Optimized non-chromosomal dynamic activation system and uses thereof

Abstract

Disclosed herein is a non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell that contains at least one loss-of-function genetic modification in a lytic enzyme to increase the stability of the ADAS and at least one gene loss-of-function in a protease to improve cargo expression, with or without an additional engineered cell wall-embedded anchor structure to optimize cargo presentation.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. Said XML copy, created on November 15, 2023, is named 51296-058WO2_Sequence_Listing_11_15_23, and is 187,929 bytes in size.

[0002] Provided herein are non-chromosomal dynamic activity systems and methods for making and using same. [Background technology]

[0003] There is a need for delivery vectors capable of targeting cells and delivering biological agents, as well as compositions containing such delivery vectors and related methods for delivering the vectors to cells and thereby modulating 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 activation systems (ADAS)) that have improved cytosolic and surface cargo expression. Summary of the Invention

[0004] In some aspects, the disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one loss-of-function genetic modification in a lytic enzyme and at least one loss-of-function genetic modification in a protease. In some aspects, the disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one lytic enzyme genomic deletion and at least one protease deletion. In some embodiments, the loss-of-function genetic modification in the lytic enzyme results in increased stability of the ADAS compared to an unmodified ADAS. In some embodiments, the loss-of-function genetic modification in the protease results in increased cargo expression in the ADAS compared to an unmodified ADAS. In some aspects, the disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one loss-of-function modification in a lytic enzyme and a cell wall-embedded anchor structure constructed to display cargo. In some aspects, the disclosure features a non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell that includes at least one lytic enzyme genomic deletion to stabilize the ADAS and an engineered cell wall-embedded anchor structure for displaying cargo.

[0005] In some embodiments, the parent bacterial cell comprises at least one loss-of-function modification 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 loss-of-function genetic modifications in a protease, hi some embodiments, the parent 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.

[0006] In some embodiments, the parent bacterial cell is modified to reduce enzymatic activity.

[0007] In some embodiments, the loss-of-function or genomic deletion in the parent bacterial cell reduces enzymatic and / or lytic activity, hi some embodiments, the parent 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 autolytic enzyme.

[0008] In some embodiments, the parent cell comprises a loss-of-function genetic modification or genomic deletion in a gene from the group consisting of lytC(cwlB), lytF(cwlE), lytE(cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0009] In some embodiments, the parental cell comprises a loss-of-function genetic modification or deletion that disrupts sporulation in a gene from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, and combinations thereof.

[0010] In some embodiments, the parent cell comprises a loss-of-function or genomic deletion of a gene for an enzyme from the group consisting of proteases, amylases, lipases, cellulases, and combinations thereof.

[0011] In some embodiments, the parent bacterial cell comprises at least one loss-of-function genetic modification in a protease. In some embodiments, the parent bacterial cell comprises at least one protease deletion. In some embodiments, the loss-of-function modification 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.

[0012] In some embodiments, the parent bacterial cell comprises at least one loss-of-function genetic modification in an amylase. In some embodiments, the parent bacterial cell comprises at least one amylase deletion. In some embodiments, the loss-of-function modification or genomic deletion is in amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, or a combination thereof.

[0013] In some embodiments, the parent bacterial cell comprises at least one loss-of-function genetic modification in a lipase. In some embodiments, the parent bacterial cell comprises at least one lipase deletion. In some embodiments, the loss-of-function modification or genomic deletion is in lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, or a combination thereof.

[0014] In some embodiments, the parent bacterial cell comprises at least one loss-of-function genetic modification in a cellulase. In some embodiments, the parent bacterial cell comprises at least one cellulase deletion. In some embodiments, the loss-of-function modification or genomic deletion is in celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof.

[0015] In some embodiments, the parent bacterial cell comprises at least one loss-of-function modification in a gene affecting a lytic enzyme, a protease, an amylase, a lipase, a cellulase, a sporulation mechanism, or a combination thereof.

[0016] In some embodiments, the parent bacterial cell comprises at least one genomic deletion of a region encoding a lytic enzyme, a protease, an amylase, a lipase, a cellulase, a gene affecting the sporulation mechanism, or a combination thereof.

[0017] In some embodiments, the parent bacterial cell is a Gram-positive bacterial cell.

[0018] In some embodiments, the parent bacterial cell is Bacillus subtilis or a Bacillus species.

[0019] In some embodiments, the parent bacterial cell is a Lactobacillus species.

[0020] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0021] In some embodiments, the parent bacterial cell is selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobacter, and the like. Rhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfota lea), Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter,Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga motoga), Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc The bacterium is derived from the genus Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0022] In some embodiments, the parental cell comprises a gene deletion of 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).

[0023] In some embodiments, the parent bacterial cell contains a loss-of-function genetic modification in a cell division topology specificity factor. In some embodiments, the parent bacterial cell contains a genomic deletion of the cell division topology specificity factor. In some embodiments, the genomic deletion is in the DivIVA, minC, minD, minE, minCD, or minCDE operon.

[0024] In some embodiments, the ADAS further comprises at least one cargo.

[0025] In some embodiments, the cargo is a protein or polypeptide, hi some embodiments, the cargo is a small molecule, a large molecule, a nucleic acid, a polynucleotide, or an enzyme.

[0026] In some embodiments, the ADAS or the parent bacterial cell is modified to increase the level of cargo in the ADAS.

[0027] In some embodiments, the cargo is an enzyme, a DNA modifying agent, a chromatin remodeling agent, a gene editing agent, a nuclear targeting agent, a binding agent, an immunogenic agent, a toxin, a mutein, a cytokine, a superkine, or any combination thereof. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editing agent is a component of a CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binding agent is an antibody or an antibody fragment. In some embodiments, the binding agent is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulatory agent. 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 conjugated to a stabilizing agent. In some embodiments, the mutein is a mutant protein of a known antigen, enzyme, cytokine, or immunogen. In some embodiments, the toxin is an agent that induces autophagy (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 delivery of the ADAS cargo to the target cell. In some embodiments, multiple ADAS cargoes are expressed.

[0028] In some embodiments, the cargo is IL-2 or superkine IL-2. In some embodiments, the surface cargo is presented via an engineered cell wall-embedded anchor structure.

[0029] In some embodiments, the cell wall-embedded anchor construct comprises antiparallel coiled-coils (APCCs) of various lengths. In some embodiments, the cell wall-embedded anchor construct comprises a shortened antiparallel coiled-coil. 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.

[0030] In some embodiments, the cell wall-embedded anchor construct contains a cell wall binding domain.

[0031] In some embodiments, the ADAS comprises a fusion promoter.

[0032] In some embodiments, the ADAS contains a proline-rich sequence that encodes a cell wall-embedded anchor structure.

[0033] In some embodiments, the ADAS encodes a self-binding coil for a cell wall-embedded anchor structure.

[0034] In some embodiments, the cell wall-embedded anchor structure is an unstructured, proline-rich construct.

[0035] In some embodiments, the cell wall-embedded anchor structure is a structured construct.

[0036] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0037] [Figure 1]Figure 1 is a bar graph showing the intensity of immunofluorescent staining of IL-2 (cargo) on MACH2762, MACH2887, MACH2888, and MACH2889 cells (defined in Tables 1-3), quantified using a microscope and normalized for cell size. Higher levels of surface-displayed cargo expression were observed in MACH2889 compared to all other strains. MACH2762 parental cells contain deletions to disrupt sporulation, lytic activity, and protease activity. MACH2889 parental cells contained the same deletion and an expression cassette to express surface IL-2. MACH2888 parental cells contained the same IL-2 cargo but without the protease deletion. MACH2887 contained the same IL-2 cargo without the protease, lytic enzyme, or sporulation deletion. As evidenced through the lack of IL-2 signaling in all but MACH2889, one or more protease deletions are required for the cargo to be expressed and surface displayed. [Figure 2A] Figure 2A is a graph showing the amount (ADAS per cell) relative to absorbance at 620 nm of HEK-Blue IL-2 cell culture supernatants exposed to multiple B. subtilis strains featuring various IL-2 surface display constructs. This effectively determines the ratio of ADAS per cell to IL-2R activation and screens the functionality and stability of various IL-2 and IL-2 mutein constructs. MACH2762, which has no display or cargo, and MACH2788, which has display but no cargo, were used as control strains. MACH2777, MACH2780, MACH2782, MACH2783, MACH2786, MACH2787, MACH2790, and MACH2791 are described in Table 3. [Figure 2B] Figure 2B is a series of schematic diagrams showing exemplary cargo display structures using various cell wall-embedded anchor constructs and a table listing the display structures and specific cargos for each strain shown in Figure 2A. IL-2 is shown as an exemplary cargo. [Figure 3A]Figure 3A is a bar graph showing systemic measurements of IFNg in mouse plasma 2 and 6 hours after intravenous administration of PBS, ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytosolic cargo), and ADAS derived from MACH2880 (with surface and cytosolic cargo). [Figure 3B] Figure 3B is a bar graph showing systemic measurements of IL-2 in mouse plasma 2 and 6 hours after intravenous administration of PBS, MACH2762-derived ADAS (no cargo), MACH2782-derived ADAS (with surface cargo), MACH2854-derived ADAS (with cytosolic cargo), and MACH2880-derived ADAS (with surface and cytosolic cargo). [Figure 3C] Figure 3C is a bar graph showing systemic measurements of IL-6 in mouse plasma 2 and 6 hours after intravenous administration of PBS, MACH2762-derived ADAS (no cargo), MACH2782-derived ADAS (with surface cargo), MACH2854-derived ADAS (with cytosolic cargo), and MACH2880-derived ADAS (with surface and cytosolic cargo). [Figure 3D] Figure 3D is a bar graph showing systemic measurements of TNFα in mouse plasma 2 and 6 hours after intravenous administration of PBS, MACH2762-derived ADAS (no cargo), MACH2782-derived ADAS (with surface cargo), MACH2854-derived ADAS (with cytosolic cargo), and MACH2880-derived ADAS (with surface and cytosolic cargo). [Figure 3E] Figure 3E is a bar graph showing dose-response measurements of IFNg secreted from human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytosolic cargo), and ADAS derived from MACH2880 (with surface and cytosolic cargo). [Figure 3F]Figure 3F is a bar graph showing dose-response measurements of secreted IL-2 from human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytosolic cargo), and ADAS derived from MACH2880 (with surface and cytosolic cargo). [Figure 3G] Figure 3G is a bar graph showing dose-response measurements of secreted IL-6 from human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytosolic cargo), and ADAS derived from MACH2880 (with surface and cytosolic cargo). [Figure 3H] Figure 3H is a bar graph showing dose-response measurements of TNFα secreted from human PBMCs 24 hours after exposure to ADAS derived from MACH2762 (no cargo), ADAS derived from MACH2782 (with surface cargo), ADAS derived from MACH2854 (with cytosolic cargo), and ADAS derived from MACH2880 (with surface and cytosolic cargo). [Figure 4] Figure 4 is a bar graph showing the level of antigen-specific CD8 T cell activation in granzyme B (GzmB)-positive live CD45+, CD8+ T cells 24 hours after administration of the indicated ADAS, as measured by flow cytometry. Measurements were performed in OT-1 mice with CD8 T cell receptors engineered to recognize ovalbumin (ova). ADAS were produced from B. subtilis strains administered subcutaneously or intravenously without cargo (MACH2762), with surface display of ovalbumin protein (MACH2829), or with cytosolic expression of ovalbumin protein and listeriolysin O (MACH2670). Strain details are provided in Tables 1-3. [Figure 5]Figure 5 is a graph showing MC38 tumor volume in mice after administration of PBS (peritumoral), anti-PD-1 (intraperitoneal), or ADAS (MACH2854-peritumoral; or MACH2862-intravenous) at 2 weeks and 10 days after tumor implantation. Strains are detailed in Tables 1-3. DETAILED DESCRIPTION OF THE INVENTION

[0038] I. Definition As used herein, the term "non-chromosomal 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 a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulatory agent, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)). In some embodiments, the ADAS is a minicell or modified minicell derived from a parent bacterial cell (e.g., a Gram-negative or Gram-positive bacterial cell). In other aspects, the ADAS is obtained from the parent cell by modifying the parent cell such that the genome is removed, and is substantially similar in size to the parent cell. In embodiments, the ADAS may be obtained from the parent bacterium using any suitable method, e.g., genetic manipulation of the parent cell or exposure to a culture medium or condition that increases the likelihood of bacterial minicell formation. An exemplary method for generating an ADAS is by disrupting the cell division machinery of the parent cell. In some embodiments, the ADAS comprises one or more endogenous or heterologous features of the parent cell surface, e.g., a cell wall, cell wall modifications, flagella or pili, and / or one or more endogenous or heterologous features of the interior volume of the parent cell, e.g., a nucleic acid, a plasmid, a protein, a small molecule, transcription machinery, or translation machinery. In other embodiments, the ADAS lacks one or more features of the parent cell. In yet other embodiments, the ADAS is loaded with or otherwise modified with features not present in the parent cell.

[0039] As used herein, the term "highly active ADAS" refers to an ADAS with a high potential for function, e.g., an ADAS capable of performing a large number of useful functions. In some embodiments, the function is a metabolic function, 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., internalization, externalization, or secretion, e.g., secretion by a bacterial secretion system (e.g., T3SS)) under suitable conditions. In certain embodiments, a highly active ADAS starts with a large pool of energy, e.g., energy in the form of ATP. In other embodiments, the ADAS has the ability to uptake or generate energy / ATP from another source. In some embodiments, a highly active ADAS is identified by, for example, having an increased ATP concentration, an increased capacity to generate ATP, an increased capacity to produce protein, an increased rate or amount of protein production, and / or an increased responsiveness to a biological signal, e.g., induction of a promoter.

[0040] As used herein, the term "parent bacterial cell" refers to a cell (e.g., a Gram-negative or Gram-positive bacterial cell) from which the ADAS is derived. The parent bacterial cell is typically a viable bacterial cell. The term "viable 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, Azoarcus, Azospirillum, Azotobacter, Bartonella The genera Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas chloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella ella), Magnetospirillum genus, Mesorhizobium genus, Methylobacterium genus, Methylococcus genus, Neisseria genus, Nitrosomonas genus, Nostoc genus, Photobacterium genus, Photorhabdus genus,Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium , Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas as), Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus Bacteria of the genus Acetobacterium acillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0041] An ADAS composition or preparation that is "substantially free" of parental and / or viable bacterial cells is defined herein as a composition that has 500 or fewer colony forming units (CFU) per mL, e.g., 400, 300, 200, 150, 100, or fewer. In some embodiments, an ADAS composition that is substantially free of parental or viable bacterial cells includes having fewer than 50 CFU / mL, fewer than 25 CFU / mL, fewer than 10 CFU / mL, fewer than 5 CFU / mL, fewer than 1 CFU / mL, fewer than 0.1 CFU / mL, or fewer than 0.001 CFU / mL, including no bacterial cells at all.

[0042] The term "cell division topology specificity factor" refers to a component of the cell division machinery in a bacterial species that is involved in determining the site of septum and functions by restricting the location of other components of the cell division machinery, for example, by restricting the location of one or more Z-ring inhibitory proteins. An exemplary cell division topology specificity factor includes minE, which was first discovered in Escherichia coli (E. coli) and has since been identified in a wide range of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). minE functions by restricting the Z-ring inhibitory proteins minC and minD to the poles of the cell. A second exemplary cell division topology specificity factor is DivIVA, which was first discovered in Bacillus subtilis (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005).

[0043] The term "Z-ring inhibitory protein" refers to a component of the cell division machinery in bacterial species that is involved in determining the site of septation and functions by inhibiting the formation of a stable FtsZ ring or by tethering such components to the membrane. In some embodiments, the location of the Z-ring inhibitory protein is regulated by cell division topology specificity factors, such as minE and DivIVA. Exemplary Z-ring inhibitory proteins include minC and minD, which were first discovered in E. coli and have since been identified in a wide range of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). In E. coli and other species, minC, minD, and minE reside at the same locus, which may be referred to as the "min operon," the minCDE operon, or the min or minCDE locus.

[0044] As used herein, the term "reduction in the level or activity of a cell topology-specific factor" refers to any overall 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 cell topology-specific factor (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) as detected by standard methods compared to the level in a reference sample (e.g., an ADAS produced from a wild-type cell or a cell 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, a decrease in level or activity refers to a decrease in level or activity in a sample that is at least about 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the level or activity of the cell topology specific factor in a reference sample, reference cell, control sample, or control cell.

[0045] As used herein, the term "percent identity" refers to the percent (%) sequence identity relative to a reference polynucleotide or polypeptide sequence after alignment by standard techniques. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences under comparison. For example, in some embodiments, percent sequence identity values ​​are generated using the sequence comparison computer program BLAST. Illustratively, the percent sequence identity of a given nucleic acid or amino acid sequence A to, or relative to, a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity relative to, or relative to, a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(ratio X / Y) where X is the number of nucleotides or amino acids scored as a perfect match by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and where Y is the total number of nucleotides or amino acids in B. In some embodiments, the sequence identity, e.g., sequence identity of a homologue of a MinE or DivIVA protein, will be at least about 40%, 50%, 60%, 70%, 80%, 85%, 90% or even 95% or more 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 more amino acid or nucleic acid identity, to a native sequence MinE (or minE) or DivIVA (or divIVA) sequence as disclosed herein.

[0046] The phrase "modulating the state of a cell," as used herein, refers to an observable change in the state (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state) of a cell (e.g., an animal, plant, or insect cell) as measured using techniques and methods known in the art for such measurements, e.g., methods that measure the level or expression of a protein, transcript, epigenetic mark, or methods that measure an increase or decrease in the activity of a biological pathway. In some embodiments, modulating the state of a cell results in a change of at least 1% compared to before administration (e.g., 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 compared to before administration; e.g., up to 100% compared to before administration). In some embodiments, modulating the state of a cell involves increasing a cellular parameter (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In some embodiments, increasing the state of the cell results in an increase in the parameter by at least 1% compared to before administration (e.g., 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 compared to before administration; e.g., up to 100% compared to before administration). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., level or expression of a protein, transcript, or activity of a biological pathway). In some embodiments, the reduction in the state of the cells results in a decrease in the parameter by at least 1% compared to before administration (e.g., 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 compared to before administration; e.g., up to 100% compared to before administration).

[0047] As used herein, the term "endogenous type 3 secretion system" or "endogenous T3SS" refers to a T3SS that is present in a cell (e.g., a parent cell), or an ADAS derived therefrom, and that is naturally encoded by the cell (e.g., a wild-type version encoded by the cell). The T3SS may be expressed by an endogenous gene in the cell and / or may be encoded and expressed by a synthetic construct in the cell. The expression or abundance of an endogenous T3SS may be increased, for example, by the addition of a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by the reduction or removal of a negative regulator of T3SS expression.

[0048] As used herein, the term "heterologous type 3 secretion system" or "heterologous T3SS" refers to a T3SS that is present in a cell (e.g., a parent cell) or an ADAS derived therefrom and that 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 no T3SS at all. In some embodiments, the T3SS is expressed in the cell by a synthetic construct.

[0049] As used herein, an "endogenous effector" of a secretion apparatus (e.g., a T3SS, T4SS, or T6SS) is a portion (e.g., a protein or polypeptide) that is naturally encoded by the cell from which the secretion apparatus (e.g., a T3SS) is derived (e.g., encoded by a wild-type version of the cell) and that is capable of being secreted by the secretion apparatus. The secretion apparatus and one or more of its endogenous effectors may be expressed in the cell in which they naturally occur, or may be heterologously expressed, e.g., expressed by a cell that does not naturally encode the endogenous effector or the secretion apparatus.

[0050] As used herein, an effector that is heterologous to a secretion apparatus ("heterologous effector") is a moiety (e.g., a protein or polypeptide) that is not naturally encoded (e.g., not encoded by the wild-type version of) the cell from which the secretion apparatus (e.g., T3SS) is derived, and that is capable of being secreted by the secretion apparatus of the cell from which the heterologous effector is derived. In some embodiments, the effector is capable of being secreted by, or is modified to be secreted by, the secretion apparatus heterologous to it. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0051] As used herein, the term "heterologous" means not native to a cell or composition in its naturally occurring state. In some embodiments, "heterologous" refers to a molecule that is not naturally found in the ADAS or parent bacterium (e.g., a gram-negative or gram-positive bacterial cell) in which it is made; for example, a cargo or payload (e.g., a nucleic acid or small molecule such as an RNA or tRNA encoding a polypeptide, protein) or a structure (e.g., a plasmid or gene editing system).

