ADAS according to Example 43, wherein the anchoring structure contains an inner cell wall-embedded anchoring domain.

[0534] Example 107. The ADAS according to Example 43, wherein the cell wall-embedded anchoring structure comprises a protease cleavage site.

[0535] Example 108. The ADAS according to any one of Examples 1-43, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or more of SEQ ID NOs: 28-48.

[0536] Example

[0537] The present invention will now be described generally, and it will be more readily understood by reference to the following examples, which are for illustrative purposes only of certain aspects and embodiments of the invention and are not intended to limit the invention. It should be understood that various other embodiments can be practiced given the general description provided above.

[0538] Example 1. Generation of ADAS.

[0539] The generation of ADAS from parental bacterial cells can be achieved using the methods disclosed in International Patent Application Publication No. WO 2020 / 123569, which is incorporated herein by reference in its entirety. Briefly, these methods and their variants are described below. In this example, ADAS is generated by disrupting one or more genes involved in regulating the partitioning function of the parental cell (i.e., ΔminC, ΔminD, ΔminCDE, ΔminCdivIVA, or ΔdivIVA). This example details the genetic means of generating ADAS strains via disruption of the min operon or overexpression of the septum machinery component FtsZ.

[0540] A. Generation of ADAS via min mutations

[0541] To disrupt the min operon, the λ-RED recombineering method was used according to the protocol designed in Datsenko and Wanner, PNAS [Proceedings of the National Academy of Sciences of the United States of America], 97(12):6640-6645, 2000. Strains containing plasmids with the λ-RED system for engineering were obtained from the Coli Genetic Stock Center (CGSC) at Yale University. Briefly, primers were designed to non-polar delete the coding sequences of Escherichia coli minC, minD, or the complete minCDE operon by encoding approximately 40 genomic homology base pairs into the 5'-ends of the primers. The 3'-ends of these primers were homologous to the plasmids pKD3 and pKD4 of the λ-RED system, and these ends provided antibiotic markers for selecting the parental bacterial strains for the genetic target mutations. The sequences of the targeted deletions are provided in Table 2. According to the method of Datsenko and Wanner, PNAS [Proceedings of the National Academy of Sciences of the United States of America], 97(12):6640-6645, 2000, after standard PCR using the primers with pKD3 as the DNA template, the purified amplicons were transformed into bacteria prepared with pKD46 (a plasmid containing the λ-RED homologous recombination system derived from a phage) via electroporation. Transformants were selected on LB agar with 35 μg / mL chloramphenicol. These resulting colonies were confirmed to have the genetic disruption (i.e., ΔminC, ΔminD, or ΔminCDE) using standard allele-specific PCR.

[0542] B. Generation of ADAS via divIVA mutation

[0543] Briefly, primers were designed to delete the coding sequences of Bacillus subtilis divIVA or divIVA and minC. The sequences of the targeted deletions are listed in Table 2, including both the WT and divIVA deletions. Combining the sigF deletion (the sequence in Table 2) to interfere with sporulation, the divIVA deletion generated the parental strain MACH2347. The strain genotypes are provided in Table 1. Introducing the erm (erythromycin) cassette together with the selected deletion primers into the growing bacteria allowed the selection of transformed colonies by growth on LB-erm5 plates. Once the transformed bacteria were cultured, the colonies with the erm cassette were treated with a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. The transformed bacteria were selected by plating on LB-Spec plates and incubating at 30 °C. Then, the selected bacteria were streaked onto LB plates without antibiotics and incubated at the high temperature of 42 °C, resulting in the loss of the temperature-sensitive plasmid. The isolated colonies were confirmed to contain the expected deletions.

[0544] C. Generation of ADAS by overexpressing ftsZ

[0545] To create ADAS from an overexpressing septum machinery, a plasmid was constructed that drives the expression of the FtsZ Z-ring protein from wild-type Escherichia coli. Briefly, computational tools were used to de novo optimize the strong ribosome binding site and the coding sequence of the E. coli FtsZ protein from scratch. This translation unit was custom-made for de novo DNA synthesis from Integrated DNA Technologies (IDT TM ), and cloned into the backbone using standard cloning techniques. The resulting plasmid pFtsZ (Table 3) has a TetR repressor, a TetA promoter repressed by the TetR protein, a kanamycin resistance marker, and a pMB1 origin of replication. When transformed into a compatible bacterium, overproduction of FtsZ protein by pFtsz can be induced by adding anhydrous tetracycline to the culture. This protein can then form spontaneous protofilaments that cause asymmetric division of the parental bacterial cells, thus generating ADAS.

[0546] Example 2. Purification of ADAS from Bacillus subtilis

[0547] Purification of ADAS from parental bacterial cells can be achieved using the methods disclosed in International Patent Application Publication No. WO 2020 / 123569, which is incorporated herein by reference in its entirety. Briefly, these methods and their variants are described below.

[0548] In this example, a method for purifying a population of ADAS from a culture of an ADAS-producing bacterial parental strain is described. This method can be used to purify any ADAS-producing strain described herein, including the strains of Example 1 and Tables 1 and 3. Purification separates ADAS from live parental bacterial cells, which are larger and contain a genome. ADAS is purified from a high cell density culture of an ADAS-producing strain via a combination of 1) high-speed or low-speed centrifugation, 2) selective growth, and 3) buffer exchange / concentration. The centrifugation procedure is used to selectively remove live parental bacterial cells and large cell debris, while enriching ADAS in the mixed suspension. The selective growth procedure reduces the number of live parental bacterial cells present in the sample by adding a compound that is directly antimicrobial (i.e., toxic to cells with a microbial genome) and / or a compound that enhances the sedimentation of live cells via centrifugation. The buffer exchange / concentration procedure transfers ADAS from a larger volume of bacterial medium to a smaller volume of 1xPBS, while removing culture additives and cell debris.

[0549] A. ADAS Purification

[0550] An ADAS-producing strain was generated using the molecular cloning procedure described in Example 1 and then cultured to high cell density in a medium. The culture can be scaled up, for example, from 1 mL to 1000 mL or more of medium.

[0551] Transfer the culture to a centrifuge tube and perform a high - speed or low - speed centrifugation procedure aimed at precipitating intact cells and large cell debris, while keeping the ADAS in the supernatant. The centrifugation procedure is carried out at 4 °C or at room temperature. In some cases, a low - speed centrifugation procedure is used, which involves a procedure of sequentially spinning at 1,000×g, 2,000×g, 3,000×g and 4,000×g for 10 minutes each on a X14R benchtop centrifuge (Beckman Coulter) or an Eppendorf TM 5424R benchtop centrifuge (Fisher Scientific). In some cases, the low - speed centrifugation procedure consists of sequentially spinning at 2,000×g for 20 minutes at 4 °C, where the supernatant from the first spin is decanted into a sterile centrifuge bottle before the second spin. In some cases, the low - speed centrifugation procedure is a single spin at 4,000×g for 40 minutes in a Sorvall TM Lynx 6000 ultracentrifuge (Thermo Scientific TM ), where the rate of rotor acceleration is set to the lowest possible setting value. In some cases, a high - speed centrifugation procedure involving sequentially pulsing at 20,000×g is used, where rotation is stopped once the desired speed is reached and the supernatant is transferred to a new high - speed bottle before the next spin. In some cases, the high - speed centrifugation procedure includes spinning at 17,000×g for 30 minutes at 4 °C, after which the pellet is resuspended in growth medium.

[0552] After low - speed centrifugation, decant the culture supernatant into a sterile culture tube and perform a selective growth process. After high - speed centrifugation, decant the culture supernatant and resuspend the pellet, and perform a selective growth process. In some cases, a concentrated antibiotic solution (e.g., spectinomycin, clindamycin, tetracycline, ceftriaxone, kanamycin, carbenicillin, gentamicin and / or ciprofloxacin) or other concentrated chemical solution (e.g., sodium chloride, sodium hydroxide, M hydrochloric acid, glucose, casein amino acids and / or D - amino acids) is added directly to the culture supernatant. In other cases, the culture supernatant is pelleted by high - speed centrifugation at 10,000×g to 20,000×g for 5 to 60 minutes and the pellet is resuspended in fresh medium containing a concentrate of an antibiotic or other chemical solution that inhibits live cells. Selective growth is carried out by incubating the ADAS at 250 rpm with stirring at 4 °C to 42 °C for 1 to 3 hours. Then transfer the ADAS to a sterile centrifuge tube and perform another round of centrifugation.

[0553] After selective growth and centrifugation, a buffer exchange / concentration procedure is performed on the supernatant. In some cases, this is done by passing the supernatant through a 0.2 µm asymmetric polyethersulfone (aPES) membrane filter (Thermo Fisher), and then through 1 to 9 volumes of 1× PBS. In some cases, the ADAS is precipitated by centrifugation at 10,000×g to 20,000×g for 5 to 60 minutes, washed in 1 to 9 volumes of 1× PBS, reprecipitated, and resuspended in 1× PBS at a concentration 1 to 100,000 times the starting culture volume. In other cases, the ADAS is precipitated by sequential high-speed pulses at 16,000×g at 1-minute intervals, then spun at 20,000×g for 20 minutes at 4°C for an extended period, after which the precipitate is resuspended and then washed several times. In some cases, the wash includes spinning at 15,000×g for 5 minutes at 4°C.