[0052] As used herein, the terms "phase-light ADAS" and "phase-light parent cell" refer to the body of an ADAS or parent cell that appears lighter or ghosted in an image (e.g., a photomicrograph taken using a light microscope) and exhibits disintegration and lysis compared to the darker ADAS or parent cell, described herein as an "intact ADAS" and an "intact parent cell," respectively (e.g., refers to a dead ADAS or dead parent cell).

[0053] As used herein, "increased stability" of an ADAS refers to an overall increase in the integrity of the ADAS. The stability of an ADAS (e.g., the stability of a particular population, strain, or variety of ADAS) can be measured as the proportion of "intact" ADAS (e.g., the percentage of intact ADAS) compared to "phase-lite" ADAS in one or more representative images of a plurality of ADAS. For example, increased stability of an ADAS derived from a modified strain containing at least one lytic enzyme deletion can be defined as the increased percentage of "intact" ADAS in a representative image of the ADAS derived from the modified strain compared to ADAS derived from a control strain (e.g., a strain without a lytic enzyme deletion) measured (e.g., scored) under the same conditions and time points. For example, if the modified ADAS has a stability greater than 40%, then more than 40% of the visible ADAS is intact rather than phase-lite. In some embodiments, ADAS stability is measured as a unitless ratio between the half-life of ADAS from an unmodified strain (e.g., a strain not containing a lytic enzyme deletion) and the half-life of ADAS from a modified strain (e.g., a strain containing a lytic enzyme deletion), measured under the same environmental conditions. In some embodiments, the fold change in optical density at wavelength 600 (OD600) of ADAS from a modified strain compared to ADAS from a control strain at one or more time points (e.g., 0, 10, 20, 23, 24, 48, 72, or more than 72 hours after ADAS enrichment) is another measure of ADAS stability. ADAS stability is modified (e.g., increased) by the genomic deletion of at least one lytic enzyme from the parental cell. In certain embodiments, an ADAS with one or more lytic enzyme deficiencies (e.g., derived from a parent cell containing one or more lytic enzyme deficiencies) has greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% greater stability, as measured by percent intact ADAS, compared to an ADAS without a lytic enzyme deficiency (e.g., derived from a parent cell that does not contain a lytic enzyme deficiency).

[0054] As used herein, "increased stability" or "optimization" of a cargo of an ADAS refers to an overall increase in the expression, presentation, function, or durability of the cargo of an ADAS compared to a control or unmodified ADAS. In some embodiments, cargo stability is measured as a unitless ratio between the half-life of the unmodified cargo and the half-life of the modified cargo, measured under the same environmental conditions. The modified cargo may be, for example, a cargo expressed by a display structure provided herein. In some aspects, cargo stability is measured as the fold change in the level of cargo expression between a modified ADAS (e.g., an ADAS derived from a modified parent cell, e.g., a parent cell modified to contain a display structure) and a control ADAS, measured under the same environmental conditions. The stability and / or optimization of cargo expression may be modified by genomic deletion of at least one of both a lytic enzyme and a protease in the parent 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 parent cell. In certain embodiments, the stability of the cargo of an ADAS derived from a parent 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 greater than the stability of the cargo of a control ADAS (e.g., an ADAS derived from a parent cell that does not contain a lytic enzyme deletion or a protease deletion). In some embodiments, stability or optimization of cargo expression is measured through cargo functionality, for example, by measuring or detecting levels of cytokine release, receptor activation, or antigen-specific cell activation.

[0055] As used herein, "lytic enzyme" refers to an enzyme (e.g., an endopeptidase, cell wall lytic enzyme, or autolytic enzyme) that mediates the breakdown of the parent bacterial cell wall and parent bacterial cell membrane. Genomic deletion of one or more of these enzymes reduces the lytic activity of the parent cell and, in some embodiments, can increase the stability and integrity of the ADAS structure, allowing for the production of more intact ADAS and / or the survival of intact ADAS for a longer period of time. Specific genomic deletions that reduce the lytic activity of the parent cell include, but are not limited to, lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, and combinations thereof.

[0056] As used herein, "sporulation" refers to the process in certain parental bacterial strains in which spores form from cells under adverse conditions. Gene deletions that can disrupt sporulation include, but are not limited to, sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, and combinations thereof.

[0057] As used herein, "protease" refers to any enzyme that degrades proteins. Genetic loss-of-function mutations (e.g., deletions) of one or more of these enzymes reduce the protease activity of the parent cell. Specific genomic deletions or loss-of-function mutations that may be contained by the parent cell of the invention 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, the parent cell of the present invention contains one or more additional enzyme deletions or loss-of-function mutations in addition to lytic enzymes and proteases. Examples of other enzymes that can be deleted or disrupted by loss-of-function mutations include, but are not limited to, amylase, lipase, and cellulase. As used herein, "amylase" refers to an enzyme that breaks down starch, polysaccharides, and complex carbohydrates into sugars. As used herein, "lipase" refers to an enzyme that is part of a group of hydrolases known to break down fats and hydrolyze glycerol esters. Examples of bacterial genes encoding amylases, lipases, and cellulases 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.

[0058] As used herein, the terms "engineered cell-wall-embedded anchor structure," "cell-wall anchor construct," and "cell-wall-embedded anchor construct" refer to a cell-wall scaffold or other functional equivalent that can provide a fixed structure for displaying surface cargo. The engineered cell-wall-embedded anchor construct can be expressed in the ADAS and / or its parent cell. Examples of possible engineered cell-wall-embedded anchor structures include, but are not limited to, antiparallel coiled-coils, cell-wall-embedded or outer membrane anchor domains with one or more linkers, either without a linker or with one or more linkers (e.g., mCherry), and cell-wall-embedded or outer membrane anchor domains without a linker. In some embodiments, (a) the cargo is tethered to the cell-wall-embedded anchor structure using a linker (e.g., a polypeptide linker), or (b) the cargo is tethered directly to the cell-wall-embedded anchor structure. In some embodiments, the cargo and one or more components of the cell-wall-embedded anchor structure are comprised by the same polypeptide chain.

[0059] As used herein, "presentation structure" or "cargo display structure" refers to any means by which a cargo is expressed and / or displayed by an ADAS (e.g., displayed on the surface or in the cytosol of the ADAS). Several surface display structures comprising a cargo and one or more components involved in displaying the cargo (e.g., a binding domain, a linker, and / or a scaffold) are shown in FIG. 2B, although these display structures should not be considered limiting. Presentation 0 corresponds to a fusion comprising a cargo displayed on a cell-wall-embedded binding domain without a linker (e.g., no linker domain connecting the cargo to the binding domain) (e.g., a fusion polypeptide comprising a cargo and a cell-wall-embedded binding domain). Presentation 1 corresponds to a cargo displayed via a cell-wall-embedded binding domain with a linker (e.g., a fusion polypeptide comprising a cargo and a cell-wall-embedded binding domain connected by a linker). Exhibit 2 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via one or more antiparallel coiled-coils (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain tethered by one or more antiparallel coiled-coils). Exhibit 3 is a construct using a structure similar to Exhibit 2, but with a shortened antiparallel coiled-coil. Exhibit 4 is a construct using a coiled linker that is tethered to the cell wall via a binding domain (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain tethered by a coiled linker). Exhibits 5 and 6 comprise cargo displayed via a linker fused to a scaffold protein (e.g., mCherry) (e.g., a fusion polypeptide comprising a cargo, scaffold protein, linker, and a cell wall-embedded binding domain). Exhibit 6 further comprises a linker tethering the cargo to the scaffold protein. In some embodiments, the presentation structure is cytosolic rather than through surface expression, e.g., the cargo is presented by one or more components (e.g., binding domains, linkers, and / or scaffolds) inside the ADAS (cytosol).The presentation structure can be "unstructured," which refers to a relatively high proportion of prolines in the structure, giving the construct a non-linear or curved shape, or "structured," such as coiled, which has a specific or more defined shape. Cargo expression can also be affected by deletions or loss-of-function mutations of various proteases and lytic enzymes, as well as by different promoters, integration sites, and cell-binding domains.

[0060] As used herein, "gene loss of function" refers to the substantial reduction or complete elimination of a protein (e.g., substantial reduction in protein function, complete loss of protein function, substantial reduction in protein expression, or complete loss of protein expression) caused by modification (e.g., genetic mutation) of a gene encoding the protein. Mutations include, but are not limited to, insertion or deletion of one or more nucleotides, non-silent codon changes, and duplications. Proteins in which gene loss of function may exist include, but are not limited to, enzymes such as lytic enzymes, proteases, amylases, lipases, or cellulases. In addition to gene loss of function through mutation, promoter inactivation or chemical inhibition can affect (e.g., reduce or eliminate) expression of a protein (e.g., lytic enzymes, proteases, amylases, lipases, or cellulases).

[0061] "Treatment" and "treating," as used herein, refer to the medical management of a subject with the intent to ameliorate, ameliorate, stabilize, prevent, or cure a disease, disorder, or condition. This term includes active treatment (treatment directed to ameliorate the disease, disorder, or condition); causal treatment (treatment directed at the cause of the associated disease, disorder, or condition); symptomatic treatment (treatment designed to alleviate the symptoms of the disease, disorder, or condition); preventative treatment (treatment directed to minimize or partially or completely inhibit the onset of the associated disease, disorder, or condition); and supportive treatment (treatment used to complement other therapies).

[0062] The term "cancer," as used herein, refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. The cancer may be locally advanced or metastatic, for example, stage I, stage II, stage III, or stage IV cancer.

[0063] II. Composition A. ADAS and Highly Active ADAS The present invention is based, at least in part, on Applicant's discovery of non-chromosomal dynamic activation systems (ADAS), including highly active ADASs that can provide broad functionality in multiple 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 do not intersect) 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 (RNPs)). In some embodiments, the ADAS is a minicell or modified minicell derived from a parent bacterial cell (e.g., a Gram-negative or Gram-positive bacterial cell). In some aspects, the ADAS is obtained from the parent bacterium using any suitable method, such as genetic manipulation of the parent cell or exposure to a culture medium or condition that increases the likelihood of bacterial minicell formation.

[0064] In some embodiments, the ADAS has a major axis cross section of between about 100 nm and 500 μm (e.g., in certain embodiments, about: 100-600 nm, e.g., 100-400 nm; or between about 0.5-10 μm and 10-500 μm). In certain embodiments, the ADAS has a minor axis cross section of 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, or up to 100% of the major axis. In certain embodiments, the ADAS has a minor axis cross section of about: 0.001-1 μm 3 , 0.3 to 5 μm 3 , 5 to 4000 μm 3 or 4000~50×107 μm 3 In some embodiments, the ADAS is substantially similar in size to the parent cell, e.g., has a size (e.g., internal volume, major axis cross-section and / or minor axis cross-section) that 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 parent cell, has the same size as the parent cell, or has a size that is about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of the size of the parent cell.

[0065] In some embodiments, the present invention provides a highly active ADAS. A "highly active" ADAS is an ADAS with a high potential for function, e.g., an ADAS capable of performing a large number of useful functions. In some embodiments, a function is defined as a metabolic function, including, for example, 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., internalization, extracellular transport, or secretion) under suitable conditions. In some embodiments, a highly active ADAS starts with a large pool of energy, e.g., in the form of adenosine triphosphate (ATP). In other embodiments, the ADAS has the ability to uptake or generate energy (e.g., ATP) from another source.

[0066] The term "ADAS provided by the present invention" encompasses all embodiments of the ADAS described herein, and in certain embodiments, includes a highly active ADAS, and a set of highly active ADASs is referred to as "highly active ADASs provided by the present invention," which are a subset of the ADASs provided by the present invention.

[0067] In one aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic activation systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1 mM, and wherein the composition is substantially free of viable bacterial cells.

[0068] In another aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic activation systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 3 mM, and wherein the composition is substantially free of viable bacterial cells.

[0069] In some embodiments, highly active ADAS has an initial ATP concentration of 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. ATP concentration can be assessed by various means, including, in certain embodiments, the BacTiter-Glo™ assay (Promega) on dissolved ADAS.

[0070] In some embodiments, high activity may also or alternatively be determined as the rate or amount of increase in ATP concentration in the ADAS over time. In some embodiments, 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% after incubation under suitable conditions, for example, after 12 hours of incubation at 37° C. In certain embodiments, a highly active ADAS has an ATP concentration of 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 have a higher ATP production rate 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 2 weeks.

[0071] In other aspects, high activity is determined as the rate of decrease in ATP concentration over time. In some embodiments, the ATP concentration of a highly active ADAS does not decrease as rapidly as an ADAS that is not highly active. In some embodiments, the decrease in ATP concentration in the ADAS or ADAS composition 24 hours after preparation is 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), as measured, for example, using the BacTiter-Glo™ Assay (Promega).

[0072] In some embodiments, high activity is additionally or alternatively determined as the longevity index of the ADAS. The longevity index is calculated as the ratio of GFP production rate at 24 hours to 30 minutes. In some embodiments, highly active ADAS have a longevity index 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 more. In more specific embodiments, the longevity index is measured in ADAS containing a functional GFP plasmid with a species-appropriate promoter, where GFP concentration is measured by a plate reader at 30 minutes and 24 hours relative to the number of ADAS, the average number of plasmids per ADAS, and the solution volume.

[0073] In some embodiments, the ADAS produces a protein, e.g., a heterologous protein. In some embodiments, high activity is determined as the rate, amount, or duration of protein production, or the rate at which expression of the protein is induced (e.g., the responsiveness of the ADAS to a signal). For example, in some embodiments, the ADAS comprises a plasmid comprising an inducible promoter and a nucleotide sequence encoding a heterologous protein, wherein contacting the ADAS with an inducer of the inducible promoter under appropriate conditions results in production of the heterologous protein. In some embodiments, production of the heterologous protein is increased by at least 1.6-fold in an ADAS contacted with an inducer, e.g., a highly active ADAS, compared to an ADAS not contacted with the inducer. For example, in some embodiments, 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 an ADAS contacted with an inducer, e.g., a highly active ADAS. In some embodiments, the rate of heterologous protein production by the highly active ADAS reaches a target level within a specific period of time, e.g., 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or more than 3 hours, after contacting the ADAS with an inducer. In some embodiments, the protein (e.g., heterologous protein) is produced at a rate of at least 0.1 femtograms per hour per highly active 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 per ADAS. In some embodiments, high activity of the ADAS is determined as the period during which the protein is produced. In some embodiments, the highly active ADAS produces a protein (e.g., a heterologous protein) for a period 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.

[0074] B. ADAS derived from parent bacteria lacking cell division topology specificity factors and highly active ADAS In some embodiments, the ADAS is derived from a bacterial parent cell, as described herein.

[0075] In some aspects, the present invention provides compositions comprising an ADAS and / or multiple ADASs derived from a parent bacterium that has a reduced level, activity or expression of a cell division topology specificity factor.

[0076] In some embodiments, the invention provides a composition comprising a plurality of ADAS, wherein the ADAS do not comprise a cell division topology specificity factor, and wherein the composition is substantially free of viable bacterial cells.

[0077] In some aspects, the invention provides a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells, produced by a process comprising: (a) producing, providing, or obtaining a plurality of parent bacteria having a reduced level or activity of a cell division topology specificity factor; (b) exposing the parent bacteria to conditions that allow for the formation of minicells, thereby producing a highly active ADAS; and (c) separating the ADAS from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.

[0078] In some embodiments of the above aspects, the cell division topology specificity factor is a polypeptide having an amino acid sequence at least 20% identical to an E. coli minE polypeptide (e.g., encoded by SEQ ID NO: 1), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the polypeptide encoded by SEQ ID NO: 1. In some embodiments, the cell division topology specificity factor comprises the amino acid sequence encoded by SEQ ID NO: 1. In some embodiments, the cell division topology specificity factor is a minE polypeptide. Exemplary species having minE polypeptides are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005.

[0079] In some embodiments, the parent bacterium is E. coli and the minE polypeptide is E. coli minE. In other embodiments, the parent bacterium is Salmonella typhimurium and the minE polypeptide is S. typhimurium minE. In still other embodiments, the parent bacterium is Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, or the like. Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Decro Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter r), Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium,Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium The genera Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, and Xanthomonas Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus Tobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter,The cell division topology specificity factor is the endogenous minE or DivIVA of the parent bacterium.

[0080] In some embodiments of the above aspects, the cell division topology specificity factor is a polypeptide having an amino acid sequence at least 20% identical to a Bacillus subtilis DivIVA polypeptide (e.g., encoded by SEQ ID NO: 6), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the polypeptide encoded by SEQ ID NO: 6. In some embodiments, the cell division topology specificity factor comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 6. In some embodiments, the cell division topology specificity factor is a DivIVA polypeptide. Exemplary species having DivIVA polypeptides are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005. In some embodiments, the parent bacterium is Bacillus subtilis and the cell division topology specificity factor is B. subtilis DivIVA.

[0081] In some embodiments, the ADAS or parent bacterium that has a reduced level or activity of a cell division topology specificity factor also has a reduced level of one or more Z-ring inhibitory proteins.

[0082] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence that is at least 20% identical to an E. coli minC polypeptide (e.g., encoded by SEQ ID NO: 2), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical 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.

[0083] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence that is at least 20% identical to an E. coli minD polypeptide (e.g., encoded by SEQ ID NO: 3), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical 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.

[0084] In some embodiments, the ADAS or parent bacterium has reduced levels, activity, or expression of at least two Z-ring inhibitory proteins. In some embodiments, the ADAS or parent bacterium has reduced expression of a minC polypeptide and a minD polypeptide. In some embodiments, the ADAS or parent bacterium has reduced expression of a minC polypeptide, a minD polypeptide, and a minE polypeptide, for example, a deletion of the minCDE operon (ΔminCDE).

[0085] In some embodiments, the reduction in the level, activity, or expression of the cell division topology specificity factor or Z-ring inhibitory protein, e.g., reduction in ADAS or reduction in the parent bacterial cell, is achieved using any suitable method. For example, in some embodiments, the reduction in level or activity occurs due to a loss-of-function mutation, e.g., 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 parent cell to an inducing condition, e.g., the inducible loss-of-function mutation is a temperature-sensitive mutation, and the inducing condition is a temperature condition.

[0086] In some embodiments, the parent cell has a deletion of the minCDE operon (ΔminCDE) or a deletion of a homologous operon.

[0087] C. ADAS with increased stability due to loss of function of lytic enzyme genes In some embodiments, the parent bacterial cell has one or more loss-of-function genetic modifications that stabilize the ADAS derived from the cell (e.g., result in increased stability of the ADAS derived from the cell). In some embodiments, the parent bacterial cell is modified to reduce enzyme activity. In certain embodiments, ADAS stability is improved through deletion of one or more enzyme genes.

[0088] In some embodiments, the parent cell comprises one or more loss-of-function genetic modifications that reduce or prevent enzymatic and / or lytic activity (e.g., reduce or prevent enzymatic or lytic activity carried out by a protein encoded by a gene comprising the loss-of-function genetic modification). In some embodiments, the parent cell comprises a genomic deletion that reduces or prevents enzymatic and / or lytic activity (e.g., reduce or prevent enzymatic or lytic activity carried out by a protein encoded by a deleted genomic region). In some embodiments, the parent bacterial cell comprises one or more genomic deletions that reduce the activity of one or more endopeptidases, cell wall lytic enzymes, and / or autolytic enzymes. In some embodiments, the parent cell comprises a loss-of-function (e.g., genomic deletion) of a gene from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0089] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in sigF. For example, in an example where a wild-type parent cell comprises a sigF sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4, a parent cell of the invention may comprise the deletion shown in a comparison of SEQ ID NOs:4 and 5. For example, the parent 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 modification to sigF may comprise a deletion of all or a portion of the coding region of the gene.

[0090] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in lytC. For example, in an example where a wild-type parent cell comprises a lytC sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, a parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 8 and 9. For example, a parent cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. A loss-of-function modification to lytC may comprise a deletion of all or a portion of the coding region of the gene.

[0091] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in aprE. For example, in an example where a wild-type parent cell comprises an aprE sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, a parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 10 and 11. For example, the parent 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 modification to aprE may comprise a deletion of all or a portion of the coding region of the gene.

[0092] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in ispA. For example, in an example where a wild-type parent cell comprises an ispA sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12, the parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 12 and 13. For example, the parent 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 modification to ispA may comprise a deletion of all or a portion of the coding region of the gene.