[0554] B. Purification of ADAS from an auxotrophic ADAS-producing parental strain

[0555] An auxotrophic (i.e., unable to synthesize organic compounds required for growth) ADAS-producing parental strain can be used to produce ADAS. Such strains can only grow when organic compounds are provided. Thus, the auxotrophic parental strain can be selected by storing or incubating the ADAS preparation in a medium lacking organic compounds, providing an additional method for reducing the parental load in the ADAS preparation.

[0556] Example 3: Genomic deletions in ADAS

[0557] Using a method similar to Example 1B, primers were designed to delete the target coding sequence (e.g., delete the genomic sequence encoding a lyase or protease). The sequences targeted for deletion are listed in Table 2, and both the wild-type (WT) sequences and the sequences showing deletion of each target gene are included. For example, SEQ ID NO:4 shows the wild-type genomic region containing SigF, and SEQ ID NO:5 shows the genomic region after a loss-of-function deletion. SEQ ID NO:6 shows the wild-type genomic region containing divIVa, and SEQ ID NO:7 shows the genomic region after a loss-of-function deletion. SEQ ID NO:8 shows the wild-type genomic region containing lytC, and SEQ ID NO:9 shows the genomic region after a loss-of-function deletion. SEQ ID NO:10 shows the wild-type genomic region containing aprE, and SEQ ID NO:11 shows the genomic region after a loss-of-function deletion. SEQ ID NO:12 shows the wild-type genomic region containing ispA, and SEQ ID NO:13 shows the genomic region after a loss-of-function deletion. SEQ ID NO:14 shows the wild-type genomic region containing wprA, and SEQ ID NO:15 shows the genomic region after a loss-of-function deletion. SEQ ID NO:16 shows the wild-type genomic region containing nprE, and SEQ ID NO:17 shows the genomic region after a loss-of-function deletion. SEQ ID NO:18 shows the wild-type genomic region containing Epr, and SEQ ID NO:19 shows the genomic region after a loss-of-function deletion. SEQ ID NO:20 shows the wild-type genomic region containing Vpr, and SEQ ID NO:21 shows the genomic region after a loss-of-function deletion. SEQ ID NO:22 shows the wild-type genomic region containing Bpr, and SEQ ID NO:23 shows the genomic region after a loss-of-function deletion. SEQ ID NO:24 shows the wild-type genomic region containing Mpr, and SEQ ID NO:25 shows the genomic region after a loss-of-function deletion. SEQ ID NO:26 shows the wild-type genomic region containing nprB, and SEQ ID NO:27 shows the genomic region after a loss-of-function deletion.

[0558] The strain genotypes are provided in Table 1. Introduction of the erm (erythromycin) cassette together with the selected deletion primers into growing bacteria allowed selection of transformed colonies by growth on LB-erm5 plates. Once the transformed bacteria were cultured, colonies of the erm cassette were treated with a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. Transformed bacteria were selected by plating on LB-Spec plates and incubating at 30 °C. The selected bacteria were then streaked onto LB plates without antibiotics and incubated at the high temperature of 42 °C, resulting in loss of the temperature-sensitive plasmid. The isolated colonies were confirmed to contain the expected deletions, and if further genomic deletions were needed, a similar method was used. As listed in Tables 1-3, deletion combinations of the gene of interest were created in Bacillus subtilis, and additional deletions or insertions used to create these strains are listed in Table 1-5. The deletions listed should not be considered restrictive.

[0559] Example 4: Cargo expression

[0560] In this example, methods for optimizing cargo expression in ADAS and evidence of functional cargo expression are shown. Briefly, these methods and their variants are described below. The parental and cargo-modified strains are listed in Tables 1 and 3. The deletions and modifications listed should not be considered restrictive.

[0561] A. Expression of surface-displayed cargo

[0562] Parental Bacillus subtilis cells were genetically modified as described in Examples 1-3. MACH2762 was further modified to express the superagonist IL-2 (MACH2889), and the presence of this cargo on the bacterial surface was assessed by immunofluorescence microscopy. Briefly, an overnight culture of the bacteria was adjusted relative to the OD 600= 2 normalization and fixation in 4% formaldehyde for 30 minutes at room temperature. The fixed cells were washed twice with PBS and blocked with PBS containing 2% bovine serum albumin (BSA) for 30 minutes at room temperature with gentle stirring. The cells were then incubated in the primary antibody (rabbit anti-IL-2, 1:400 in 50 μL), followed by incubation in the secondary antibody (goat anti-rabbit Alexa-594, 1:400 in 50 μL). Between incubations, the cells were washed twice in PBS. The finally stained bacteria were resuspended in PBS, placed on a 2% agarose pad in PBS, and imaged on a Leica DMi8 Thunder inverted microscope equipped with a 63X phase objective lens. Fluorescent images were captured with a Leica DFC9000 camera and a rhodamine filter set, and the IL-2 signal per cell was quantified by dividing the background-subtracted fluorescent signal by the total cell area of 20 - 100 cells of each strain in multiple fields of view. Extensive IL-2 staining was observed on the surface of MACH2889, but not on MACH2762, indicating that the optimized cargo expression construct utilized in MACH2889 is sufficient for surface display of the superfactor IL-2. This superfactor IL-2 surface display module was introduced into unmodified Bacillus subtilis (MACH2887) and Bacillus subtilis lacking sigF and lytC (MACH2888), and neither of these two strains showed an increase in superfactor IL-2 staining relative to the MACH2762 negative control, indicating that the absence of one or more proteases is required for surface display of the superfactor IL-2 ( Figure 1 , Table 3).