[0093] In some embodiments, the parent cell comprises a loss-of-function gene in wprA. For example, in an example where a wild-type parent cell comprises a wprA sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14, a parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 14 and 15. For example, a parent cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. A loss-of-function modification to wprA may comprise a deletion of all or a portion of the coding region of the gene.

[0094] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in nprE. For example, in an example where a wild-type parent cell comprises an nprE sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16, a parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 16 and 17. For example, the parent 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 modification to nprE may comprise a deletion of all or a portion of the coding region of the gene.

[0095] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in Epr. For example, in an example where a wild-type parent cell comprises an Epr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18, the parent cell of the invention can comprise a deletion as shown in a comparison of SEQ ID NOs: 18 and 19. For example, the parent cell can comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. The loss-of-function modification to Epr can comprise a deletion of all or part of the coding region of the gene.

[0096] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in Vpr. For example, in an example where a wild-type parent cell comprises a Vpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20, the parent cell of the invention can comprise a deletion as shown in a comparison of SEQ ID NOs: 20 and 21. For example, the parent cell can comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21. The loss-of-function modification to Vpr can comprise a deletion of all or a portion of the coding region of the gene.

[0097] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in Bpr. For example, in an example where a wild-type parent cell comprises a Bpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22, the parent cell of the invention can comprise a deletion as shown in a comparison of SEQ ID NOs: 22 and 23. For example, the parent cell can comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23. The loss-of-function modification to Bpr can comprise a deletion of all or a portion of the coding region of the gene.

[0098] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in Mpr. For example, in an example where a wild-type parent cell comprises an Mpr sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24, the parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 24 and 25. For example, the parent 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 modification to Mpr may comprise a deletion of all or a portion of the coding region of the gene.

[0099] In some embodiments, the parent cell comprises a loss-of-function modification of the gene in nprB. For example, in an example where a wild-type parent cell comprises an nprB sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26, a parent cell of the invention may comprise a deletion as shown in a comparison of SEQ ID NOs: 26 and 27. For example, the parent 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 modification to nprB may comprise a deletion of all or a portion of the coding region of the gene.

[0100] One of skill in the art would be readily able to identify modifications to a nucleotide sequence encoding a protein (e.g., a protein having enzymatic and / or lytic activity, e.g., a lytic enzyme) that would result in loss of function of the protein.

[0101] In some embodiments, the parent cell contains a loss-of-function gene for lytC.

[0102] In some embodiments, the parent cell contains a genomic deletion of lytC.

[0103] In some embodiments, the parent bacterial cell comprises a loss-of-function (e.g., genomic deletion) of at least one gene encoding a lytic enzyme, a gene affecting the sporulation mechanism, or a cell division topology factor, and combinations thereof (e.g., comprises two or more loss-of-function genetic modifications affecting one or more lytic enzymes, one or more genes affecting the sporulation mechanism, and / or one or more cell division topology factors).

[0104] In some embodiments, the parent bacterial cell comprises loss-of-function (e.g., genomic deletions) of the following genes: lytC, sigF, and divIVa. In some embodiments, the parent bacterial cell comprises genomic deletions of lytC, sigF, and divIVa.

[0105] In some embodiments, the parent bacterial cell is a Gram-positive bacterial cell.

[0106] In some embodiments, the parent bacterial cell is Bacillus subtilis or of the genus Bacillus (e.g., a Bacillus species).

[0107] In some embodiments, the parent bacterial cells are of the genus Lactobacillus (eg, a Lactobacillus species).

[0108] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0109] In some embodiments, the parent bacterial cell is selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobacter, and the like. Rhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfota lea), Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter,Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga motoga), Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc The bacteria are those of the genus Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0110] In some embodiments, the parental cell contains gene deletions 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 genome sections in the parental bacterial cell can lead to the reduction of many lytic elements through the removal of prophage and prophage-like segments. These deletions are essential because deletions or loss-of-function mutations of certain lytic enzymes (e.g., lytC) are required to establish an intact and stable ADAS.

[0111] D. ADAS derived from parent cells with disrupted sporulation In some embodiments, the parental cells comprise a genetic deletion or loss-of-function modification (e.g., a loss-of-function mutation) that disrupts sporulation (e.g., reduces or prevents sporulation). In some embodiments, the loss-of-function modification is selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, and combinations thereof. The genomic deletions used to disrupt sporulation and the genomic deletions used to reduce enzymatic activity in the ADAS are not essential or dependent on each other, but each provides unique benefits to the formation and use of the ADAS. Disrupting sporulation is beneficial for forming an ADAS because it maintains the parental cell population and eliminates spores that may be difficult to distinguish from the ADAS. Thus, in some aspects, the parental cells provided herein comprise both one or more cargo presentation structures and one or more modifications (e.g., loss-of-function mutations) that disrupt sporulation.

[0112] E. ADAS including cargo In some embodiments, the ADAS provided by the present invention comprises a cargo contained within the ADAS. In some embodiments, the cargo is any moiety disposed within the ADAS (e.g., encapsulated by the ADAS) or displayed on the surface of the ADAS (e.g., conjugated to). In some embodiments, the cargo comprises a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulatory drug, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP), or a combination thereof. In some aspects, the cargo is delivered by a secretion apparatus (e.g., a T3SS). In other aspects, the cargo is not delivered by a T3SS. In some aspects, delivery of the cargo into the cytoplasm of the target eukaryotic cell is enhanced by the heterologous pore-forming toxin listeriolysin O (e.g., listeriolysin O is carried by the ADAS, conjugated to the cargo, or administered separately). In some embodiments, the cargo is secreted from the ADAS.

[0113] 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 a combination 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 translation of a protein or polypeptide, for example, an siRNA or an antisense oligonucleotide (ASO).

[0114] In some embodiments, the cargo is an agent capable of modulating 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 capable of modulating the host microbiome is a probiotic agent.

[0115] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme produces a target product by modifying a substrate. 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 environment to which the ADAS is delivered.

[0116] In certain embodiments, the cargo is modified for improved stability (e.g., increased stability when carried by an ADAS) compared to an unmodified version of the cargo. The "stability" of a cargo may be measured as a unitless ratio between the half-life of the unmodified form of the cargo and the half-life of the modified cargo, measured under the same environmental conditions. In some embodiments, the environment is experimentally controlled, such as simulated body fluid, RNase-free water, cytoplasm, extracellular space, or "ADASplasm" (i.e., the contents of the internal volume of the ADAS, e.g., after dissolution). In some applications, the environment is an agricultural environment, such as various field soils, river water, or seawater. In other embodiments, the environment is a real 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 substantially degrade the cargo (e.g., (a) degrades the unmodified cargo but not substantially degrades the modified cargo, or (b) does not substantially degrade either the modified or unmodified cargo).

[0117] In some embodiments, the ADAS is modified to improve cargo stability and / or optimize cargo expression. In some embodiments, the ADAS is derived from a parent cell that has been modified to improve cargo stability in the ADAS and / or optimize cargo expression in the ADAS. "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 carried by the ADAS when compared to a control or unmodified ADAS. In some embodiments, cargo stability is measured as a unitless ratio between the half-life of the unmodified cargo and the half-life of the modified cargo, measured under the same environmental conditions. In other aspects, cargo stability is measured as the fold change in the level of cargo expression between a modified ADAS (e.g., an ADAS derived from a modified parent cell, e.g., a parent cell modified to include a presentation structure) and a control ADAS, measured under the same environmental conditions.

[0118] In some embodiments, the stability and / or optimization of cargo expression is modified (e.g., increased) by genomic deletion or other loss-of-function mutation of at least one of both the lytic enzyme and the protease in the parent bacterial cell.

[0119] In some embodiments, more than one lytic enzyme, protease, or other enzyme is deleted or mutated in the parent cell, hi some embodiments, additional enzyme deletions or other loss-of-function mutations in addition to the lytic enzymes and proteases are present in the parent cell.

[0120] In some embodiments, enzymes having oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, lipase, kinase, phosphatase, protease, nuclease, amylase, cellulase and / or synthetase activity are deleted or mutated in the parent cell. In some embodiments, the deletion or loss-of-function modification 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 parent cell contains a deletion or loss-of-function modification in one, two, three, four, five, six, seven, eight, nine, ten, or more of the listed genes).

[0121] In some embodiments, the stability of the cargo in an ADAS derived from a parent cell having (i) one or more deletions or loss-of-function mutations in a lytic enzyme 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 greater than the stability of the cargo in a control ADAS. In some embodiments, the stability or optimization of cargo expression is measured through cargo functionality, for example, through downstream cytokine levels, receptor activation, or antigen-specific cellular activation. In some embodiments, the measured amount of cargo functionality from an ADAS derived from a modified parent cell is 1, 2, 3, 4, 5, 10, 50, 100, 500, 1000 or more times higher than the same measured amount from a control or unmodified ADAS (e.g., an ADAS derived from an unmodified parent cell).

[0122] In some embodiments, the parent cell contains one or more deletions or other loss-of-function mutations in a protease, hi some embodiments, the one or more protease deletions or loss-of-function modifications increase the stability of the cargo of the ADAS (e.g., increase cargo expression, presentation, function, or durability, e.g., optimize cargo expression) compared to an ADAS produced from a parent cell that does not contain the deletions or loss-of-function mutations. In some embodiments, the deleted 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 a combination thereof (e.g., the parent cell comprises a deletion of a loss-of-function modification in one, two, three, four, five, six, seven, eight, nine, ten, or more of the listed genes).

[0123] In some embodiments, the parental bacterial cell comprises 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 comprises (i) deletions of sigF, divIVA, and lytc, and (ii) deletions of one, two, three, or all four of the proteases aprE, ispA, wprA, and nprE. In some embodiments, the parental bacterial cell comprises (i) gene loss-of-function mutations in sigF, divIVA, and lytc, and (ii) loss-of-function mutations in one, two, three, or all four of the genes aprE, ispA, wprA, and nprE.

[0124] In some embodiments, the ADAS expresses a cytosolic cargo, a surface-displayed cargo, or a combination thereof, hi some embodiments, the cargo is an antigen, a cytokine, a mutein, a protein, a polypeptide, or any combination thereof.

[0125] In some embodiments, the ADAS comprises an engineered cell-wall-embedded anchor structure for a surface-displayed cargo. In some embodiments, the engineered cell-wall-embedded anchor structure is a cell-wall scaffold, a membrane anchor domain, an outer membrane anchor domain, an inner membrane anchor domain, a cell-wall anchor domain, or functional equivalents thereof. In some embodiments, the engineered cell-wall-embedded anchor structure comprises one or more of an antiparallel coiled-coil, a cell-wall or outer membrane anchor domain with one or more linkers, which may be coiled or not, a separate cargo protein (e.g., mCherry) with no linker or with one or more linkers, a cell-wall or outer membrane anchor domain with no linker, or a combination thereof. In some embodiments, the cell-wall anchor construct is a truncated antiparallel coiled-coil. In some embodiments, the cell-wall anchor construct is a linker. In some embodiments, the cell-wall anchor construct is a coiled linker. In some embodiments, the cell-wall anchor construct is a fusion construct. In some embodiments, the engineered cell-wall-embedded anchor structure has a COOH-terminal end embedded in the cell wall. In some embodiments, the COOH-terminus is comprised in a sorting signal consisting of the COOH-terminus, an LPXTG motif, a hydrophobic domain, a tail of a majority of positively charged residues, or any combination thereof.

[0126] In some embodiments, the parent 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 parent bacterial cell comprises a loss-of-function mutation in a gene in nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

[0127] In some embodiments, the parent 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 parent bacterial cell comprises a loss-of-function mutation of a gene in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

[0128] In certain embodiments, the cargo comprises a protein. In certain embodiments, the protein has a stability greater than about 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 1000000. 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, e.g., a paracrine, endocrine, or autocrine.

[0129] In some embodiments, the cargo comprises a plant hormone such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.

[0130] In some embodiments, the cargo is an anti-inflammatory or pro-inflammatory agent, such as a cytokine (e.g., a heterologously expressed anti- or pro-inflammatory cytokine or mutein 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).

[0131] In certain embodiments, the cargo is an immunomodulatory agent. Examples of immunomodulatory agents include immunostimulants; checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4); chemotherapeutic agents; immunosuppressants; antigens; superantigens; and small molecules (e.g., cyclosporin A, cyclic dinucleotides (CDNs), or STING agonists (e.g., MK-1454, c-di-AMP cyclase (DacA))). In some embodiments, the immunomodulatory agent is a moiety that induces tolerance in a subject, such as an allergen, an autoantigen (e.g., a disease-associated autoantigen), or a microorganism-specific antigen. In some embodiments, the immunomodulatory agent is a vaccine, e.g., 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, e.g., SARS-CoV-2. In some embodiments, the cargo is an adjuvant, e.g., an immunomodulatory molecule or a molecule that alters the compartmentalization, presentation, or profile of one or more costimulatory molecules associated with the 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 the innate immune pathway upstream of the adaptive immune response). In other instances, the adjuvant enhances presentation of the antigen on immune cells or immune moieties (e.g., MHC class 1) of the target organism. In some instances, the adjuvant is listeriolysin O (LLO). In some instances, the cargo comprises 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 assists in delivery of the ADAS cargo to the target cell. In some embodiments, the ADAS comprises an antigen and one or more adjuvants.

[0132] In some embodiments, the cargo is an agent for the treatment or prevention of cancer, e.g., an agent that reduces the likelihood that a patient will develop cancer or an agent that treats cancer (e.g., an agent that increases progression-free survival and / or overall survival of an individual with cancer). Cancer or tumor includes, but is not limited to, any disease or disorder characterized by uncontrolled cell growth that would be considered a neoplasia, malignancy, metastasis, or cancer. Cancer may be primary or metastatic cancer. Specific cancers that can be treated according to the present invention include, but are not limited to, those listed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). Cancers include biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colorectal cancer; uterine cancer; esophageal cancer; gastric cancer; hematologic malignancies, including acute lymphocytic and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia-lymphoma; intraepithelial neoplasia, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; and oral cancer, including squamous cell carcinoma. Cancers commonly encountered include, but are not limited to, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, 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, and choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma, and medullary carcinoma; and kidney cancer, including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer.

[0133] 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 (MSI H) / mismatch repair (MMR) deficient solid cancers. In some embodiments, the NSCLC lacks an EGFR sensitizing mutation and / or an ALK translocation. In some embodiments, the microsatellite-high (MSI H) / mismatch repair (MMR) deficient solid cancer is 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, reproductive tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues. In some embodiments, the cancer is colorectal cancer.

[0134] Drugs for preventing cancer include, but are not limited to, anti-inflammatory agents, growth inhibitors, cytokines, and tumor antigens. Drugs 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 that target cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as listerialysin o), cytotoxins, inflammasome inhibitors, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0135] In some embodiments, the cargo is an anti-cancer therapeutic. In some embodiments, the cargo includes one or more wild-type or engineered antigens (or antibodies to antigens). In some embodiments, the antigen is derived from a tumor, e.g., a tumor-specific antigen, a tumor-associated antigen, a tumor neo-antigen, or a combination thereof. In some embodiments, the cargo is an antigenic, tumor antigenic, or protein, such as 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, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAG E-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 epitopes include BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, MC38 cancer epitopes, or combinations thereof.In some embodiments, the expressed cargo is an antigenic, tumor antigenic or protein, and may be 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, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, F These include AP, 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.

[0136] 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, 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 cargoes.

[0137] In some embodiments, the expressed cargo is antigenic, tumor antigenic or protein, including cyclic dinucleotides, STING activators such as cGAMP, c-di-AMP, IRF-3 and its variants.

[0138] In some embodiments, the tumor antigenic polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof, hi one embodiment, the tumor antigenic polypeptide comprises a lung cancer antigen.

[0139] In some embodiments, the antigen is an autoantigenic polypeptide or an immunogenic variant or fragment thereof. In some embodiments, the autoantigenic polypeptide comprises an antigen that is typically expressed on a cell and recognized as an autoantigen by the immune system. In some embodiments, the autoantigenic polypeptide comprises a multiple sclerosis antigenic polypeptide, a rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof.

[0140] 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 to wild-type (WT) IL-2 (e.g., human IL-2) or an IL-2 mutein (e.g., as set forth in SEQ ID NO:59 or SEQ ID NO:60; see Table 5), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:59 or SEQ ID NO:60. 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.

[0141] In another aspect, provided herein is a method for treating or preventing cancer in a subject, comprising administering to the subject a therapeutic composition comprising an ADAS described in the above aspects of the invention (e.g., an ADAS comprising a cargo that is an agent for the treatment or prevention of cancer. Thus, in another aspect, provided herein is an ADAS therapeutic composition comprising a plurality of ADAS comprising cargos that are agents for the treatment or prevention of cancer. The composition can, for example, comprise a physiologically acceptable carrier.

[0142] In some aspects, the ADAS (e.g., an ADAS therapeutic composition) is administered orally, intravenously, intradermally, intramuscularly, intraperitoneally, peritumorally, intranasally, intraocularly, or intrarectally, and / or subcutaneously. In certain embodiments, the ADAS (e.g., an ADAS therapeutic composition) is administered orally, intravenously, intramuscularly, and / or subcutaneously. In some embodiments, the ADAS (e.g., an ADAS therapeutic composition) is administered to the subject once, twice, three times, four times, or more times. In some embodiments, the amount of 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 × 109 , 4×10 9 , 6×10 9 , 8×10 9 or 1 x 10 10 For example, the administration is at least 1 x 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 x 10 10 The method includes administering an ADAS to a subject.

[0143] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme is an enzyme that performs a 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, e.g., 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, oxidoreductase, or a combination thereof.

[0144] In some embodiments, the enzyme produces a target product by altering a substrate. In some embodiments, the substrate is present in an ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in a target cell or 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.

[0145] In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally via a linker to the outer membrane.

[0146] Instead, in some embodiments, the cargo is a nucleic acid encoding any of the enzymes described herein.

[0147] 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).

[0148] In some embodiments, the cargo is an anti-infective agent, eg, an anti-microbial agent, eg, an anti-infective or anti-microbial peptide, protein and / or natural product.

[0149] In some embodiments, the cargo is a protein that modulates a 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.

[0150] For ADASs that include 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 RNA has a stability greater than about: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, or 10000000, e.g., in the ADAS plasma. In certain embodiments, the RNA cargo can be stabilized by the addition of a tRNA scaffold, such as a step-loop structure (e.g., non-human tRNALys3 and E. coli tRNAMet (Nat. Methods, Ponchon 2007)). Both are well characterized and have been recombinantly expressed. However, various other types, such as aptamers, lncRNAs, and ribozymes, may be used as well. The RNA can also be stabilized if the ADAS is obtained from a parent strain that is null (or hypomorphic) for one or more ribonucleases.

[0151] In some detailed embodiments, the RNA is a protein-coding mRNA. In more detailed embodiments, the protein-coding mRNA encodes an enzyme (e.g., an enzyme that confers liver enzyme activity, such as human PBGD (hPBGD) mRNA) or encodes an antigen that elicits an immune response (e.g., elicits strong and durable neutralizing antibody titers), such as, for example, mRNA encoding CMV glycoprotein gB and / or pentameric complex (PC). In certain detailed embodiments, the RNA is a small non-coding RNA, such as shRNA, ASO, tRNA, dsRNA, or a combination thereof.

[0152] In certain embodiments, the ADAS provided by the present invention comprises a cargo comprising at least one component of a gene editing system. Components of a "gene editing system" include (or encode) a protein (or a nucleic acid encoding said protein) and a nucleic acid associated with the function of such a protein, e.g., a guide RNA, that can modify a DNA sequence of interest, such as a genomic DNA sequence, along with suitable associated nucleic acids, for example, by inserting or deleting the sequence of interest or by changing the methylation status of the sequence of interest. Exemplary gene editing systems include those based on Cas systems, such as Cas9, Cpfl, or other RNA-targeting systems with their companion RNAs (e.g., sequence-complementary CRISPR guide RNAs), as well as zinc finger nucleases and TAL effectors conjugated to nucleases.

[0153] Other embodiments of the ADAS provided by the present invention include plasmids, which contain DNA as cargo, and optionally, the DNA contains a protein-coding sequence. Exemplary DNA cargoes include, in certain embodiments, plasmids encoding an RNA sequence of interest (see examples above), which may be flanked on both sides by tRNA inserts, for example. A variety of DNA cargoes are encompassed by the present invention, including those that produce ADAS (e.g., genome-degrading exonucleases that drive FTZ overexpression); long-life plasmids (ATP synthase expression, rhodopsin expression); those that express stabilized non-coding RNAs, tRNAs, lncRNAs; those that express secretion apparatus tag proteins, NleE2 effector domains, and localization tags; secretion apparatus T3 / 4SS, T5SS, T6SS; logic circuits; conditionally expressed secretion apparatus; and combinations thereof. In some embodiments, the logic circuit includes an inducible expression or repression cassette, such as the IPTG-inducible Plac promoter and the hrpR portion of the AND gate, and the heat-inducible promoter pL (derived from λ phage, which is normally repressed by a thermolabile protein) and the hrpS portion of the AND gate. To engineer an OR gate, the system described by Rosado et al., PLoS Genetics, 2018, can be used. Briefly, a cis-repressed mRNA encoding RFP under a constitutive promoter can be used. This repression can then be removed in the presence of RAJ11 sRNA. A plasmid containing the IPTG-inducible promoter PLac and the heat-inducible promoter pL, both of which drive expression of RAJ11 sRNA, can then be used. The output would then be RFP expression, which is observed in response to either input. These systems can be adapted for various sensor-type functions.

[0154] In some embodiments, the ADAS provided by the present invention comprises a transporter in the membrane, hi certain embodiments, the transporter is specific for glucose, sodium, potassium, a metal ion, an anionic solute, a cationic solute, or water.

[0155] In some embodiments, the membrane of an ADAS provided by the present invention comprises an enzyme. In particular embodiments, the enzyme is a protease, an oxidoreductase, or a combination thereof. In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally via a linker, to the outer membrane.

[0156] F.ADAS presentation structure In some embodiments, the cargo carried by the ADAS is present within the ADAS, hi some embodiments, the cargo is expressed cytosolically (e.g., expressed in the cytosol of a parent cell in which the ADAS is produced and / or expressed within the ADAS).

[0157] 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 parent cell from which the ADAS is derived.

[0158] In some embodiments, the cargo carried by the ADAS is secreted by the ADAS.

[0159] In some embodiments, the engineered cell-wall-embedded anchor structure is used to present surface-expressed cargo. An engineered cell-wall-embedded anchor structure (also referred to as a cell-wall anchor construct or cell-wall-embedded anchor construct) is a construct (e.g., a cell-wall scaffold or other functional equivalent) that provides a fixed structure for presenting surface cargo. As a general rule, the engineered cell-wall-embedded anchor structure comprises at least one element that is associated with (e.g., embedded in) the cell wall of the ADAS, and the element can be associated with (e.g., conjugated to) a cargo such that the cargo is presented on the exterior of the ADAS.

[0160] The cell wall-embedded anchor structure can be a component of a presentation structure, which refers to any means by which cargo is expressed and / or presented by the ADAS (e.g., presented on the surface of the ADAS).

[0161] The presentation structure comprises at least one cargo. Further exemplary components of the presentation structure include, but are not limited to, cell wall-embedded anchor structures (e.g., binding domains) and scaffold proteins. In some embodiments, the components of the presentation structure are connected by one or more linkers. In other embodiments, one or more components of the presentation structure are directly connected (e.g., connected without the use of a linker).

[0162] For example, in some embodiments, the constructed cell-wall-embedded anchor structure comprises a cell-wall-binding domain and one or more antiparallel coiled-coils (e.g., the one or more antiparallel coiled-coils are tethered to a cargo); the cell-wall-embedded anchor structure comprises a cell-wall-embedded binding domain with one or more linkers, coiled or non-coiled (e.g., the one or more linkers are tethered to a cargo); the cell-wall-embedded anchor structure comprises a separate cargo protein or scaffold protein (e.g., mCherry) fused to a cell-wall-binding domain with no linker or with one or more linkers (e.g., the separate cargo protein or scaffold protein is tethered to the cargo directly or via one or more linkers); or the constructed cell-wall-embedded anchor structure comprises a cell-wall-embedded binding domain with no linker (e.g., the binding domain is tethered directly to the cargo).

[0163] In some embodiments, the presentation structure comprises one or more polypeptides, and one or more elements of the presentation structure (e.g., cargo, cell wall-embedded anchor structure, scaffold protein, and / or linker) are comprised by the same polypeptide chain. In some embodiments, all elements of the presentation structure are comprised by the same polypeptide chain. Exemplary presentation structures are provided in Figure 2B and in the Examples, although these examples should not be considered limiting.

[0164] In some embodiments, the presentation structure is encoded by a nucleotide sequence. In some embodiments, the nucleotide sequence is contained in the parent cell from which the ADAS is derived. Thus, in one aspect, the present disclosure features a nucleotide sequence that encodes any one component of the presentation structure provided herein. In another aspect, the present disclosure features a parent cell that includes a nucleotide sequence that encodes any one component of the presentation structure provided herein.

[0165] In some embodiments, the presentation structure is encoded by a nucleotide sequence comprising 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., 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., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:56.

[0166] In some embodiments, the presentation structure 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., 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.

[0167] In some embodiments, the presentation structure is encoded by a nucleotide sequence encoding a ribosome binding site (RBS). In some embodiments, the binding site is an 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., 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 an aprE-RBS.

[0168] In some embodiments, the presentation structure 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., 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 an aprE-SS polypeptide.

[0169] In some embodiments, the presentation structure is encoded by nucleotide sequences that are separated by a cargo sequence (e.g., the cargo sequence and one or more elements of the presentation structure (e.g., a cell wall-embedded anchor structure, a scaffold protein, and / or a linker) are encoded by a single nucleotide sequence, with the cargo sequence occurring within or between nucleotide sequences encoding the other element(s) of the presentation structure). Thus, in some embodiments, the presentation structure is a polypeptide in which amino acid sequences corresponding to non-cargo components are separated by amino acid sequences corresponding to the cargo. In some embodiments, the presentation structure sequence is separated by the cargo sequence at the sites marked with "^" in Table 5. One of skill in the art will be able to identify other suitable insertion sites for the cargo sequence (e.g., sites that would not disrupt the function of other elements of the presentation structure).

[0170] In some embodiments, the presentation structure comprises a non-covalent cell-binding domain (e.g., comprises a non-covalent cell-binding domain as an alternative to or in addition to a 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., 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.

[0171] In some embodiments, the presentation structure comprises a covalent cell-binding domain (e.g., comprises a covalent cell-binding domain as an alternative to or in addition to a 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., 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.

[0172] In some embodiments, the presentation structure 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., 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.

[0173] In some embodiments, the parent bacterial cell from which the ADAS is derived contains at least one genomic deletion of a protease, and the nucleotide sequence encoding the display structure is integrated into the deleted protease site. In some embodiments, the parent bacterial cell from which the ADAS is derived contains at least one genomic deletion of an amylase, and the nucleotide sequence encoding the display structure is integrated into the deleted amylase site. In some embodiments, the integration site is amyE (e.g., amyE has been deleted from the genome of the parent cell). In some embodiments, the deleted sequence of the amyE locus has at least 20% identity to SEQ ID NO: 50, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 50. In some embodiments, the parent bacterial cell from which the ADAS is derived contains at least one genomic deletion of a phosphodiesterase, and the nucleotide sequence encoding the display structure is integrated into the deleted phosphodiesterase site. In some embodiments, the integration site is pdeH. In some embodiments, the deleted sequence of the pdeH locus is at least 20% identical to SEQ ID NO:494, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO:49.

[0174] In some embodiments, the presentation structure includes a linker, the linker is a linker without a defined structure. In some embodiments, the linker is proline-rich (e.g., greater than 25%, 27%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% proline). In some embodiments, the presentation structure includes a linker, the linker is a linker with a defined structure (e.g., a coiled linker, one or more antiparallel coiled coils, etc.).

[0175] In some embodiments, the presentation structure (e.g., one or more linkers comprised by the presentation structure) lacks a protease site, thereby reducing proteolytic cleavage of the cargo from the ADAS. In other embodiments, the presentation structure (e.g., one or more linkers comprised by the presentation structure) contains a protease site, allowing release of the cargo from the ADAS surface. In some embodiments, the protease site is specific to a eukaryotic organism (e.g., a plant, an animal, a mouse, a human, etc.), e.g., the organism to which the ADAS is to be delivered. In some embodiments, the protease site is specific to a human tissue or target site (e.g., lung, blood, gastrointestinal tract, etc.), e.g., the tissue or target site to which the ADAS is to be delivered.

[0176] In some embodiments, display 0 is used as the cargo presentation structure. In some embodiments, display 1 is used as the cargo presentation structure. In some embodiments, display 2 is used as the cargo presentation structure. In some embodiments, display 3 is used as the cargo presentation structure. In some embodiments, display 4 is used as the cargo presentation structure. In some embodiments, display 6 is used as the cargo presentation structure.

[0177] Presentation 0 In some embodiments, the presentation structure is the construct shown in Presentation 0 (see FIG. 2B). Thus, in some embodiments, the present invention provides a presentation structure polypeptide according to Presentation 0; a nucleotide sequence encoding the same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0178] Briefly, presentation 0 is a fusion (e.g., a fusion polypeptide) comprising a cargo displayed on a cell wall-embedded binding domain without a linker (e.g., there is no linker domain connecting the cargo to the binding domain) (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain).

[0179] 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 to SEQ ID NO:61, 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.

[0180] Thus, in some embodiments, the presentation structure is a polypeptide comprising a cell wall-embedded binding domain (eg, a binding domain encoded by SEQ ID NO: 61 or a variant or derivative thereof) and cargo.

[0181] Presentation 1 In some embodiments, the presentation structure is the construct shown in Presentation 1 (see FIG. 2B). Thus, in some embodiments, the present invention provides a presentation structure polypeptide according to Presentation 1; a nucleotide sequence encoding same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0182] Briefly, presentation 1 is a fusion (e.g., a fusion polypeptide) comprising a cargo displayed on a cell wall-embedded binding domain with a linker (e.g., a linker domain connecting the cargo to the binding domain) (e.g., a fusion polypeptide comprising a cargo, a linker, and a cell wall-embedded binding domain).

[0183] 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., 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., a polypeptide linker, e.g., a polypeptide linker with or without a defined structure).

[0184] In some embodiments, Representation 1 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 62, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 62. In some embodiments, Representation 1 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 62.

[0185] In some embodiments, the presentation structure is a Presentation 1 linker polypeptide.

[0186] Thus, in some embodiments, the presentation 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), a linker, and cargo. In some embodiments, the cell-wall-embedded binding domain and linker are encoded by SEQ ID NO: 62 or a variant or derivative thereof.

[0187] Presentation 2 In some embodiments, the presentation structure is the construct shown in Presentation 2 (see Figure 2B). Thus, in some embodiments, the invention provides a presentation structure polypeptide according to Presentation 2; a nucleotide sequence encoding it; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0188] Briefly, Exhibit 2 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via one or more antiparallel coiled-coils (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain tethered by one or more antiparallel coiled-coils).

[0189] In some embodiments, the presentation structure comprises an antiparallel coiled-coil (APCC).

[0190] In some embodiments, Exhibit 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., 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, Exhibit 1 comprises the amino acid sequence encoded by SEQ ID NO: 61, and further comprises one or more antiparallel coiled-coils.

[0191] In some embodiments, display 2 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 63, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 63. In some embodiments, display 2 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 63. SEQ ID NO: 63 as described herein includes an "X" marker that indicates the position where a nucleotide sequence encoding a cargo will be included. Thus, one of skill in the art will understand that a comparison of a given sequence to SEQ ID NO: 63 can be performed 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 antiparallel coiled-coils).

[0192] In some embodiments, the presentation structure is a presentation 2 linker polypeptide.

[0193] In some embodiments, the cell wall-embedded anchor construct is a truncated antiparallel coiled-coil.

[0194] Thus, in some embodiments, the presentation 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 antiparallel coiled-coils, and cargo. In some embodiments, the cell-wall-embedded binding domain and the one or more antiparallel coiled-coils are encoded by SEQ ID NO: 63 or a variant or derivative thereof.

[0195] Presentation 3 In some embodiments, the presentation structure is the construct shown in Presentation 3 (see FIG. 2B). Thus, in some embodiments, the invention provides a presentation structure polypeptide according to Presentation 3; a nucleotide sequence encoding the same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0196] Briefly, Exhibit 3 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via one or more antiparallel coiled-coils (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain tethered by one or more antiparallel coiled-coils), where the antiparallel coiled-coil is shorter than in Exhibit Structure 2. Thus, Exhibit 3 is similar in structure to Exhibit 2, but has a shortened antiparallel coiled-coil.

[0197] In some embodiments, Representation 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., 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 truncated antiparallel coiled-coils. In some embodiments, Representation 3 comprises the amino acid sequence encoded by SEQ ID NO: 61, and further comprises one or more truncated antiparallel coiled-coils.

[0198] In some embodiments, Exhibit 3 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:64, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:64. In some embodiments, Exhibit 3 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:64. SEQ ID NO:64 as described herein includes an "X" marker that indicates the position where a nucleotide sequence encoding a cargo will be included. Thus, one of skill in the art will understand that a comparison of a given sequence to SEQ ID NO:64 can be performed 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 truncated antiparallel coiled-coils).

[0199] In some embodiments, the linker (e.g., an antiparallel coiled-coil) lacks a protease site, reducing proteolytic cleavage of the cargo from the ADAS. In some embodiments, the linker contains a protease site to allow 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 tissue or target sites (e.g., lung, blood, gastrointestinal tract, etc.).

[0200] Thus, in some embodiments, the presentation 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 truncated antiparallel coiled-coils, and cargo. In some embodiments, the cell-wall-embedded binding domain and one or more truncated antiparallel coiled-coils are encoded by SEQ ID NO: 64 or a variant or derivative thereof.

[0201] Presentation 4 In some embodiments, the presentation structure is the construct shown in Presentation 4 (see FIG. 2B). Thus, in some embodiments, the invention provides a presentation structure polypeptide according to Presentation 4; a nucleotide sequence encoding same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0202] Briefly, Exhibit 4 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via a coiled linker (e.g., the cell wall-embedded anchor construct contains a coiled linker) (e.g., a fusion polypeptide comprising a cargo and a cell wall-embedded binding domain tethered by a coiled linker).

[0203] In some embodiments, Representation 4 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 61, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 61, and further comprises a coiled linker. In some embodiments, Representation 4 comprises the amino acid sequence encoded by SEQ ID NO: 61, and further comprises a coiled linker.

[0204] In some embodiments, Representation 4 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 65, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 65. In some embodiments, Representation 4 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:65.

[0205] Thus, in some embodiments, the presentation 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), a coiled linker, and cargo. In some embodiments, the cell-wall-embedded binding domain and the coiled linker are encoded by SEQ ID NO: 65 or a variant or derivative thereof.

[0206] Presentation 5 In some embodiments, the presentation structure is the construct shown in Presentation 5 (see Figure 2B).

[0207] Thus, in some aspects, the invention provides a presented structural polypeptide according to Presentation 5; a nucleotide sequence encoding the same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0208] Briefly, Exhibit 5 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via a scaffold protein (e.g., mCherry), and a linker connecting the cell wall-embedded binding domain to the scaffold protein (e.g., a fusion polypeptide comprising cargo, scaffold protein, linker, and cell wall-embedded binding domain).

[0209] In some embodiments, Representation 5 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 61, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 61, and further comprises a linker and a scaffold protein. In some embodiments, Representation 5 comprises the amino acid sequence encoded by SEQ ID NO: 61, and further comprises a linker and a scaffold protein.

[0210] In some embodiments, Display 5 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 66, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 66. In some embodiments, Display 5 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 66. SEQ ID NO: 66 as described herein includes an "X" marker that indicates the position where a nucleotide sequence encoding a cargo will be included. Thus, one of skill in the art will understand that a comparison of a given sequence to SEQ ID NO: 66 can be performed 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).

[0211] Thus, in some embodiments, the presentation 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), a linker, a scaffold protein, and a cargo. In some embodiments, the cell-wall-embedded binding domain, linker, and scaffold protein are encoded by SEQ ID NO: 66 or a variant or derivative thereof.

[0212] presentation 6 In some embodiments, the presentation structure is the construct shown in Presentation 6 (see Figure 2B).

[0213] Thus, in some aspects, the invention provides a presented structural polypeptide according to Presentation 6; a nucleotide sequence encoding the same; a parent bacterial cell comprising the polypeptide and / or nucleotide sequence; and an ADAS comprising the polypeptide and / or nucleotide sequence.

[0214] Briefly, Exhibit 6 is a construct comprising a cargo tethered to a cell wall-embedded binding domain (anchor) via a scaffold protein (e.g., mCherry), where a first linker tethers the cargo to the scaffold protein and a second linker tethers the cell wall-embedded binding domain to the scaffold protein (e.g., a fusion polypeptide comprising the cargo, first linker, scaffold protein, second linker, and cell wall-embedded binding domain).

[0215] In some embodiments, Representation 6 comprises a cell wall-embedded binding domain encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO: 61, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 61, and further comprises a first linker, a second linker, and a scaffold protein. In some embodiments, Representation 6 comprises the nucleotide sequence of SEQ ID NO: 61, and further comprises a first linker, a second linker, and a scaffold protein.

[0216] In some embodiments, Display 6 comprises a polypeptide encoded by a nucleotide sequence having at least 20% identity to SEQ ID NO:67, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:67. In some embodiments, Display 6 comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO:67. SEQ ID NO:67 as described herein includes an "X" marker that indicates the position where a nucleotide sequence encoding a cargo will be included. Thus, one of skill in the art will understand that a comparison of a given sequence to SEQ ID NO:67 can be performed 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).

[0217] Thus, in some embodiments, the presentation 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), a first linker, a scaffold protein, a second linker, and a cargo. In some embodiments, the cell-wall-embedded binding domain, linker, and scaffold protein are encoded by SEQ ID NO: 67 or a variant or derivative thereof.

[0218] In some embodiments, the presentation structure is cytosolic rather than through surface expression, e.g., the cargo is presented by one or more components (e.g., binding domains, linkers, and / or scaffolds) inside the ADAS (cytosol).

[0219] The presentation structures can be "unstructured," which refers to a relatively high proportion of prolines in the structure, imparting a non-linear or curved shape to the construct, or they can be structured, such as coiled, and have a specific or more defined shape.

[0220] In some embodiments, the cell wall-embedded anchor structure comprises a cell wall binding domain.

[0221] In some embodiments, the ADAS comprises a fusion promoter.

[0222] In some embodiments, the ADAS contains a proline-rich sequence that encodes a cell wall-embedded anchor structure.

[0223] In some embodiments, the ADAS contains a self-binding coil for a cell wall-embedded anchor structure.

[0224] In some embodiments, the cell wall-embedded anchor structure is an unstructured, proline-rich construct.

[0225] In some embodiments, the cell wall-embedded anchor structure is a structured construct.

[0226] In some embodiments, the constructed cell wall-embedded anchor structure has an N-terminal end embedded in the cell wall. In some embodiments, the anchor structure contains an internal cell wall-embedded anchor domain. In some embodiments, the cell wall-embedded anchor structure incorporates a protease cleavage site. In some embodiments, the presentation structure includes a protease cleavage site.

[0227] In some embodiments, at least one element of the presentation structure (eg, the cargo, the cell wall-embedded anchor structure, the scaffold protein, and / or the linker) is heterologous to the parent cell from which the ADAS is derived.

[0228] G. ADAS containing secretory apparatus In certain embodiments, the ADAS provided by the present invention comprises a bacterial secretion apparatus (e.g., an endogenous bacterial secretion apparatus or a heterologous secretion apparatus). A "bacterial secretion apparatus" is a protein or protein complex that can export cargo from the cytoplasm of a bacterial cell (or, e.g., an ADAS derived therefrom) into the extracellular space, the periplasmic space of Gram-negative bacteria, or the intracellular space of another cell. In some embodiments, the bacterial secretion apparatus operates by an active (e.g., ATP-dependent or PMF-dependent) process, and in certain embodiments, the bacterial secretion apparatus comprises a tube or spike that spans from the host cell (or ADAS) to the target cell. In other embodiments, the bacterial secretion apparatus is a transmembrane channel. Exemplary bacterial secretion apparatuses include the T3SS and T4SS (and T3 / T4SS, as defined below), which are tube-containing structures through which cargo travels through the interior of a protein tube, and the T6SS, which carries cargo at the end of a spike. Other exemplary bacterial secretion apparatus include the transmembrane T1SS, T2SS, T5SS, T7SS, Sec, and Tat.