[0563] B. Functionality of surface-displayed cargo

[0564] MACH2762 was further modified to express variants of human IL-2, including wild-type (WT), S1, and S2, on the cell surface via a unique construct of an anchoring structure embedded in the cell wall. The resulting ADAS were screened for their ability to stimulate the IL-2 receptor in vitro using the HEK-Blue IL-2 reporter cell line (Invivogen) exposed to different doses (number of ADAS per cell). Figure 2A)。The reporter cell line was engineered with a secreted alkaline phosphatase (SeAP) reporter gene, which is produced in proportion to IL-2 receptor activation and can be quantified by measuring absorbance at 620 nm according to the QuantiBlue assay protocol. ADASs without IL-2 display (MACH2762 and MACH2788) failed to activate the IL-2 receptor at any dose, while ADASs with IL-2 expression and surface display modules showed dose-dependent activation of the IL-2 receptor. Higher doses of the IL-2-displaying ADAS led to increased activation of the IL-2 receptor, up to an absorbance of approximately 1.25, at which point the assay reached saturation and the effect of increased IL-2 receptor activation was masked due to limited sensitivity. At ADAS doses that did not saturate the assay (620 nm absorbance < 1.25), equivalent doses of ADAS were observed to have variable ability to activate the IL-2 receptor, which was partially dependent on both the IL-2 variant and the display construct ( Figure 2B ). These data indicate that different cargos (e.g., IL-2 variants) and display constructs (0-6) allow for tunable target engagement through multiple potential mechanisms, including but not limited to modulating cargo accessibility or stability.

[0565] In vivo cytokine analysis via mouse plasma and ex vivo cytokine analysis of human peripheral blood mononuclear cell (PBMC) culture supernatants further demonstrated the functionality of surface-displayed IL-2 alone or in combination with additional immunomodulatory cargo to achieve synergistic effects. For plasma cytokine analysis, ADASs from Bacillus subtilis (MACH2762) or a similar parental strain were engineered to surface-display the superagonist IL-2 (MACH2782), cytoplasmically deliver cyclic dinucleotides (CDNs) cyclic di-AMP and cyclic di-GMP via the pore-forming activity of listeriolysin O (MACH2854), or a combination of these features (MACH2880). In vivo cytokine release assays were performed by collecting peripheral blood 2 and 6 hours after intravenous administration of 1E9 ADASs in 100 μL of phosphate-buffered saline (PBS) or an equal volume of PBS (to serve as a negative control). The drawn blood was processed to obtain plasma, diluted, and analyzed on a V-PLEX mouse cytokine 19-plex kit (Meso Scale Diagnostics) according to the manufacturer's protocol to calculate the plasma concentrations of the cytokines IFNγ, IL-2, IL-6, and TNFα ( Figures 3A - 3D)。The data indicate the in vivo systemic immune responses to surface and cytosolic ADAS cargoes, and combinations of the two. Surface display of IL-2 alone (MACH2782) resulted in the highest induction of murine IL-2, IL-6, and TNFα at 2 hours post-injection; however, the introduced CDN cargo and Listeria monocytogenes hemolysin O-mediated cytosolic delivery (MACH2880) induced the highest levels of IFNγ at 6 hours post-injection. For ex vivo cytokine analysis, PBMCs from healthy human donors were incubated with different doses of the above series of ADAS in cell culture for 24 hours, at which point the culture supernatants were harvested. Cytokines released into the supernatants were quantified via a Human XL Cytokine Luminex performance panel (Luminex Corporation) according to the manufacturer's protocol. At low effective doses of ADAS (e.g., 1 ADAS / PBMC), there was little difference between the cytokine profiles elicited by control ADAS (MACH2762) compared to surface-displayed superfactor IL-2 (MACH2782), cytosolic CDN delivery (MACH2854), or ADAS (MACH2880) deploying both immunomodulatory payloads. Overall, ADAS with surface-displayed superfactor IL-2 (MACH2782 and MACH2880) stimulated the highest anti-cancer cytokine levels( Figures 3E - 3G ), with the exception that the highest levels of TNFα were observed when MACH2854 was administered at a dose of 1,000 ADAS / PBMC( Figure 3H ). The data indicate human responses to surface and cytosolic ADAS cargoes, and combinations of the two. Overall, these data demonstrate that ADAS engineered with surface-displayed IL-2, cytosolic CDN delivery, or a combination of the two stimulated significantly higher anti-cancer cytokine levels in vivo and ex vivo compared to control ADAS or negative control treatments. Additionally, these complementary experimental systems provide evidence that the immune responses to modified ADAS are consistent between murine and human models, and that at least two different immunomodulatory cargoes can be deployed from a single ADAS.