[0229] In some aspects, the disclosure features a non-chromosomal active activation system (ADAS) derived from a parent bacterial cell, the ADAS comprising a bacterial type 3 secretion system (T3SS) that is heterologous to the parent bacterial cell.

[0230] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0231] In some embodiments, the parent bacterial cell does not contain an endogenous T3SS.

[0232] In some embodiments, the parent bacterial cell is an E. coli cell. In some embodiments, the E. coli cell is a Nissle E. coli cell.

[0233] In some embodiments, the parent bacterial cells are probiotic cells.

[0234] 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 E. 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.

[0235] In some embodiments, the parent bacterial cell comprises one or more heterologous nucleotide sequences encoding components of a T3SS. In some embodiments, the one or more nucleotide sequences encoding components of a T3SS are carried on a vector. In some embodiments, the parent bacterial cell is transiently transformed with a vector. In some embodiments, the parent bacterial cell is stably transformed with a vector. In some embodiments, the parent bacterial cell further comprises a moiety that increases the level of T3SS in ADAS. In some embodiments, the moiety is a transcriptional activator of the one or more heterologous nucleotide sequences encoding components of a T3SS.

[0236] In another aspect, the disclosure features a non-chromosomal dynamic activation system (ADAS), T1P-ADAS, derived from a parent bacterial cell, the T1P-ADAS comprising a bacterial type 3 secretion system (T3SS) endogenous to the parent bacterial cell, wherein the parent bacterial cell has been modified to reduce levels of an endogenous protein or polypeptide capable of being secreted by the T3SS.

[0237] In some embodiments, the parent bacterial cell is modified by deleting a transcriptional activator of an endogenous protein or polypeptide that has the ability to be secreted by a T3SS.

[0238] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0239] In some embodiments, the parent bacterial cell is a Salmonella species, a Vibrio species, an Escherichia species, a Yersinia species, or a Shigella species. In some embodiments, the Salmonella species is Salmonella enterica. In some embodiments, the Vibrio species is Vibrio parahaemolyticus. In some embodiments, the Escherichia species is enteropathogenic E. coli (EPEC). In some embodiments, the Yersinia species is Yersinia enterocolitica. In some embodiments, the Shigella species is Shigella flexneri.

[0240] In some embodiments, the parent bacterial cell further comprises a moiety that increases the level of a T3SS in the ADAS, hi some embodiments, the moiety is a transcriptional activator of a nucleotide sequence encoding a component of the T3SS.

[0241] In some embodiments, the parent bacterial cell has been modified to reduce the level of a negative regulator of a component of the T3SS, hi some embodiments, the chromosomal locus encoding the negative regulator has been deleted from the parent bacterial cell.

[0242] In some embodiments, the parent bacterial cells have been modified to reduce levels of one or more of: LPS; non-essential metabolic proteins; non-T3SS-associated toxins; endotoxins; flagella; and pili.

[0243] 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.

[0244] In some embodiments, the cargo is a protein or polypeptide.

[0245] In some embodiments, the cargo is endogenously secreted by a T3SS.

[0246] In some embodiments, the ADAS or the parent bacterial cell is modified to increase the level of cargo in the ADAS.

[0247] In some embodiments, the cargo is not endogenously secreted by a T3SS.

[0248] In some embodiments, the cargo is endogenously secreted by a T3SS of a species other than the ADAS T3SS species.

[0249] In some embodiments, the cargo is endogenously secreted by a type 4 secretion system (T4SS) or a type 6 secretion system (T6SS).

[0250] In some embodiments, the cargo is modified for delivery by the T3SS.

[0251] In some embodiments, the cargo is an enzyme, a DNA modifying agent, a chromatin remodeling agent, a gene editing agent, a nuclear targeting agent, a binding agent, an immunogenic agent, or a toxin. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editing agent is a component of a CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binding agent is an antibody or antibody fragment. In some embodiments, the binding agent is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulatory agent. In some embodiments, the immunogenic agent is an immunosuppressant.

[0252] In some embodiments, the cargo is modified by the addition of a secretion signal.

[0253] In another aspect, the disclosure features a method for delivering cargo to the cytoplasm of a target cell, the method including contacting the target cell with the ADAS of any one of the above aspects.

[0254] In some embodiments, the ADAS comprises a cargo, where the cargo comprises a moiety that directs export by the bacterial secretion apparatus, e.g., in some embodiments, this moiety is Pho / D, Tat, or a synthetic peptide signal.

[0255] In certain embodiments, the ADAS provided by the present invention is a double-membrane ADAS. In more particular embodiments, the double-membrane ADAS further comprises a bacterial secretion apparatus. In even more particular embodiments, the bacterial secretion apparatus is selected from a T3SS, a T4SS, a T3 / 4SS, or a T6SS, optionally with an attenuated or non-functional effector that does not affect the fitness of the target cell.

[0256] The ADAS provided by the present invention, in some embodiments, comprises a bacterial secretion apparatus.

[0257] In some embodiments, the bacterial secretion apparatus is capable of exporting cargo across the ADAS outer membrane into a target cell, such as an animal, fungal, bacterial, or plant cell, such as a T3SS, T4SS, T3 / T4SS, or T6SS.

[0258] In more specific embodiments, the bacterial secretion apparatus is a T3 / 4SS. A "T3 / 4SS" refers to a secretion apparatus based on a T3SS or a T4SS, including hybrid apparatus and unmodified versions, that links the two to form a protein tube that delivers one or more effectors between a bacterium (or ADAS) and a target cell. The target cell can be an animal, plant, fungus, or bacterium. In some embodiments, the T3 / 4SS comprises an effector, which may be a modified effector. Examples of T3SS apparatus include the Salmonella SPI-1 apparatus, the Escherichia coli ETT1 apparatus, the Xanthamonas citri / campestri T3SS apparatus, and the Pseudomonas syringae T3SS apparatus. Examples of T4SS apparatus include the Agrobacterium Ti plasmid apparatus and the Helicobacter pylori T4SS. In particular embodiments, the T3 / 4SS has a modified effector function, e.g., an effector selected from SopD2, SopE, Bop, Map, Tir, EspB, EspF, NleC, NleH2, or NleE2. In more particular embodiments, the modified effector function is a function of intracellular targeting, such as translocation into the nucleus, Golgi, mitochondria, actin, microvilli, ZO-1, microtubules, or cytoplasm. In even more particular embodiments, the modified effector function is nuclear targeting based on NleE2 from Escherichia coli (E. coli). In other particular embodiments, the modified effector function is a function of filopodia formation, disruption of tight junctions, loss of microvilli, or inactivation of SGLT-1.

[0259] In other embodiments, the ADAS provided by the present invention that includes a bacterial secretion apparatus includes a T6SS. In some embodiments, the T6SS includes an effector that targets and kills a bacterium in its natural host. In certain detailed embodiments, the T6SS is derived from the P. putida K1-T6SS, and optionally, the effector includes the amino acid sequence of Tke2 (Accession No. AUZ59427.1), or a functional fragment thereof. In other embodiments, the T6SS includes an effector that targets and kills a fungus in its natural host, for example, the T6SS is derived from Serratia marcescens, and the effector includes the amino acid sequence of Tfe1 (Genbank: SMDB11_RS05530) or Tfe2 (Genbank: SMDB11_RS05390).

[0260] In other embodiments of the ADAS provided by the present invention that include a bacterial secretion apparatus, the bacterial secretion apparatus is capable of exporting cargo outside the cell. In certain more specific embodiments, the bacterial secretion apparatus is a T1SS, a T2SS, a T5SS, a T7SS, Sec, or Tat.

[0261] H. ADAS lacking protease, RNase, and / or LPS In another aspect, the invention provides compositions further comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS have reduced protease levels or activity compared to an ADAS produced from a wild-type parent bacterium. In some aspects, the ADAS are produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one protease. In some embodiments, the parent cell contains one or more protease deletions. In some embodiments, the parent cell contains one or more loss-of-function mutations in a protease gene. In some embodiments, the protease deletions or other loss-of-function mutations increase the stability of the ADAS cargo (e.g., increase cargo expression, presentation, function, or durability, e.g., optimize cargo expression) compared to an ADAS produced from a parent cell that does not contain the deletions or loss-of-function mutations. In some embodiments, the protease deletion or loss of function is a deletion or loss of function of one or more of 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., a deletion or loss of function in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the listed proteases).

[0262] In some embodiments, the parental cell comprises 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 deletions. In some embodiments, the parental cell comprises 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 loss-of-function mutations in 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 one, two, three, or all four, or a combination, of aprE, ispA, wprA, and nprE.

[0263] In another aspect, the invention provides compositions comprising multiple ADASs (e.g., highly active ADASs), wherein the ADASs have reduced RNase levels or activity compared to ADASs produced from a wild-type parent bacterium. In some aspects, the ADASs are produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one RNase. In some embodiments, the RNase is an endoribonuclease or an exoribonuclease.

[0264] In another aspect, the present invention provides a composition comprising a plurality of ADAS, wherein the ADAS has been modified to have reduced lipopolysaccharide (LPS) activity. In some embodiments, the modification is a mutation in lipid A biosynthesis myristoyltransferase (msbB).

[0265] In certain embodiments, the ADAS provided by the present invention lacks one or more metabolically non-essential proteins. "Metabolically non-essential proteins" include, but are not limited to, pili, flagella, unwanted secretion apparatus, transposases, effectors, phage elements, or regulatory elements thereof, such as flhC or OmpA. In some embodiments, the ADAS provided by the present invention lacks one or more RNases, proteases, or combinations thereof, and in particular embodiments, lacks 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.

[0266] I. ADAS with targeting moieties In another embodiment, the present invention provides a composition comprising a plurality of ADAS, wherein the ADAS comprises a targeting moiety. 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 parent cell (e.g., a surface ligand inherited 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 ADAS modification methods described herein. In some embodiments, the targeting moiety promotes tissue-associated targeting of the ADAS to a certain tissue or cell type.

[0267] In certain embodiments, the nanobody is a nanobody directed 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 yet other embodiments, the tumor-targeting peptide is an RGD motif or a CendR peptide.

[0268] J. ADAS derived from commensal or pathogenic parent strains In another embodiment, the present invention provides a composition comprising multiple ADAS (e.g., highly active ADAS), wherein the ADAS are derived from a parent bacterium that is a mammalian pathogen or a mammalian commensal bacterium. In some instances, the mammalian commensal bacterium is a Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus, or Actinomyces species, or the mammalian pathogenic bacterium is enterohemorrhagic Escherichia coli (EHEC), Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori.

[0269] In another embodiment, the present invention provides a composition comprising multiple ADAS (e.g., highly active ADAS), wherein the ADAS are derived from a parent bacterium that is a plant pathogen or a plant commensal bacterium. In some instances, the plant commensal bacterium is Bacillus subtilis or Pseudomonas putida, or the plant pathogenic bacterium is Xanthomonas species or Pseudomonas syringae.

[0270] K. ADAS derived from auxotrophic parent strain In another embodiment, the present invention provides a composition comprising multiple ADAS (e.g., hyperactive ADAS), where the ADAS is derived from an auxotrophic parent bacterium, i.e., a parent bacterium that is unable to synthesize an organic compound required for growth. Such bacteria can only grow if the organic compound is provided.

[0271] L. ADAS with additional parts In certain embodiments, the ADAS comprises a functional ATP synthase, and in some embodiments, a membrane-embedded proton pump. The ADAS can be derived from a variety of sources, including parent bacterial strains ("parent strains") engineered or derived to produce genome-free closed membrane systems, bacteria from which the genome has been excised, bacterial cell preparation extracts (e.g., by mechanical or other means), or total synthetic, optionally including fractions of bacterial cell preparations. In some embodiments, a highly active ADAS has a stoichiometric activity of at least: 10,000 nm 2 per 1, 5000nm 2 1 per 3500nm 2 per 1,000nm 2 The ATP synthase concentration is 1 per 100 μg / mL.

[0272] The ADAS provided by the present invention can include a variety of additional components, including, for example, photovoltaic pumps, retinals and retinal-producing cassettes, metabolic enzymes, targeting agents, cargo, bacterial secretion apparatuses and transporters, including combinations of the foregoing, including specific detailed embodiments described below. In certain embodiments, the ADAS lacks metabolically non-essential genes and / or other elements, such as specific enzymes, nucleases, or proteases.

[0273] In certain embodiments, the ADAS provided by the present invention comprises an ATP synthase and optionally lacks a regulatory domain, such as lacking the epsilon domain. The deletion can be achieved by various means. In certain embodiments, the deletion is by inducible deletion of the native ε domain. In certain embodiments, the deletion can be achieved by flanking LoxP sites and inducible Cre expression or CRISPR knockout, or can be inducible (placed on a plasmid under the tTa tet transactivator in an ATP synthase knockout strain).

[0274] In some embodiments, the ADAS can include a photovoltaic proton pump. In certain embodiments, the photovoltaic proton pump is proteorhodopsin. In more particular embodiments, the proteorhodopsin comprises the amino acid sequence of proteorhodopsin from the uncultured marine bacterial clade SAR86, GenBank accession number AAS73014.1. In other embodiments, the photovoltaic proton pump is Gloeobacter rhodopsin. In certain embodiments, the photovoltaic proton pump is bacteriorhodopsin, deltarhodopsin, or halorhodopsin from Halobacterium salinarum, Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica, or Haloarcula marismortui.

[0275] 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 machinery) or a nucleic acid encoding the same.

[0276] 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, for example, phosphofructokinase (Pfk-A) comprising the amino acid sequence of UniProt Accession No. P0A796 or a functional fragment thereof. In other embodiments, the glycolytic pathway protein is, for example, triosephosphate isomerase (tpi) comprising the amino acid sequence of UniProt Accession No. P0A858 or a functional fragment thereof.

[0277] M. ADAS Compositions and Formulations The present invention provides compositions or preparations containing an ADAS provided by the present invention, including, inter alia, a highly active ADAS preparation provided by the present invention, where optionally the ADAS preparation is substantially free of viable cells. Collectively, these may be referred to as "composition(s) provided by the present invention," and may contain any ADAS provided by the present invention, and any combination of ADAS provided by the present invention.

[0278] For example, in some embodiments, compositions provided herein contain 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, which comprises the bacterial secretion apparatus. In certain embodiments, the bacterial secretion apparatus is one of a T3SS, a T4SS, a T3 / 4SS, or a T6SS.

[0279] In some embodiments, the compositions provided herein include an ADAS that contains a T3SS, wherein the ADAS has a wavelength of about: 40,000, 35,000, 30,000, 25,000, 19,600, 15,000, 10,000, or 5,000 nm 2 and an average T3SS membrane density greater than 1. In certain detailed embodiments, the ADAS is derived from a S. typhimurium or E. coli parent strain.

[0280] Certain embodiments of the compositions provided by the present invention include an ADAS containing a T3SS, wherein the ADAS has an average molecular weight of about: 300,000, 250,000, 200,000, 150,000, 100,000, 50,000, 20,000, 10,000, 5,000 nm 2 In certain detailed embodiments, the ADAS is derived from an Agrobacterium tumefaciens parent strain.

[0281] In another aspect, 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 contain a bacterial secretion apparatus including a T3, T4, T3 / 4SS, T6SS, and optionally one or more of: exogenous carbohydrate, phosphate-producing synthase, light-responsive protein, import protein, enzyme, functional cargo, organism-specific effector, fusion protein.

[0282] As will be readily apparent, the compositions and preparations provided by the present invention can contain any ADAS provided by the present invention, such as a hyperactive ADAS or an ADAS lacking the minE gene product.

[0283] The compositions provided by the present invention can be prepared into any suitable formulation. For example, the formulation may be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosol, or spray administration. In some embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.

[0284] In some embodiments, the ADAS compositions described herein comprise less than 100 colony forming units (CFU / mL) viable bacterial cells, e.g., less than 50 CFU / mL, less than 20 CFU / mL, less than 10 CFU / mL, less than 1 CFU / mL, or less than 0.1 CFU / mL.

[0285] In some embodiments, the invention provides an ADAS composition, wherein the ADAS is lyophilized and reconstituted, and the reconstituted ADAS has an ATP concentration that is at least 90% of the ATP concentration of the non-lyophilized ADAS, e.g., at least 95%, 98% or at least equal to the ATP concentration of the non-lyophilized ADAS.

[0286] In some embodiments, the invention provides an ADAS composition, wherein the ADAS is stored, e.g., stored at 4° C., and after storage, the ADAS has an ATP concentration that is at least 90%, e.g., at least 95%, 98%, or at least equal to the ATP concentration of the non-storage ADAS. In some embodiments, storage is for at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 6 months, or at least 1 year.

[0287] In some embodiments, the ADAS is maintained or in some "resting" state and then rapidly activated.

[0288] In some embodiments, the ADAS composition is formulated for delivery to an animal, for example, formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosol, or nebulized administration.

[0289] In some embodiments, the ADAS composition is formulated for delivery to plants. In some aspects, the composition includes an adjuvant, such as a surfactant (e.g., a nonionic surfactant, a surfactant and a nitrogen source, an organosilicon surfactant, or a high surfactant oil concentrate), a crop oil concentrate, a vegetable oil concentrate, a modified vegetable oil, a nitrogen source, a deposition (drift control) and / or retention agent (with or without ammonium sulfate and / or an antifoam agent), a compatibility agent, a buffering agent and / or an acidifying agent, a water conditioning agent, a basic blend, a spreader-sticker and / or a weighting agent, an adjuvant and a foliar fertilizer, a defoamer, a foam marker, a scent, or a tank cleaner and / or a neutralizing agent. In some embodiments, the adjuvant is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (13th ed.), Purdue University).

[0290] In some embodiments, the ADAS composition is formulated for delivery to an invertebrate (e.g., an arthropod (e.g., an insect or arachnid), a nematode, a protozoan, or an annelid). In some embodiments, the ADAS composition is formulated for delivery to an insect.

[0291] In some embodiments, the composition is formulated as a liquid, solid, aerosol, paste, gel, or gaseous composition.

[0292] N. ADAS containing enzymes In one aspect, the invention features a composition including multiple ADASs, where the ADASs include enzymes that convert 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 a target cell or 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.

[0293] O. ADAS including cancer drugs In one aspect, the invention features a composition comprising a plurality of ADAS, wherein the ADAS comprises a cancer therapeutic agent. Exemplary cancer therapeutic agents are described, for example, in Section II(E) above, and include, but are not limited to, anti-inflammatory agents, growth inhibitory agents, chemotherapeutic agents, immunotherapeutic agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments that target cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines comprising cancer neoantigens), agents that induce autophagy (e.g., activators such as listerialysin o), cytotoxins, inflammasome inhibitors, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0294] In some embodiments, the cancer therapeutic agent is displayed on an assembled cell wall-embedded anchor structure. In some embodiments, the cancer therapeutic agent is displayed on a presentation structure (e.g., as described above in Section II(F)).

[0295] III. ADAS manufacturing method A. Creation of ADAS and highly active ADAS In some embodiments, production of an ADAS features a method for producing a composition comprising a plurality of ADASs, the composition being substantially free of viable bacterial cells, the method comprising: (a) producing, providing, or obtaining a plurality of parent bacteria having a reduced level or activity of a cell division topology specificity factor; (b) exposing the parent bacteria to conditions that allow for the formation of minicells, thereby producing a highly active ADAS; and (c) separating the highly active ADASs from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.

[0296] Parent bacteria include any suitable bacterial species from which an ADAS can be generated (e.g., a species that can be modified to produce an ADAS using the methods described herein). The following provides a non-limiting list of suitable genera from which an ADAS can be derived: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Baltic Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas nas), Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum (M agnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium,Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus us), Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella lla), Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus ), Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium or Thermoanaerobacter bacteria.

[0297] In some embodiments, any of the methods for producing an ADAS composition, e.g., a highly active ADAS composition, described in Section I herein is used. For example, methods for producing a highly active ADAS; a method for producing an ADAS lacking a cell division topology specificity factor and optionally lacking a Z-ring inhibitory protein (e.g., a method for producing an ADAS from a ΔminCDE parent bacterium); and a method for producing any of the ADASs mentioned herein, wherein the ADAS comprises a cargo.

[0298] In some embodiments, the ADAS (e.g., a highly active ADAS) is produced from a parent strain that is a plant bacterium, such as a plant commensal bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogenic bacterium (e.g., Xanthomonas species or Pseudomonas syringae), or a bacterium capable of plant rhizosphere colonization and / or root nodulation, such as a Rhizobia bacterium.