[0566] To test whether ADAS can be used to trigger antigen-specific immune cell activation in vivo, we generated parental strains of Bacillus subtilis that produce ADAS modified with different forms of ovalbumin (a model protein antigen) and tested their ability to stimulate antigen-specific CD8 T cell responses in vivo. OT-1 mice (a transgenic mouse strain from The Jackson Laboratory with a CD8 T cell receptor engineered to recognize an immunogenic peptide within ovalbumin) were administered 50 μL of subcutaneous PBS, 100 μg of subcutaneous ovalbumin-containing PBS, or ADAS obtained from MACH2762 (control ADAS - 1×10 9 ADAS in 50 μL of PBS), MACH2670 (intracellular ovalbumin and listeriolysin - 1×10 9 ADAS in 50 μL of PBS), or MACH2829 (surface ovalbumin - 1E9 ADAS intravenously in 100 μL of PBS). Twenty-four hours later, the mice were euthanized and the spleens and draining lymph nodes of each mouse were harvested and pooled. The pooled organs were processed into single-cell suspensions, stained with a mixture of fluorescent antibodies targeting proteins on the surface of immune cells, subjected to a combinatorial fixation and permeabilization procedure, stained with a second set of fluorescent antibodies targeting proteins within immune cells, and analyzed by flow cytometry. When exposed to their target antigen, CD8 T cells produce additional granzyme B (a key protein important for the effector function of cytotoxic T lymphocytes). To measure the extent of antigen-specific CD8 T cell activation observed in our study, we first identified the CD8 T cell population by gating on live cells that were positive for CD45 and CD8. Next, we used a negative control group that had not been exposed to the antigen to establish a threshold for intracellular granzyme B. Finally, we applied this gating strategy to all study groups ( Figure 4 ). We observed a significant increase in the percentage of granzyme B-positive CD8 T cells in animals administered ovalbumin (positive control, MACH2670, or MACH2829), indicating that ADAS modified with cytoplasmic ovalbumin and listeriolysin or ADAS displaying ovalbumin from the cell surface was sufficient to stimulate a high level of antigen-specific CD8 T cell activation 24 hours after a single administration. In animals administered the control ADAS, we did not observe an increase in the percentage of granzyme B-positive cells. Overall, these data indicate that ADAS modified with heterologous protein antigens can activate antigen-specific CD8 T cells in vivo via multiple antigen expression strategies and multiple administration routes.

[0567] Example 5: Cancer Therapeutic Agent

[0568] 500,000 MC38 cells were implanted into the flanks of mice and allowed to grow for 9 days, at which time the tumor volume was measured with calipers. The mice were randomly divided into approximately 10 treatment groups such that the average tumor volume per group was approximately 100 cubic millimeters. On day 10, the mice received an injection of one of four test articles: 200 μg of intraperitoneal anti-PD-1, 50 μL of peritumoral PBS, 50 μL of ADAS obtained from MACH2854 (carrying LLO, DTT-MC38, and cyclic di-AMP cargo) in PBS at a concentration of 4 × 10 9 peritumoral, or 100 μL of ADAS obtained from MACH2862 (carrying LLO, surface FLAG-MC38, cytoplasmic DTT-MC38, and surface superfactor hIL-2 cargo) in PBS at a concentration of 6 × 10 8 intravenous. On day 14 after implantation, the tumor volume was measured again ( Figure 5 ). When compared by t-test, the tumors of animals treated with anti-PD-1 (positive control) were significantly smaller than those treated with PBS (negative control). The tumors of animals treated with ADAS were significantly smaller than those of the PBS group, indicating the functional efficacy of the ADAS expressing the cargo as a potential therapeutic agent via multiple immunostimulatory strategies and multiple routes of administration. The parental strains and cargo-modified strains are listed in Tables 1 and 3.

[0569] Table 1. Strains

[0570]

[0571] Table 2. Deleted sequences

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620] Table 3. Cargo expression constructs

[0621]

[0622]

[0623] Table 4. Additional integration loci

[0624]

[0625]

[0626]

[0627] Table 5. Sequence elements

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637] Although the foregoing invention has been described in detail for purposes of clarity of understanding by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby incorporated by reference in their entireties. Other embodiments are within the claims.