[0299] In some embodiments, the ADAS (e.g., a highly active ADAS) is produced from a parent strain that is a symbiont of an invertebrate, such as a symbiont of an arthropod (e.g., an insect or arachnid), a nematode, a protozoan, or an annelid. In embodiments, the invertebrate is a plant or animal pest or pathogen.

[0300] In some embodiments, the ADAS (eg, a highly active ADAS) is generated from a parent strain that is competent for genetic transformation, such as an Agrobacterium.

[0301] In some embodiments, the ADAS (e.g., a highly active ADAS) is produced from a parent strain that is a human bacterium or an extremophile, such as a commensal human bacterium (e.g., E. coli, Staphylococcus spp., Bifidobacterium spp., Micrococcus spp., Lactobacillus spp., or Actinomyces spp.) or a pathogenic human bacterium (e.g., Escherichia coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori).

[0302] In some embodiments, the ADAS and / or parent strain is a functionalized derivative of any of the foregoing, including, for example, functional cassettes that direct the bacterium to one or more of: secrete antibiotics, digest plastics, secrete insecticides, survive in extreme environments, create nanoparticles, incorporate into other organisms, respond to the environment, and produce a reporter signal.

[0303] In some embodiments, the parent bacterium includes functionalized derivatives of any of the foregoing, e.g., those that include functional cassettes, such as functional cassettes that induce the bacterium to do one or more of the following: secrete antimicrobials, digest plastics, secrete pesticides, survive extreme environments, make nanoparticles, integrate into other organisms, respond to the environment, and produce reporter signals.

[0304] In some embodiments, the ADAS is derived from a parent strain that has been engineered or induced to overexpress ATP synthase. In some more particular embodiments, the ATP synthase is heterologous to the parent strain. In certain particular embodiments, the parent strain contains a functional F o It is modified to express F1ATP synthase.

[0305] In certain embodiments, the ADAS provided by the present invention is obtained from a parent strain cultured under conditions selected from 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.

[0306] The highly active ADAS of any one of the preceding claims, made from an extremophile microorganism, comprising a functionalized derivative of any of the foregoing, e.g., a functional cassette, e.g., a functional cassette that induces the bacterium to do one or more of the following: secrete antimicrobials, digest plastics, secrete insecticides, survive extreme environments, make nanoparticles, integrate into other organisms, respond to the environment, and produce a reporter signal.

[0307] Due to bacterial diversity, ADAS can be produced with membranes modified to improve, for example, the biodistribution of the ADAS upon administration to target cells. In certain embodiments, the membrane is modified to be less immunogenic or immunostimulatory in plants or animals. For example, in certain embodiments, the ADAS is obtained from a parent strain, where the immunostimulatory capacity of the parent strain has been reduced or eliminated by post-production treatment with detergents, enzymes, or functionalization with PEG. In certain embodiments, the ADAS is produced from a parent strain, and the membrane is modified by knocking out the LPS synthesis pathway in the parent strain, for example, by knocking out msbB. In other detailed embodiments, the ADAS is produced from a parent strain that produces cell wall-defective particles upon exposure to hyperosmotic conditions.

[0308] In some embodiments, the method involves transforming the parent strain with an inducible DNase system, such as exoI (NCBI GeneID: 946529) and sbcD (NCBI GeneID: 945049) nucleases, or I-CeuI (e.g., Swissprot: P32761.1) nuclease. In more particular embodiments, the method involves using a single-, double-, triple-, or quadruple-auxotrophic strain and carrying the complementing genes on a plasmid encoding the inducible nuclease.

[0309] In some embodiments, the parent strain is cultured under conditions selected from 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 (4.5-6.5), or a combination thereof.

[0310] In certain embodiments, the parent strain lacks flagella and unwanted secretion apparatus, and optionally the flagella and unwanted secretion apparatus are removed using λRed recombineering.

[0311] In some embodiments, flagellar regulatory components are excised from the parental strain genome by insertion of a plasmid containing a CRISPR domain targeting flagellar regulatory genes, such as flhD and flhC.

[0312] In certain embodiments, a method is provided for producing a highly active ADAS, in which an ADAS containing a plasmid containing a rhodopsin-encoding gene is cultured in the presence of light. In more specific embodiments, the rhodopsin is proteorhodopsin from SAR86 uncultivated bacteria having the amino acid sequence of GenBank accession number AAS73014.1, or a functional fragment thereof. In even more specific embodiments, the culture is supplemented with retinal. In other more specific embodiments, the rhodopsin is proteorhodopsin and the plasmid further contains a gene that synthesizes retinal (such a plasmid is the pACYC-RDS plasmid from Kim et al., Microb Cell Fact, 2012).

[0313] In certain particular embodiments, the parental strain contains a nucleic acid sequence encoding a Nanobody that is in turn expressed on the membrane of the ADAS.

[0314] In some embodiments of the methods provided by the present invention, the parent strain contains nucleic acid sequences encoding one or more bacterial secretion apparatus operons. Exemplary plasmids include the Salmonella SPI-1 T3SS, the Shigella flexneri T3SS, the Agrobacterium (Agro) Ti plasmid, and the P. putida K1-T6SS apparatus.

[0315] In certain embodiments, the parent strain comprises a cargo. In some embodiments, the parent strain contains a nucleic acid sequence encoding a set of genes that synthesize a small molecule cargo.

[0316] IV. Purification of ADAS and ADAS Compositions In some embodiments of the methods and compositions provided herein, the ADAS is purified from a composition (e.g., a culture) comprising viable bacteria, e.g., parent bacteria. For example, the invention features a method for producing a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, the method including: (a) generating, providing, or obtaining a plurality of parent bacteria that exhibit a reduced level or activity of a cell division topology specificity factor; (b) exposing the parent bacteria to conditions that allow the formation of minicells, thereby producing the ADAS; and (c) separating the ADAS from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.

[0317] Purification separates the ADAS from viable parent bacterial cells, which contain the genome and may be larger. Separation of the highly active ADAS from the parent bacteria can be accomplished using a number of methods, as described herein. Exemplary purification methods described herein include centrifugation, selective growth, and buffer exchange / concentration processes.

[0318] In some aspects, provided herein are ADAS compositions, and methods for comparing such compositions, that are substantially free of parental and / or viable bacterial cells, e.g., have no more than 500 colony forming units (CFU) per mL, e.g., 400, 300, 200, 150, or 100 CFU per mL, or fewer than 50, fewer than 25, fewer than 10, fewer than 5, fewer than 1, or fewer than 0.1 CFU per mL. In some embodiments, an ADAS composition that is substantially free of parental bacterial cells does not contain any bacterial cells.

[0319] Auxotrophic parent strains can be used to produce the ADAS provided by the invention. As described in more detail below, such production methods are useful for purifying the ADAS. For example, in some embodiments, after production of the ADAS, the parent bacterial cells are removed by growing in a medium lacking nutrients (e.g., amino acids) essential for the viability of the parent bacteria. In some embodiments, the ADAS provided by the invention lacks nutrients such as arginine (e.g., knockout of argA, strains JW2786-1 and NK5992), cysteine ​​(e.g., arginine), arginine (e.g., knockout of argA, strains JW2786-1 and NK5992), cysteine ​​(e.g., arginine), arginine (e.g., arginine ... knockout of cysE (such as strains JW3582-2 and JM15), knockout of glutamine, e.g., glnA (such as strains JW3841-1 and M5004), knockout of glycine, e.g., glyA (such as strains JW2535-1 and AT2457), knockout of histidine, e.g., hisB (such as strains JW2004-1 and SB3930), knockout of isoleucine, e.g., ilvA (such as strains JW3745-2 and AB1255), knockout of leucine, e.g., leuB (such as strains JW5807-2 and CV514), lysine, e.g., knockout of lysA (such as strains JW2806-1 and KL334); methionine, e.g., knockout of metA (such as strains JW3973-1 and DL41); phenylalanine, e.g., knockout of pheA (such as strains JW2580-1 and KA197); proline, e.g., knockout of proA (such as strains JW0233-2 and NK5525); serine, e.g., knockout of serA (such as strains JW2880-1 and JC158); threonine, e.g., knockout of thrC (such as strains JW0003-2 and Gif 41), tryptophan, e.g., a knockout of trpC (such as strains JW1254-2 and CAG18455), tyrosine, e.g., a knockout of tyrA (such as strains JW2581-1 and N3087), or valine / isoleucine / leucine, e.g., a knockout of ilvd (such as strains JW5605-1 and CAG18431).

[0320] In certain embodiments, the method comprises using a single-, double-, triple-, or quadruple-auxotrophic parent strain, optionally wherein the parent strain further comprises a plasmid expressing ftsZ.

[0321] How to use V.ADAS A. ADAS Delivery Methods In one aspect, the invention features a method for delivering an ADAS (e.g., a highly active ADAS) to a target cell, the method including: (a) providing a composition comprising any of a plurality of ADAS provided herein, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a). In some embodiments, the plurality of ADAS is a plurality of highly active ADAS, and the ADAS has an initial ATP concentration of at least 1.25 mM.

[0322] In another aspect, the invention features a method for delivering an ADAS to a target cell, the method including: (a) providing a composition including any one of the ADASs provided herein; and (b) contacting the target cell with the composition of step (a).

[0323] In some embodiments, the target cell is, for example, an animal cell, a plant cell, or a fungal cell.

[0324] B. Cargo Delivery Method In another aspect, the invention features a method for delivering 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 including: (a) providing a composition comprising a plurality of any of the ADAS provided herein, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a). In some embodiments, the plurality of ADAS is a plurality of high-activity ADAS, and the ADAS has an initial ATP concentration of at least 1.25 mM.

[0325] In another aspect, the invention features a method for delivering 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 immunomodulatory agent, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method including: (a) providing a composition including any of a plurality of ADAS provided herein; and (b) contacting the target cell with the composition of step (a).

[0326] In another aspect, the invention features a method for delivering 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 including: (a) providing a composition comprising any of a plurality of ADAS provided herein, wherein the ADAS is derived from a parent bacterium having a reduction in the level or activity of a cell division topology specificity factor, the ADAS comprising the cargo, and the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a).

[0327] In some embodiments, the target cell to which the cargo is delivered is, for example, an animal cell, a plant cell, or a fungal cell.

[0328] In another aspect, the disclosure features a method for delivering a therapeutic agent, e.g., a cancer therapeutic agent, to a subject, the method including administering to the subject an ADAS or a composition including the same derived from a parent bacterial cell that includes at least one loss-of-function genetic modification in a lytic enzyme and at least one loss-of-function genetic modification in a protease, wherein the ADAS has anti-cancer properties.

[0329] In another aspect, the disclosure features a method for delivering a therapeutic agent, e.g., a cancer therapeutic agent, to a subject, the method including administering to the subject an ADAS derived from a parent bacterial cell that includes an engineered cell wall-embedded anchor structure for cargo presentation, or a composition including the same, wherein the ADAS has anti-cancer properties.

[0330] C. Methods for modulating the state of a cell In one aspect, the invention features a method for regulating the state of an animal cell, the method comprising: (a) providing a composition comprising any of the plurality of non-chromosomal dynamic activation systems (ADAS) provided herein, wherein the composition is substantially free of viable 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, and the ADASs have an initial ATP concentration of at least 1.25 mM.

[0331] In another aspect, the invention features a method for regulating the state of a plant cell, the method including: (a) providing a composition comprising any of the plurality of non-chromosomal dynamic activation systems (ADAS) provided herein, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant cell with the composition of step (a), thereby regulating the state of the plant cell. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, and the ADASs have an initial ATP concentration of at least 1.25 mM.

[0332] In another aspect, the invention features a method for regulating the state of an insect cell, the method including: (a) providing a composition comprising any of the plurality of non-chromosomal dynamic activation systems (ADAS) provided herein, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby regulating the state of the insect cell. In some embodiments, the plurality of ADASs are a plurality of high-activity ADASs, and the ADASs have an initial ATP concentration of at least 1.25 mM.

[0333] In another aspect, the invention features a method for modulating the state of an animal cell, the method including: (a) providing a composition including any of a plurality of ADAS provided herein, where the ADAS is derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, and the composition is substantially free of viable bacterial cells; and (b) contacting the animal cell with the composition of step (a), thereby modulating the state of the animal cell.

[0334] In another aspect, the invention features a method for regulating the state of a plant cell, the method including: (a) providing a composition including any of a plurality of ADAS provided herein, where the ADAS is derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, and the composition is substantially free of viable bacterial cells; and (b) contacting the plant cell with the composition of step (a), thereby regulating the state of the plant cell.

[0335] In another aspect, the invention features a method for regulating the state of an insect cell, the method including: (a) providing a composition including any of a plurality of ADAS provided herein, where the ADAS is derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, and the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby regulating the state of the insect cell.

[0336] In one aspect, the invention features a method for regulating a state of an animal cell, the method including: (a) providing a composition including any of the non-chromosomal dynamic activation systems (ADAS) provided herein; and (b) contacting the animal cell with the composition of step (a), thereby regulating the state of the animal cell.

[0337] In another aspect, the invention features a method for regulating the state of a plant cell, the method including: (a) providing a composition including any of the non-chromosomal dynamic activity systems (ADAS) provided herein; and (b) contacting the plant cell with the composition of step (a), thereby regulating the state of the plant cell.

[0338] In another aspect, the invention features a method for regulating the state of an insect cell, the method including: (a) providing a composition including any of the non-chromosomal dynamic activation systems (ADAS) provided herein; and (b) contacting an insect cell with the composition of step (a), thereby regulating the state of the insect cell.

[0339] In some embodiments, modulating is any observable change in the state (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state) of a cell (e.g., an animal, plant, or insect cell) as measured using techniques and methods known in the art for such measurement, e.g., methods that measure the level or expression of a protein, transcript, epigenetic mark, or methods that measure an increase or decrease in the activity of a biological pathway. In some embodiments, modulating the state of a cell involves increasing a cellular parameter (e.g., the level or expression of a protein, transcript, or activity of a biological pathway). In other embodiments, modulating the state involves decreasing a cellular parameter (e.g., the level or expression of a protein, transcript, or activity of a biological pathway).

[0340] D. Methods for Treating Animals, Plants, Insects, or Fungi In some aspects, the invention features a method for treating an animal in need thereof, the method comprising: (a) providing a composition comprising any of the plurality of non-chromosomal dynamic activation systems (ADAS) provided herein, wherein the composition is substantially free of viable 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, and the ADAS have an initial ATP concentration of at least 1.25 mM.

[0341] In some embodiments, the invention features the use of any of the ADAS provided herein in the manufacture of a medicament for the treatment of an animal, a plant, an insect, or a fungus.

[0342] In some embodiments, the invention features the use of any of the ADAS provided herein in the manufacture of a medicament for treating an animal, wherein the composition is substantially free of viable bacterial cells. In some embodiments, the ADAS has an initial ATP concentration of at least 1.25 mM.

[0343] In another aspect, the invention features a method for treating an animal in need thereof, the method including: (a) providing a composition comprising any of a plurality of ADAS provided herein, where the ADAS is derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, and the composition is substantially free of viable bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.

[0344] In some aspects, the invention features a method for treating an animal in need thereof, the method including: (a) providing a composition comprising any of the non-chromosomal dynamic activation systems (ADAS) provided herein; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.

[0345] In some embodiments, the animal in need of treatment has a disease, for example, cancer. In some embodiments, the ADAS carries a chemotherapeutic or immunotherapeutic cargo.

[0346] In some embodiments, the cargo is an anti-cancer therapeutic. In some embodiments, the cargo includes one or more wild-type or engineered antigens (or antibodies to antigens). In some embodiments, the antigen is derived from a tumor, e.g., a tumor-specific antigen, a tumor-associated antigen, a tumor neo-antigen, or a combination thereof. In some embodiments, the cargo is an antigenic, tumor antigenic, or protein, such as 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, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAG E-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 epitopes include BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, MC38 cancer epitopes, or combinations thereof.In some embodiments, the expressed cargo is an antigenic, tumor antigenic or protein, and may be 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, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, F Examples of such proteins include AP, 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.

[0347] 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 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 cargoes.

[0348] In some embodiments, the expressed cargo is antigenic, tumor antigenic or protein, including cyclic dinucleotides, STING activators such as cGAMP, c-di-AMP, IRF-3 and its variants.

[0349] In some embodiments, the cargo (e.g., an anti-cancer therapeutic or immunotherapy) is cisplatin, cyclophosphamide, 5-fluorouracil, bleomycin, an alkylating agent, an antimetabolite, an anthracycline, a topoisomerase inhibitor, a mitotic inhibitor, or a corticosteroid.

[0350] In some aspects, the invention features a method for treating a plant in need thereof, the method including: (a) providing a composition comprising any of the non-chromosomal dynamic activation systems (ADAS) provided herein, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant or a pest (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant. In some embodiments, the ADAS has an initial ATP concentration of at least 1.25 mM.

[0351] In another aspect, the invention features a method for treating a plant in need thereof, the method including: (a) providing a composition comprising any of a plurality of ADAS provided herein, where the ADAS is derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, and the composition is substantially free of viable bacterial cells; and (b) contacting the plant or a pest (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant.

[0352] In some aspects, the invention features a method for treating a plant in need thereof, the method including: (a) providing a composition comprising any of the non-chromosomal dynamic activation systems (ADAS) provided herein; and (b) contacting the plant or a pest (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant.

[0353] In an additional aspect, the present invention provides methods for modulating target cells. Target cells can be any cell, including animal cells (e.g., humans and farmed or domesticated animals, including non-human animals, including pests), plant cells (including those derived 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 allowing an ADAS provided by the present invention or a composition provided by the present invention to access the target cell in an effective amount. In some embodiments, access to the target cell can be direct, e.g., the target cell is directly regulated by the ADAS, such as by secretion of an agent in close proximity to the target cell or injection of an agent into the target cell, or indirect. Indirect modulation of the target cell can be by targeting another cell, e.g., by modulating a neighboring cell that may be commensal or pathogenic to the target cell. The neighboring cells, like the target cells, may be in vitro or in vivo, i.e., in an organism that may be commensal or pathogenic. These methods are collectively referred to as "methods of use provided by the present invention." In related aspects, the present invention provides targeted uses of the ADAS and compositions provided by the present invention according to the methods of use provided by the present invention.

[0354] For example, in some embodiments, the present invention provides a method for regulating the condition of an animal cell by making accessible to the animal cell an effective amount of an ADAS provided by the present invention or a composition provided by the present invention. In certain embodiments, the ADAS or composition is made accessible to the animal cell in vivo in an animal, e.g., a mammal, e.g., a human. In some embodiments, the animal cell is exposed to bacteria in a healthy animal. 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 genital epithelial cell, or a urinary tract cell. In even more specific embodiments, the animal cell is an intestinal epithelial cell, such as an intestinal epithelial cell from a human subject with inflammatory bowel disease, such as Crohn's disease or colitis. In even more particular embodiments, the animal cells are intestinal epithelial cells from a subject with inflammatory bowel disease, and the ADAS comprises a bacterial secretion apparatus and a cargo comprising an anti-inflammatory agent, for example, an antibody or antibody fragment that targets tumor necrosis factor (TNF) (e.g., an anti-TNF antibody); an antibody or antibody fragment that targets IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment that targets IL-23 (e.g., an anti-IL-23 antibody).

[0355] In other embodiments, the animal cells are exposed to the bacteria in a diseased state. In certain embodiments, the animal cells are pathogenic, such as a tumor. In other embodiments, the animal cells are exposed to the bacteria in a diseased state, such as a wound, ulcer, tumor, or inflammatory disorder.

[0356] In certain embodiments, the ADAS is derived from an animal commensal parent strain. In other embodiments, the ADAS is derived from an animal pathogenic parent strain.

[0357] In certain detailed embodiments, animal cells are contacted with an effective amount of an ADAS comprising a T3 / 4 SS or T6 SS and a cargo, and the cargo is delivered to the animal cells. In some specific embodiments, the animal cells are provided with access to an effective amount of an ADAS comprising a cargo and a secretion machinery, and the cargo is secreted extracellularly and contacted with the animal cells.

[0358] In some embodiments, the state of an animal cell is modulated by providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention with access to bacterial or fungal cells in the vicinity of the animal cell. That is, these methods involve indirectly modulating the state of the animal cell. In particular embodiments, the bacterial or fungal cell is pathogenic. In more particular embodiments, the fitness of the pathogenic bacterial or fungal cell is reduced. In other particular embodiments, the bacterial or fungal cell is commensal. In more particular embodiments, the fitness of the commensal bacterial or fungal cell is increased. In even more particular embodiments, the fitness of the commensal bacterial or fungal strain is increased by reducing the fitness of competing bacterial or fungal populations, which may be neutral, commensal, or pathogenic.

[0359] In certain detailed embodiments, bacterial or fungal cells in the vicinity of the animal cells are contacted with an effective amount of an ADAS comprising a T3 / 4 SS or T6 SS and a cargo, and the cargo is delivered to the bacterial or fungal cells. In other detailed embodiments, bacterial or fungal cells in the vicinity of the animal cells are made accessible to an effective amount of an ADAS that extracellularly secretes the cargo that contacts the bacterial or fungal cells.

[0360] In certain embodiments, the ADAS is derived from a parent strain that is a competitor of the bacterial or fungal cell. In other embodiments, the ADAS is derived from a parent strain that is a mutualist of the bacterial or fungal cell.

[0361] As will be apparent, the various methods of use provided by the present invention for modulating the state of animal cells can be readily adapted to corresponding methods for modulating the state of plant, fungal, or bacterial cells. For purposes of illustration, methods for modulating plant or fungal cells will be described in more detail.

[0362] Thus, in a related aspect, the present invention provides a method for regulating the condition of a plant or fungal cell by allowing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention to access: a) the plant or fungal cell, b) a neighboring bacterial cell or a neighboring fungal cell in proximity to the plant or fungal cell, or c) an invertebrate (e.g., an arthropod (e.g., an insect or arachnid), nematode, protozoan, or annelid) cell in proximity to the plant or fungal cell.

[0363] In certain embodiments, ADAS can be made accessible to plant cells in plants, for example, row crops including corn, wheat, soybean and rice, and vegetables including solanaceae such as tomato and pepper; Cucurbitaceae plants such as melon and cucumber; Brassicas such as cabbage and broccoli; Leafy vegetables such as kale and lettuce; Root vegetables and tubers such as potato and carrot; Large-seeded vegetables such as beans and corn; and crop plants such as mushrooms.In some embodiments, plant or fungal cells are exposed to bacteria in healthy plants or fungi.In other embodiments, plant or fungal cells are exposed to bacteria in diseased states.

[0364] In certain embodiments, the plant or fungal cell is dividing, such as a meristematic cell, or pathogenic, such as a tumor. In some embodiments, the plant or fungal cell is exposed to bacteria in a diseased state, such as a wound, i.e., the plant or fungal cell is not part of a human food source.

[0365] For certain embodiments, the ADAS is derived from a commensal parent strain. In other embodiments, the ADAS is derived from a plant or fungal pathogenic parent strain.

[0366] In some embodiments, the ADAS comprises a T3 / 4 SS or a T6 SS and a cargo, and the cargo is delivered to the plant or fungal cell. In other embodiments, the plant or fungal cell is provided with access to an effective amount of an ADAS comprising a bacterial secretion apparatus and a cargo, and the bacterial secretion apparatus secretes the cargo extracellularly, thereby contacting the plant or fungal cell with the cargo.

[0367] In some embodiments, the methods involve allowing an effective amount of the ADAS or composition to access neighboring bacteria or fungal cells in the vicinity of the plant or fungal cell. In more detailed embodiments, the neighboring bacteria or fungal cells are pathogenic, and optionally the fitness of the pathogenic neighboring bacteria or fungal cells is reduced. In other more detailed embodiments, the neighboring bacteria or fungal cells are commensal, and optionally the fitness of the commensal neighboring bacteria or fungal cells is increased. In even more detailed embodiments, the fitness is increased through the reduction of competing bacteria or fungi, which may be neutral, commensal, or pathogenic.

[0368] In some embodiments, adjacent bacterial or fungal cells are contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and a cargo, and the cargo is delivered to the adjacent bacterial or fungal cell.

[0369] In other embodiments, adjacent bacterial or fungal cells are provided with access to an effective amount of an ADAS comprising a bacterial secretion apparatus and a cargo, and the bacterial secretion apparatus secretes the cargo extracellularly, thereby contacting the adjacent bacterial or fungal cells with the cargo.

[0370] In some embodiments, the ADAS is derived from a parent strain that is a competitor of a neighboring bacterial or fungal cell. In other embodiments, the ADAS is derived from a parent strain that is a mutualistic bacterium of a neighboring bacterial or fungal cell.

[0371] In particular embodiments, the methods include allowing an effective amount of the ADAS or composition to access invertebrate (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cells that are in the vicinity of the plant or fungus. In more particular embodiments, the invertebrate is pathogenic. In even more particular embodiments, the fitness of the pathogenic invertebrate is reduced. In even more particular embodiments, the fitness of the pathogenic invertebrate is reduced through modulation of symbionts of the invertebrate. In other particular embodiments, the invertebrate is commensal. In more particular embodiments, the fitness of the commensal invertebrate is increased. In even more particular embodiments, the fitness is increased through reduction of competing bacteria or fungi, which may be neutral, commensal, or pathogenic.

[0372] In yet another aspect, the present invention provides a method for removing one or more undesirable substances from an environment, 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 (e.g., proteins, polymers, nanoparticles, binders, or combinations thereof) that absorb, chelate, or degrade the one or more undesirable substances. "Environment" is defined as non-cellular targets, such as oceans, soil, Superfund sites, skin, ponds, the intestinal lumen, and food in containers.

[0373] In certain embodiments, the undesirable material comprises a heavy metal such as mercury, and the ADAS comprises one or more molecules (e.g., proteins, polymers, nanoparticles, binders, or combinations thereof) that bind to the heavy metal, e.g., MerR for mercury. In some embodiments, the undesirable material comprises a plastic such as PET, and the ADAS comprises one or more plastic-degrading enzymes, such as PETase. In certain embodiments, the undesirable material comprises one or more small organic molecules, and the ADAS comprises one or more enzymes capable of metabolizing the one or more small organic molecules.

[0374] E. RNA delivery method In another aspect, the present invention provides a composition containing a 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 is suitable for delivery to a target cell via the T4SS. In a specific embodiment, the RNA-binding protein is Cas9 fused to VirE2 and VirF, the RNA cargo is a guide RNA, and optionally, the T4SS is a Ti system from Agrobacterium. In other embodiments, the RNA-binding protein is p19 from Carnation Italian Ringspot Virus fused to VirE2 or VirF, the RNA cargo is an siRNA, and optionally, the T4SS is a Ti system from Agrobacterium.

[0375] In related aspects, the invention provides methods for producing these particular compositions, which involve transfecting Agrobacterium cells with a plasmid containing Cas9 fused to VirE2 and VirF and an RNA cargo.

[0376] In a further related aspect, the invention provides a method for delivering RNA to a plant or animal cell, comprising contacting said plant or animal cell with a bacterium or ADAS, wherein the bacterium or ADAS comprises a T4SS, an RNA-binding protein cargo, and an RNA cargo, and wherein the RNA is delivered to the plant or animal cell. In more particular embodiments, the RNA-binding protein cargo is also delivered to the plant or animal cell.

[0377] VI. Other Embodiments Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments: Embodiment 1. A non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one loss-of-function genetic modification in a lytic enzyme and at least one loss-of-function genetic modification in a protease. Embodiment 2. The ADAS of embodiment 1, wherein the loss-of-function genetic modification in the protease results in increased expression of cargo by the ADAS compared to an ADAS from a parent bacterial cell that does not contain the modification. Embodiment 3. The ADAS of embodiment 1, wherein the loss-of-function genetic modification in the lytic enzyme results in increased stability of the ADAS compared to an ADAS derived from a parent bacterial cell that does not contain the modification. Embodiment 4. The ADAS of embodiment 1, wherein the loss-of-function genetic modification is a non-silent codon change, a deletion, an insertion, a mutation, or any combination thereof. Embodiment 5 The ADAS of embodiment 1, wherein the loss-of-function genetic modification is a deletion. Embodiment 6. The ADAS of embodiment 1, wherein the lytic enzyme is an endopeptidase, a cell wall lytic enzyme and / or an autolytic enzyme. Embodiment 7. The ADAS of embodiment 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. Embodiment 8 The ADAS of embodiment 6, wherein the loss-of-function genetic modification is in lytC. Embodiment 9 The ADAS of embodiment 1, wherein the parent bacterial cell further comprises a loss-of-function modification in a cell division topology specificity factor. Embodiment 10. The ADAS of embodiment 9, wherein the cell division topology specificity factor is a deletion or loss-of-function modification of a gene in the DivIVA, minC, minD, minE, minCD, or minCDE operon. Embodiment 11 The ADAS of embodiment 1, wherein the parent bacterial cell is Gram-positive. Embodiment 12 The ADAS of embodiment 1, wherein the parent bacterial cell is Gram-negative. Embodiment 13 The ADAS of embodiment 1, wherein the parent bacterial cell further comprises a loss-of-function genetic modification that disrupts sporulation. Embodiment 14. The ADAS of embodiment 13, wherein the loss-of-function genetic modification that disrupts sporulation is a loss-of-function modification in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, or a combination thereof. Embodiment 15 The ADAS of embodiment 13, wherein the loss-of-function genetic modification in a sporulation gene is in SigF. Embodiment 16. The parent bacterium is selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, and the like. dyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus , Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia,Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus rmus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium m), Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc c), Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium or Thermoanaerobacter. Embodiment 17 The ADAS of embodiment 11, wherein the parent bacterial cell is Bacillus subtilis. Embodiment 18. The ADAS of embodiment 1, wherein the loss-of-function genetic modification 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. Embodiment 19. The ADAS of embodiment 1, wherein the parent cell comprises genetic loss-of-function modifications 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. Embodiment 20. The ADAS of embodiment 19, wherein the gene loss-of-function modifications are in sigF, divIVA, lytc, and the protease deletions are in aprE, ispA, wprA, nprB, Mpr, Vpr, Epr, Bpr, and nprE, or a combination thereof. Embodiment 21. The ADAS of any one of embodiments 1 to 20, wherein the ADAS expresses one or more cargoes. Embodiment 22 The ADAS of embodiment 21, wherein the ADAS expresses cytosolic cargo, surface-displayed cargo, secreted cargo, or a combination thereof. Embodiment 23. The ADAS of embodiment 22, wherein the cargo is an antigen, cytokine, mutein, protein, polypeptide, small molecule, large molecule, nucleic acid, polynucleotide, enzyme, or any combination thereof. Embodiment 24. A non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell that contains an engineered cell wall-embedded anchor structure for cargo presentation. Embodiment 25 The ADAS of embodiment 24, wherein the parent bacterial cell further comprises one or more loss-of-function genetic modifications in lytic enzymes. Embodiment 26 The ADAS of embodiment 25, wherein the parent bacterial cell further comprises one or more loss-of-function genetic modifications in a protease. Embodiment 27. The ADAS of embodiment 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. Embodiment 28 The ADAS of embodiment 25, wherein the loss-of-function genetic modification in the lytic enzyme is in lytC. Embodiment 29 The ADAS of embodiment 26, wherein the parent bacterial cell further comprises a loss-of-function modification in a cell division topology specificity factor. Embodiment 30. The ADAS of embodiment 29, wherein the cell division topology specificity factor is a DivIVA, minC, minD, minE, minCD, or minCDE operon. Embodiment 31 The ADAS of embodiment 24, wherein the parent bacterial cell is Gram-positive. Embodiment 32 The ADAS of embodiment 24, wherein the parent bacterial cell is Gram-negative. Embodiment 33 The ADAS of embodiment 24, wherein the parent bacterial cell further comprises a loss-of-function genetic modification that disrupts sporulation. Embodiment 34. The ADAS of embodiment 33, wherein the loss-of-function genetic modification that disrupts sporulation is a loss-of-function modification in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, or a combination thereof. Embodiment 35 The ADAS of embodiment 33, wherein the loss-of-function modification in the sporulation gene is in SigF. Embodiment 36. The parent bacterial cell is selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea a), Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus (Methylococcus), Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter,Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga oga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium bacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc 25. The ADAS of embodiment 24, wherein the ADAS is a bacterium of the genus Staphylococcus, Streptococcus, Symbiobacterium or Thermoanaerobacter. Embodiment 37 The ADAS of embodiment 31, wherein the parent bacterial cell is Bacillus subtilis. Embodiment 38. The ADAS of any one of embodiments 26 to 30, wherein the loss-of-function genetic modification 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. Embodiment 39. The ADAS of embodiment 26, wherein the parent cell comprises loss-of-function genetic modifications 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. Embodiment 40. The ADAS of embodiment 39, wherein the parent bacterial cell comprises a loss-of-function genetic modification in each of sigF, divIVA, and lytc, and in one or more of the protease genes aprE, ispA, wprA, nprE, epr, vpr, bpr, mpr, nprB, or a combination thereof. Embodiment 41. The ADAS of any one of embodiments 24 to 40, wherein the ADAS expresses one or more of a cytosolic cargo, a surface-displayed cargo, a secreted cargo, or a combination thereof. Embodiment 42. The ADAS of embodiment 41, wherein the cargo is an antigen, tumor antigen, cytokine, mutein, protein, polypeptide, small molecule, large molecule, nucleic acid, polynucleotide, enzyme, IL-2, IL-2 superkine, or any combination thereof. Embodiment 43. The ADAS of embodiment 42, wherein the ADAS expresses cargo via an engineered cell wall-embedded anchor structure. Embodiment 44. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor structure is a membrane anchor domain, an outer membrane anchor domain, an inner membrane anchor domain, a cell wall anchor domain or a functional equivalent thereof. Embodiment 45. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor structure is an antiparallel coiled coil. Embodiment 46. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor structure has a COOH terminal portion embedded in the cell wall. Embodiment 47 The ADAS of embodiment 46, wherein the COOH-terminus is comprised in a sorting signal comprising a COOH-terminus and an LPXTG motif, a hydrophobic domain, a tail of a majority of positively charged residues, or any combination thereof. Embodiment 48. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor structure is a truncated antiparallel coiled-coil, a linker, a coiled linker, or a fusion construct. Embodiment 49 The ADAS of any one of embodiments 1 to 48, wherein the parent bacterial cell further comprises at least one additional loss-of-function genetic modification in the enzyme. Embodiment 50 The ADAS of embodiment 49, wherein the parent bacterial cell comprises a loss-of-function genetic modification in one or more amylases. Embodiment 51. The ADAS of embodiment 50, wherein the amylase is amyE, amyR2, amyl, amyS, amyX, bbmA, malA, malS, susG, amyA, treS, pulA, or a combination thereof. Embodiment 52. The ADAS of embodiment 51, wherein the parent bacterial cell comprises a loss-of-function genetic modification in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof. Embodiment 53 The ADAS of embodiment 49, wherein the parent bacterial cell further comprises a loss-of-function genetic modification in one or more lipases. Embodiment 54. The ADAS of embodiment 53, wherein the lipase is lip, lipA, estA, estB, lipC, lip1, lip2, ytpA, hlyC, plhC, or a combination thereof. Embodiment 55. The ADAS of embodiment 49, wherein the parent bacterial cell further comprises a loss-of-function gene in one or more cellulases. Embodiment 56. The ADAS of embodiment 55, wherein the cellulase is celE, celS, bcsZ, eglS, celZ, cel-3, celI, celCCA, celVI, engXCA, engB, celG, celH, or a combination thereof. Embodiment 57. The ADAS of embodiment 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 greater than the stability of the cargo in a control ADAS. Embodiment 58. The ADAS of embodiment 43, wherein the ADAS is administered in an amount of more than 1, more than 10, more than 100, more than 1000 or more than 2000 ADAS per cell. Embodiment 59. The ADAS of embodiment 43, wherein the parent 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. Embodiment 60. The ADAS of any one of embodiments 1 to 59, wherein the ADAS is to be delivered via subcutaneous, intraperitoneal, intravenous, intramuscular, oral, aerosol or nebulized administration. Embodiment 61. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 62. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 63. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 64. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 65. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 66. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 67. The ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor 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. Embodiment 68. The ADAS of any one of embodiments 43 to 67, wherein the cell wall-embedded anchor structure contains a cell wall binding domain. Embodiment 69 The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure contains a fusion promoter. Embodiment 70. The ADAS of embodiment 69, wherein the promoter is PrrnI-Pveg. Embodiment 71. The ADAS of embodiment 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. Embodiment 72. The ADAS of embodiment 43, wherein the ADAS contains a proline-rich sequence that encodes a cell wall-embedded anchor structure. Embodiment 73. The ADAS of embodiment 43, wherein the ADAS contains a self-binding coil for a cell wall-embedded anchor structure. Embodiment 74 The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure is an unstructured proline-rich construct. Embodiment 75. The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure is a construct with a defined structure. Embodiment 76 The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure contains a promoter. Embodiment 77. The ADAS of embodiment 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. Embodiment 78. The ADAS of embodiment 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. Embodiment 79. The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure sequence contains a ribosome binding site. Embodiment 80. The ADAS of embodiment 79, wherein the binding site is an 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. Embodiment 81 The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure sequence contains a secretion signal. Embodiment 82. The ADAS of embodiment 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. Embodiment 83 The ADAS of any one of embodiments 43 to 67, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is separated by a nucleotide sequence encoding the cargo. Embodiment 84. The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure uses a non-covalent cell-binding domain. Embodiment 85. The ADAS of embodiment 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. Embodiment 86. The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure uses a covalent cell-binding domain. Embodiment 87. The ADAS of embodiment 86, wherein the binding domain is a 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. Embodiment 88 The ADAS of embodiment 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure comprises a terminator sequence. Embodiment 89 The ADAS of embodiment 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is integrated into the deleted protease site. Embodiment 90 The ADAS of embodiment 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is integrated into the deleted amylase site. Embodiment 91 The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure is incorporated into a deleted phosphodiesterase site. Embodiment 92. The ADAS of embodiment 72, wherein the proline-rich sequence is a linker that is at least 25%, 27%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% proline. Embodiment 93. A method for treating or preventing cancer in a subject, comprising: administering to the subject one or more doses of the ADAS of any one of embodiments 1 to 92 or a composition comprising same. Embodiment 94. The ADAS of embodiment 93, wherein the ADAS has anti-cancer properties. Embodiment 95. The method of embodiment 94, wherein the ADAS is administered by oral, intravenous, intradermal, intramuscular, intranasal, intraocular, intrarectal, intraperitoneal, intratumoral, aerosol and / or subcutaneous administration, or any combination thereof. Embodiment 96. The method of embodiment 95, wherein the ADAS is administered to the subject at a dose of at least 1×10, 1×10, 1×10, 1×10, 5×10, 6×10, 8×10, 1×10, 2×10, 4×10, 6×10, 8×10, or 1×10 ADAS. Embodiment 97. The method of embodiment 94, wherein 1, 2, 3, 4 or more than 4 doses of the ADAS are administered to the subject. Embodiment 98. The method of embodiment 97, wherein one or two doses of the ADAS composition are administered to the subject. Embodiment 99. The ADAS of embodiment 94, wherein the cargo comprises one or more tumor antigens. Embodiment 100. The ADAS of embodiment 99, wherein the cargo further comprises a known anti-cancer cytokine. Embodiment 101. The ADAS of embodiment 100, wherein the cytokine is IL-2 or an IL-2 mutein. Embodiment 102. The ADAS of embodiment 101, wherein the ADAS cargo further comprises a membrane pore-forming toxin. Embodiment 103. The ADAS of embodiment 43, wherein the ADAS cargo comprises a pore-forming toxin and at least one other cargo component. Embodiment 104. The ADAS of embodiment 102 or 103, wherein the toxin is listeriolysin O. Embodiment 105. An ADAS of embodiment 43, wherein the constructed cell wall-embedded anchor structure has an N-terminal portion embedded in the cell wall. Embodiment 106. The ADAS of embodiment 43, wherein the anchor structure contains an internal cell wall-embedded anchor domain. Embodiment 107. The ADAS of embodiment 43, wherein the cell wall-embedded anchor structure incorporates a protease cleavage site. Embodiment 108. The ADAS of any one of embodiments 1 to 43, wherein the parent 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 to 48. [Example]

[0378] Having generally described the invention above, the invention will be more readily understood by reference to the following examples, which are included solely for purposes of illustration of certain aspects and embodiments of the invention and are not intended to be limiting thereof. It will be appreciated that various other embodiments may be practiced in light of the general description provided above.

[0379] Example 1. Production of ADAS. Production of ADAS from parent bacterial cells can be achieved using the methods disclosed in WO 2020 / 123569, which is incorporated herein by reference in its entirety. Briefly, these methods and variations thereof are described below.