Claims

1. A non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function alteration in a lytic enzyme and at least one loss-of-function alteration in a protease.

2. The ADAS of claim 1, wherein the loss-of-function alteration in the protease results in increased expression of cargo by the ADAS relative to an ADAS derived from a parental bacterial cell that does not contain the alteration.

3. The ADAS of claim 1, wherein the loss-of-function alteration in the lytic enzyme results in increased stability of the ADAS relative to an ADAS derived from a parental bacterial cell that does not contain the alteration.

4. The ADAS of claim 1, wherein the loss-of-function alteration is a non-silent codon change, deletion, insertion, mutation, or any combination thereof.

5. The ADAS of claim 1, wherein the loss-of-function alteration is a deletion.

6. The ADAS of claim 1, wherein the lytic enzyme is an endopeptidase, cell wall lytic enzyme, and / or autolysin.

7. The ADAS of claim 6, wherein the lytic enzyme is selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

8. The ADAS of claim 6, wherein the loss-of-function alteration is in lytC.

9. The ADAS of claim 1, wherein the parental bacterial cell further comprises a loss-of-function alteration in a cell division topological specificity factor.

10. The ADAS of claim 9, wherein the cell division topological specificity factor is a genetic deletion or loss-of-function alteration of DivIVA, minC, minD, minE, minCD, or the minCDE operon.

11. The ADAS of claim 1, wherein the parental bacterial cell is Gram-positive.

12. The ADAS of claim 1, wherein the parental bacterial cell is Gram-negative.

13. The ADAS of claim 1, wherein the parental bacterial cell further comprises a loss-of-function alteration that disrupts sporulation.

14. The ADAS of claim 13, wherein the loss-of-function alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, or combinations thereof.

15. The ADAS of claim 13, wherein the loss-of-function alteration in the sporulation gene is in SigF.

16. The ADAS according to claim 1, wherein the parental bacterium is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

17. The ADAS according to claim 11, wherein the parental bacterial cell is Bacillus subtilis.

18. The ADAS according to claim 1, wherein the loss-of-genetic-function alteration in the protease is aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or a combination thereof.

19. The ADAS according to claim 1, wherein the parental cell comprises loss-of-genetic-function alterations in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine proteases.

20. The ADAS according to claim 19, wherein the loss-of-genetic-function alteration is in sigF, divIVA, lytC, and is a protease deletion of aprE, ispA, wprA, nprB, Mpr, Vpr, Epr, Bpr, and nprE, or a combination thereof.

21. The ADAS according to any one of claims 1-20, wherein the ADAS expresses one or more cargos.

22. The ADAS according to claim 21, wherein the ADAS expresses cytoplasmic cargos, surface-displayed cargos, secreted cargos, or a combination thereof.

23. The ADAS according to claim 22, wherein the cargo is an antigen, a cytokine, a mutant protein, a protein, a polypeptide, a small molecule, a macromolecule, a nucleic acid, a polynucleotide, an enzyme, or any combination thereof.

24. A non-chromosomal Dynamic Activity System (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising a cell wall-embedded anchoring structure for displaying a cargo.

25. The ADAS according to claim 24, wherein the parental bacterial cell further comprises one or more loss-of-function genetic alterations in a lytic enzyme.

26. The ADAS according to claim 25, wherein the parental bacterial cell further comprises one or more loss-of-function genetic alterations in a protease.

27. The ADAS according to claim 25, wherein the lytic enzyme is selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

28. The ADAS according to claim 25, wherein the loss-of-function genetic alteration in the lytic enzyme is in lytC.

29. The ADAS according to claim 26, wherein the parental bacterial cell further comprises a loss-of-function alteration in a cell division topological specificity factor.

30. The ADAS according to claim 29, wherein the cell division topological specificity factor is DivIVA, minC, minD, minE, minCD, or the minCDE operon.

31. The ADAS according to claim 24, wherein the parental bacterial cell is Gram-positive.

32. The ADAS according to claim 24, wherein the parental bacterial cell is Gram-negative.

33. The ADAS according to claim 24, wherein the parental bacterial cell further comprises a loss-of-function genetic alteration that disrupts sporulation.

34. The ADAS according to claim 33, wherein the loss-of-function genetic alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof.

35. The ADAS according to claim 33, wherein the loss-of-function alteration in the sporulation gene is in SigF.

36. The ADAS according to claim 24, wherein the parental bacterial cell is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phormidium, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

37. The ADAS according to claim 31, wherein the parental bacterial cell is Bacillus subtilis.

38. The ADAS according to any one of claims 26 - 30, wherein the loss-of-function genetic alteration in the protease gene is aprE, ispA, wprA, nprE, nprB, Mpr, Vpr, Epr, Bpr, HtrA, AprX, MlpA, Map, Pcp, PepT, AmpS, LonA / B, ClpE, tesA, degS, ompT, clpP, ptrA, lon, ctpB, mamE, ibpA, prc, spa, lasA, sepA, espE, hslV, htpX, iga, tri, ravZ, gluP, rasP, htrB, gpr, aprx, prsW, clpX, prsW, or a combination thereof.

39. The ADAS according to claim 26, wherein the parental cell comprises loss-of-function genetic alterations in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or more proteases.

40. The ADAS according to claim 39, wherein the parental bacterial cell contains each of sigF, divIVA, and lytC, and a loss-of-function alteration in one or more of the protease genes aprE, ispA, wprA, nprE, epr, vpr, bpr, mpr, nprB, or a combination thereof.

41. The ADAS according to any one of claims 24-40, wherein the ADAS expresses one or more of a cytoplasmic cargo, a surface-displayed cargo, a secreted cargo, or any combination thereof.

42. The ADAS according to claim 41, wherein the cargo is an antigen, a tumor antigen, a cytokine, a mutant protein, a protein, a polypeptide, a small molecule, a macromolecule, a nucleic acid, a polynucleotide, an enzyme, IL-2, an IL-2 superagonist, or any combination thereof.

43. The ADAS according to claim 42, wherein the ADAS expresses the cargo via a constructed cell wall-embedded anchoring structure.

44. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is a membrane-anchoring domain, an outer membrane-anchoring domain, an inner membrane-anchoring domain, a cell wall-anchoring domain, or a functional equivalent thereof.

45. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is an antiparallel coiled coil.

46. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure has a COOH terminus embedded in the cell wall.

47. The ADAS according to claim 46, wherein the COOH terminus is included in a sorting signal that comprises the COOH terminus and an LPXTG motif, a hydrophobic domain, a tail of mostly positively charged residues, or any combination thereof.

48. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is a shortened antiparallel coiled coil, a linker, a coiled linker, or a fusion construct.

49. The ADAS according to any one of claims 1-48, wherein the parental bacterial cell further contains at least one additional loss-of-function alteration in an enzyme.

50. The ADAS according to claim 49, wherein the parental bacterial cell contains a loss-of-function alteration in one or more amylases.

51. The ADAS according to claim 50, wherein the amylase is amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, or a combination thereof.

52. The ADAS according to claim 51, wherein the parental bacterial cell contains a loss-of-function alteration in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

53. The ADAS according to claim 49, wherein the parental bacterial cell further comprises a loss-of-function alteration in one or more lipases.

54. The ADAS according to claim 53, wherein the lipase is lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, or a combination thereof.

55. The ADAS according to claim 49, wherein the parental bacterial cell further comprises a loss of genetic function in one or more cellulases.

56. The ADAS according to claim 55, wherein the cellulase is among celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof.

57. The ADAS according to claim 43, wherein the stability of the cargo is 2-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, or more than 300-fold the stability of the cargo in the control ADAS.

58. The ADAS according to claim 43, wherein the ADAS is administered at a dose of more than 1, more than 10, more than 100, more than 1000, or more than 2000 ADAS / cell.

59. The ADAS according to claim 43, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or more of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.

60. The ADAS according to any one of claims 1-59, wherein the ADAS is delivered by subcutaneous, intraperitoneal, intravenous, intramuscular, oral, aerosolized, or nebulized administration.

61. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

61.

62. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

62.

63. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

63.

64. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

64.

65. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

65.

66. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

66.

67. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

67.

68. The ADAS according to any one of claims 43-67, wherein the cell wall-embedded anchoring structure contains a cell wall-binding domain.

69. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure contains a fusion promoter.

70. The ADAS according to claim 69, wherein the promoter is PrrnI-Pveg.

71. The ADAS according to claim 70, wherein the PrrnI-Pveg promoter is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

54.

72. The ADAS according to claim 43, wherein the ADAS contains a proline-rich sequence encoding the cell wall-embedded anchoring structure.

73. The ADAS according to claim 43, wherein the ADAS contains a self-binding helix for the cell wall-embedded anchoring structure.

74. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure is an unstructured and proline-rich construct.

75. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure is a structured construct.

76. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure contains a promoter.

77. The ADAS according to claim 76, wherein the promoter is Pveg and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

55.

78. The ADAS according to claim 76, wherein the promoter is PaprE and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

56.

79. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure sequence contains a ribosome binding site.

80. The ADAS according to claim 79, wherein the binding site is aprE-RBS and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

57.

81. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure sequence contains a secretion signal.

82. The ADAS according to claim 81, wherein the secretion signal is aprE-SS and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

58.

83. The ADAS according to any one of claims 43-67, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is interrupted by the nucleotide sequence encoding the cargo.

84. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure uses a non-covalent cell-binding domain.

85. The ADAS according to claim 84, wherein the binding domain is LysM and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

68.

86. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure uses a covalent cell-binding domain.

87. The ADAS according to claim 86, wherein the binding domain is CWAD and is encoded by a nucleotide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:

69.

88. The ADAS according to claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure includes a terminator sequence.

89. The ADAS according to claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is integrated into a deleted protease site.

90. The ADAS according to claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchoring structure is integrated into the deleted amylase locus.

91. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure is integrated into the deleted phosphodiesterase locus.

92. The ADAS according to claim 72, wherein the proline-rich sequence is a linker having at least 25%, 27%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% proline.

93. A method for treating or preventing cancer in a subject, the method comprising: administering to the subject one or more doses of the ADAS according to any one of claims 1-92 or a composition comprising the ADAS.

94. The ADAS according to claim 93, wherein the ADAS has anti-cancer properties.

95. The method according to claim 94, wherein the ADAS is administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly, rectally, intraperitoneally, intratumorally, by aerosolization, and / or subcutaneously or any combination thereof.

96. The method according to claim 95, wherein the ADAS is administered to the subject at a dose of at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2×10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 ADAS.

97. The method according to claim 94, wherein one, two, three, four, or more than four doses of the ADAS are administered to the subject.

98. The method according to claim 97, wherein one or two doses of the ADAS composition are administered to the subject.

99. The ADAS according to claim 94, wherein the cargo comprises one or more tumor antigens.

100. The ADAS according to claim 99, wherein the cargo further comprises a known anti-cancer cytokine.

101. The ADAS according to claim 100, wherein the cytokine is IL-2 or an IL-2 mutant protein.

102. The ADAS according to claim 101, wherein the ADAS cargo further comprises a pore-forming toxin.

103. The ADAS according to claim 43, wherein the ADAS cargo comprises a pore-forming toxin and at least one other cargo component.

104. The ADAS according to claim 102 or 103, wherein the toxin is listeriolysin O.

105. The ADAS according to claim 43, wherein the constructed cell wall-embedded anchoring structure has an N-terminus embedded in the cell wall.

106. The ADAS according to claim 43, wherein the anchoring structure contains an inner cell wall-embedded anchoring domain.

107. The ADAS according to claim 43, wherein the cell wall-embedded anchoring structure comprises a protease cleavage site.

108. The ADAS according to any one of claims 1-43, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or more of SEQ ID NO: 28-48.

109. A method of delivering a cancer therapeutic agent to a subject, the method comprising administering to the subject an ADAS derived from a parental bacterial cell, or a composition comprising the ADAS, the parental bacterial cell comprising at least one loss-of-function alteration in a lytic enzyme and at least one loss-of-function alteration in a protease, wherein the ADAS has anti-cancer properties.

110. The method of claim 109, wherein the ADAS comprises a cargo.

111. A method of delivering a cancer therapeutic agent to a subject, the method comprising administering to the subject an ADAS derived from a parental bacterial cell, or a composition comprising the ADAS, the parental bacterial cell comprising a cell wall-embedded anchor structure for displaying a cargo, wherein the ADAS has anti-cancer properties.

112. The method of claim 109 or 111, wherein the ADAS is administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly, rectally, intraperitoneally, intratumorally, aerosolized, and / or subcutaneously or any combination thereof.

113. The method according to claim 112, wherein the ADAS is administered to the subject at a dose of at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2×10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 ADAS.

114. The method of claim 113, wherein one, two, three, four, or more than four doses of the ADAS are administered to the subject.

115. The method of claim 114, wherein one or two doses of the ADAS composition are administered to the subject.

116. The ADAS of claim 109 or 111, wherein the cargo comprises one or more tumor antigens.

117. The ADAS of claim 109 or 111, wherein the cargo comprises a known anti-cancer cytokine.

118. The ADAS of claim 117, wherein the cytokine is IL-2 or an IL-2 mutant protein.

119. The ADAS of claim 118, wherein the ADAS cargo further comprises a pore-forming toxin.

120. The ADAS of claim 109 or 111, wherein the ADAS cargo comprises a pore-forming toxin and at least one other cargo component.

121. The ADAS of claim 119 or 120, wherein the toxin is listeriolysin O.

122. The ADAS of claim 111, wherein the cell wall-embedded anchor structure of the construct has an N-terminus embedded in the cell wall.

123. The ADAS of claim 111, wherein the anchor structure contains an inner cell wall-embedded anchor domain.

124. The ADAS of claim 111, wherein the cell wall-embedded anchor structure comprises a protease cleavage site.

125. The ADAS of any one of claims 109-124, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or more of SEQ ID NOs: 28-48.

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