[0380] In this example, ADAS is produced by disruption of one or more genes involved in regulating parental cell division functions, i.e., ΔminC, ΔminD, ΔminCDE, ΔminCdivIVA, or ΔdivIVA. This example details genetic means to generate ADAS-producing strains via disruption of the min operon or overexpression of the septation machinery component FtsZ.

[0381] A. Production of ADAS via min mutation To disrupt the min operon, the Lambda-RED recombineering method was employed according to the protocol described in Datsenko and Wanner, PNAS, 97(12):6640-6645, 2000. Strains engineered to contain Lambda-RED-based plasmids were obtained from the Coli Genetic Stock Center (CGSC) at Yale University. Briefly, primers were designed to nonpolarly delete the coding sequence of the entire E. coli minC, minD, or minCDE operon by encoding approximately 40 base pairs of genomic homology at the 5' end of the primers. The 3' ends of these primers are homologous to the Lambda-RED-based plasmids pKD3 and pKD4, which provide antibiotic markers used to select parental bacterial strains inheriting the targeted mutation. The sequences targeted for deletion are provided in Table 2. After performing standard PCR using primers with pKD3 as the DNA template, the purified amplicon was transformed via electroporation into bacteria prepared with pKD46, a plasmid containing the phage-derived Lambda-RED homologous recombination system, according to the method of Datsenko and Wanner, PNAS, 97(12):6640-6645, 2000. Transformants were selected on LB agar containing 35 pg / mL chloramphenicol. The resulting colonies were confirmed to contain the gene disruption (i.e., AminC, AminD, or AminCDE) using standard allele-specific PCR.

[0382] Production of ADAS via B. divIVA mutations Briefly, primers were designed to delete the coding sequences of B. subtilis divIVA or divIVA and minC. The sequences targeted for deletion are listed in Table 2, including both WT and divIVA deletions. Parent strain MACH2347 was generated by deleting divIVA along with a deletion of sigF (sequence in Table 2), which prevents sporulation. The strain genotype is provided in Table 1. Introducing an erm (erythromycin) cassette into growing bacteria along with the selected deletion primers allowed for selection of transformed colonies through growth on LB-erm5 plates. Once transformed bacteria were cultured, the erm cassette was cured from the colonies using a temperature-sensitive plasmid conferring resistance to spectinomycin (Spec). Transformed bacteria were selected by plating on LB-Spec plates and incubated at 30°C. Selected bacteria were then streaked onto LB plates containing no antibiotic and incubated at 42°C to induce loss of the temperature-sensitive plasmid. Isolated colonies were confirmed to contain the intended deletion.

[0383] Production of ADAS by overexpression of C. ftsZ To generate ADAS from overexpression of the septation machinery, we constructed a plasmid driving expression of the FtsZ Z-ring protein from wild-type Escherichia coli (E. coli). Briefly, the strong ribosome binding site and coding sequence of the E. coli FtsZ protein were de novo optimized using De Novo DNA computational tools. This translation unit was ordered for de novo DNA synthesis from Integrated DNA Technologies (IDT™) and cloned into a backbone using standard cloning techniques. The resulting plasmid, pFtsZ (Table 3), features the TetR repressor, the TetA promoter repressed by the TetR protein, a kanamycin resistance marker, and the pMB1 origin of replication. When transformed into competent bacteria, pFtsz can be induced to overproduce FtsZ protein via the addition of anhydrotetracycline to the culture. Furthermore, this protein is capable of spontaneous protofilament formation, which leads to asymmetric division of the parent bacterial cell, thereby producing ADAS.

[0384] Example 2. Purification of ADAS from Bacillus subtilis Purification of ADAS from parent bacterial cells can be achieved using the methods disclosed in WO 2020 / 123569, which is incorporated herein by reference in its entirety. Briefly, these methods and variations thereof are described below.

[0385] This example describes a method for purifying a population of ADAS from a culture of an ADAS-producing bacterial parent strain. This method can be used to purify any of the ADAS-producing strains described herein, including the strains in Example 1 and Tables 1 and 3. Purification separates ADAS from larger, genome-containing, viable parent bacterial cells. ADAS was purified from high-cell-density cultures of ADAS-producing strains through a combination of 1) high- or low-speed centrifugation, 2) selective growth, and 3) buffer exchange / concentration. The centrifugation procedure was used to selectively reduce viable parent bacterial cells and large cellular debris while concentrating ADAS in a mixed suspension. The selective growth procedure was used to reduce the number of viable parent bacterial cells present in the sample through the addition of compounds that are directly antimicrobial (i.e., toxic to cells carrying the microbial genome) and / or compounds that enhance the sedimentation of viable cells via centrifugation. The buffer exchange / concentration procedure was used to transfer ADAS from a larger volume of bacterial culture medium to a smaller volume of 1x PBS while removing culture additives and cellular debris.

[0386] A.ADAS purification ADAS-producing strains were generated using the molecular cloning procedure described in Example 1 and then grown to high cell density in culture medium. Cultures can be expanded, for example, from 1 mL to 1000 mL or more of culture medium.

[0387] The culture was transferred to a centrifuge tube and subjected to a high- or low-speed centrifugation procedure aimed at pelleting intact cells and large cell debris while maintaining ADAS in the supernatant. The centrifugation procedure was performed at 4°C or room temperature. In some cases, a low-speed centrifugation procedure involving a series of consecutive 10-minute spins at 1,000 x g, 2,000 x g, 3,000 x g, and 4,000 x g was used and performed in an Allegra® X14R tabletop centrifuge (Beckman Coulter) or an Eppendorf™ 5424 R tabletop centrifuge (Fisher Scientific). In some cases, the low-speed centrifugation procedure consisted of consecutive 2,000 x g spins for 20 minutes at 4°C, with the supernatant from the first spin being transferred to a sterile centrifuge bottle before the second spin. In some cases, the low-speed centrifugation procedure consisted of a single 40-minute spin at 4,000 × g in a Sorvall™ Lynx 6000 Superspeed Centrifuge (Thermo Scientific™), with the rotor acceleration speed set to the lowest possible setting. In some cases, a high-speed centrifugation procedure was used with successive pulses of 20,000 × g; the spinning was stopped as soon as the required speed was reached, and the supernatant was transferred to a new high-speed bottle before the next spin. In some cases, the high-speed centrifugation procedure consisted of a 30-minute spin at 4°C and 17,000 × g, after which the pellet was resuspended in growth medium.

[0388] After low-speed centrifugation, the culture supernatant was transferred to a sterile culture tube and subjected to a selective growth process. After high-speed centrifugation, the culture supernatant was transferred, and the pellet was resuspended and subjected to a selective growth process. In some cases, concentrated antibiotic solutions (e.g., spectinomycin, clindamycin, tetracycline, ceftriaxone, kanamycin, carbenicillin, gentamicin, and / or ciprofloxacin) or other concentrated chemical solutions (e.g., sodium chloride, sodium hydroxide, M hydrochloric acid, glucose, cas-amino acids, and / or D-amino acids) were added directly to the culture supernatant. In other cases, the culture supernatant was pelleted via high-speed centrifugation at 10,000 × g to 20,000 × g for 5 to 60 minutes, and the pellet was resuspended in fresh culture medium containing an antibiotic or other chemical solution at a concentration inhibitory to viable cells. Selective growth was performed by incubating the ADAS at 4 °C to 42 °C for 1 to 3 hours with agitation at 250 rpm. The ADAS was then transferred to a sterile centrifuge tube and centrifuged one additional time.

[0389] After selective growth and centrifugation, the supernatant was subjected to a buffer exchange / concentration procedure. In some cases, this was accomplished by passing the supernatant through a 0.2 pm asymmetric polyethersulfone (aPES) membrane filter (Thermo Fisher) followed by 1–9 volumes of 1x PBS. In some cases, ADAS were pelleted via centrifugation at 10,000–20,000×g for 5–60 minutes, washed in 1–9 volumes of 1x PBS, pelleted again, and resuspended in 1x PBS at a concentration of 1–100,000× from the starting culture volume. In other cases, ADAS were pelleted via successive 1-minute fast pulses at 16,000×g, followed by a longer high-speed spin at 20,000×g for 20 minutes at 4°C, after which the pellet was resuspended and then washed several times. In some cases, the wash consisted of a 5-minute spin at 15,000×g at 4°C.

[0390] B. Purification of ADAS from the Auxotrophic ADAS-Producing Parent Strain ADAS-producing parent strains that are auxotrophic, i.e., unable to synthesize an organic compound required for growth, are useful for ADAS production. Such strains can grow only if the organic compound is provided. Auxotrophic parent strains may therefore be selected for by storing or incubating ADAS preparations in media lacking the organic compound, thus providing an additional method for reducing the parental load in ADAS preparations.

[0391] Example 3: Genomic deletions in ADAS Using methods similar to those described in Example 1B, primers were designed to delete coding sequences of interest (e.g., deletion of genomic sequences encoding lytic enzymes or proteases). Sequences targeted for deletion are listed in Table 2, which includes both wild-type (WT) sequences and sequences representing deletions of each gene of interest. For example, SEQ ID NO: 4 represents the wild-type genomic region containing SigF, and SEQ ID NO: 5 represents the genomic region after a loss-of-function deletion. SEQ ID NO: 6 represents the wild-type genomic region containing divIVa, and SEQ ID NO: 7 represents the genomic region after a loss-of-function deletion. SEQ ID NO: 8 represents the wild-type genomic region containing lytC, and SEQ ID NO: 9 represents the genomic region after a loss-of-function deletion. SEQ ID NO: 10 represents the wild-type genomic region containing aprE, and SEQ ID NO: 11 represents the genomic region after a loss-of-function deletion. SEQ ID NO: 12 represents the wild-type genomic region containing ispA, and SEQ ID NO: 13 represents 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 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 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 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 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 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 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 loss-of-function deletion.

[0392] Strain genotypes are provided in Table 1. Introducing an erm (erythromycin) cassette into growing bacteria along with selected deletion primers allowed for selection of transformed colonies through growth on LB-erm5 plates. Once the transformed bacteria were cultured, the erm cassette was cured from the colonies using a temperature-sensitive plasmid conferring resistance to spectinomycin (Spec). Transformed bacteria were selected by plating onto LB-Spec plates and incubated at 30°C. Selected bacteria were then streaked onto LB plates containing no antibiotic and incubated at an elevated temperature of 42°C, leading to loss of the temperature-sensitive plasmid. Isolated colonies were confirmed to contain the intended deletions, and a similar process was used if additional genomic deletions were required. Deletion combinations of genes of interest were generated in B. subtilis as listed in Tables 1-3, and additional deletions or insertions to generate these strains are listed in Tables 1-5. The listed deletions should not be considered limiting.

[0393] Example 4: Cargo Expression In this example, methods for optimizing cargo expression in ADAS and demonstration of functional cargo expression are presented. Briefly, these methods and variations thereof are described below. The parent and cargo-modified strains are listed in Tables 1 and 3. The listed deletions and modifications should not be considered limiting.

[0394] A. Expression of surface-displayed cargo Parent B. subtilis cells were genetically modified as described in Examples 1-3. MACH2762 was further modified to express the superkine IL-2 (MACH2889), and the presence of this cargo on the bacterial surface was determined via immunofluorescence microscopy. Briefly, overnight cultures of bacteria were incubated at OD 600The cells were normalized to a r = 2 and fixed in 4% formaldehyde for 30 minutes at room temperature. Fixed cells were washed twice with PBS and blocked in 2% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature with gentle agitation. Cells were then incubated in primary antibody (rabbit anti-IL-2 at 1:400 in 50 μL) followed by secondary antibody (goat anti-rabbit Alexa-594 at 1:400 in 50 μL). Between incubations, cells were washed twice with PBS. The final stained bacteria were resuspended in PBS, placed on a pad of 2% agarose in PBS, and imaged with a Leica DMi8 Thunder inverted microscope equipped with a 63X phase-contrast objective. Fluorescent images were captured with a Leica DFC9000 camera and a rhodamine filter set. IL-2 signal per cell was quantified by dividing the background-subtracted fluorescent signal by the total cell area for 20–100 cells per strain across multiple fields of view. Extensive IL-2 staining was observed on the surface of MACH2889 but not MACH2762, indicating that the optimized cargo expression construct utilized in MACH2889 was sufficient for surface display of superkine IL-2. When this superkine IL-2 surface display module was introduced into unmodified B. subtilis (MACH2887) and B. subtilis lacking sigF and lytC (MACH2888), neither strain showed an increase in superkine IL-2 staining compared to the MACH2762 negative control, indicating that deletion of one or more proteases is required for surface display of superkine IL-2 (Figure 1, Table 3).

[0395] B. Functionality of Surface-Displayed Cargo MACH2762 was further modified to express human IL-2 variants, including wild-type (WT), S1, and S2, on the cell surface via separate constructs 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 various amounts (ADAS per cell) of ADAS (Figure 2A). This reporter cell line was engineered with a secreted alkaline phosphatase (SeAP) reporter, whose production is proportional to IL-2 receptor activation, which can be quantified by measuring absorbance at 620 nm using the QuantiBlue detection protocol. While the ADAS that did not display IL-2 (MACH2762 and MACH2788) were unable to activate the IL-2 receptor at any dose, the ADAS with the IL-2 expression and surface display modules showed dose-dependent activation of the IL-2 receptor. Higher doses of ADAS presenting IL-2 led to increased IL-2 receptor activation up to an absorbance of approximately 1.25, at which point the assay saturates and the effect of increased IL-2 receptor activation becomes obscured by limited sensitivity. At doses of ADAS that did not saturate the assay (absorbance at 620 nm <1.25), equivalent doses of ADAS were observed to have variable abilities to activate the IL-2 receptor, depending in part on the IL-2 variant and presentation structure (Figure 2B). These data suggest that different cargos (e.g., IL-2 variants) and presentation structures (0–6) enable tunable target engagement through multiple possible mechanisms, including, but not limited to, modulating cargo access or stability.

[0396] The functionality of surface-displayed IL-2 alone or in combination with additional immunomodulatory cargo for synergistic effects was further demonstrated through in vivo cytokine profiling of mouse plasma and ex vivo cytokine profiling of human peripheral blood mononuclear cell culture supernatants (PBMCs). For plasma cytokine profiling, ADAS derived from B. subtilis (MACH2762) or a similar parent strain were engineered for surface display of the superkine IL-2 (MACH2782), cytosolic delivery of the cyclic dinucleotides (CDNs) cyclic-di-AMP and cyclic-di-GMP via the membrane 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 h after intravenous administration of 1E9 ADAS in 100 μL of phosphate-buffered saline (PBS) or an equal volume of PBS, which served as a negative control. The collected blood was processed and diluted to obtain plasma, and analyzed using the V-PLEX Mouse cytokine 19-plex kit (Meso Scale Diagnostics) according to the manufacturer's protocol to measure plasma concentrations of the cytokines IFNg, IL-2, IL-6, and TNFa (Figures 3A–3D). The data demonstrate the in vivo systemic immune response to both surface and cytosolic ADAS cargoes, as well as the combination of the two. Surface display of IL-2 alone (MACH2782) led to the highest induction of murine IL-2, IL-6, and TNFa at 2 h post-injection; however, incorporation of the CDN cargo and cytosolic delivery mediated by listeriolysin O (MACH2880) induced the highest levels of IFNg at 6 h post-injection. For ex vivo cytokine profiling, PBMCs from healthy human donors were incubated in cell culture with various doses of the ADAS series described above for 24 h, at which point culture supernatants were harvested. Cytokines released into the supernatant were quantified via Human XL Cytokine Luminex Performance Panel (Luminex) according to the manufacturer's protocol.At low effective doses of ADAS (e.g., 1 ADAS per PBMC), there was little difference between the cytokine profiles elicited by control ADAS (MACH2762) compared with ADAS deploying surface-displayed superkine IL-2 (MACH2782), cytosolic CDN delivery (MACH2854), or both immunomodulatory payloads (MACH2880). Overall, ADAS with surface-displayed superkine IL-2 (MACH2782 and MACH2880) stimulated the highest levels of anticancer cytokines, with the exception of TNFα (Figures 3E–3G), which was observed at its highest level when MACH2854 was administered at 1,000 ADAS per PBMC (Figure 3H). The data demonstrate human responses to both surface and cytosolic ADAS cargoes, as well as the combination of the two. Collectively, these data demonstrate that ADAS engineered with surface-displayed IL-2, cytosolic CDN delivery, or a combination of the two stimulate substantial levels of anticancer cytokines in vivo and ex vivo compared with control ADAS or negative control treatment. Furthermore, these complementary experimental systems provide evidence that the immune response to modified ADAS is consistent between mouse and human models, demonstrating that at least two distinct immunomodulatory cargoes can be deployed from a single ADAS.

[0397] To test whether ADAS can be used to elicit antigen-specific immune cell activation in vivo, we generated parent strains of B. subtilis producing ADAS modified with different forms of the model protein antigen, ovalbumin, and tested their ability to stimulate antigen-specific CD8 T cell responses in vivo. OT-1 mice (The Jackson Laboratory), a transgenic mouse line with a CD8 T cell receptor engineered to recognize an immunogenic peptide within the ovalbumin protein, were injected with 50 μL of subcutaneous PBS, 100 μg of subcutaneous ovalbumin in PBS, or MACH2762 (control ADAS - 1 × 10 subcutaneously in 50 μL PBS). 9ADAS), MACH2670 (ovalbumin and listeriolysin intravenously) - 1 x 10 subcutaneously in 50 μL PBS 9 Mice were administered ADAS derived from ovalbumin (ADAS) or MACH2829 (surface ovalbumin—1E9 ADAS intravenously in 100 μL PBS). 24 hours later, mice were euthanized, and the spleens and draining lymph nodes of each mouse were harvested and combined. The combined 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 combined fixation and permeabilization procedure, stained with a second panel of fluorescent antibodies targeting proteins within immune cells, and analyzed by flow cytometry. Upon exposure to their target antigen, CD8 T cells additionally produce 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 positive for CD45 and CD8. Next, we used a negative control group that was not exposed to 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 cytosolic ovalbumin and listeriolysin or ADAS displaying ovalbumin on the cell surface were sufficient to stimulate high levels of antigen-specific CD8 T cell activation 24 h after a single administration. No increase in the percentage of granzyme B-positive cells was observed in animals administered control ADAS. Collectively, these data demonstrate that ADAS modified with heterologous protein antigens are capable of activating antigen-specific CD8 T cells in vivo via multiple antigen expression strategies and multiple routes of administration.

[0398] Example 5: Cancer Therapeutic Agents 500,000 MC38 cells were implanted into the flanks of mice and allowed to grow for 9 days, at which point tumor volumes were measured with calipers and mice were randomized into approximately 10 treatment groups, with a mean tumor volume of approximately 100 cubic millimeters per group. On day 10, mice received an injection of one of four test articles: 200 μg intraperitoneal anti-PD-1, 50 μL peritumoral PBS, 4×10 derived from MACH2854 in 50 μL PBS. 9 6 × 10 derived from peritumoral ADAS (carrying LLO, DTT-MC38, and cyclic di-AMP cargo) or MACH2862 in 100 μl PBS 8 Intravenous ADAS (carrying LLO, surface FLAG-MC38, cytosolic DTT-MC38, and surface superkine hIL-2 cargo) was administered. Tumor volumes were measured again 14 days after implantation (Figure 5). Animals treated with anti-PD-1 (positive control) had significantly smaller tumors than those treated with PBS (negative control) when compared by T-test. Animals treated with ADAS had significantly smaller tumors than the PBS group, demonstrating the functional efficacy of cargo-expressing ADAS as a potential therapeutic agent via multiple immune stimulatory strategies and multiple routes of administration. The parental and cargo-modified strains are listed in Tables 1 and 3.

[0399] [Table 1]

[0400] [Table 2]

[0401] [Table 3]

[0402] [Table 4]

[0403] [Table 5]

[0404] Table 6

[0405] Table 7

[0406] Table 8

[0407] Table 9

[0408] Table 10

[0409] Table 11

[0410] Table 12

[0411] Table 13

[0412] Table 14

[0413] Table 15

[0414] Table 16

[0415] Table 17

[0416] Table 18

[0417] Table 19

[0418] Table 20

[0419] Table 21

[0420] Table 22

[0421] Table 23

[0422] Table 24

[0423] Table 25

[0424] Table 26

[0425] Table 27

[0426] Table 28

[0427] Table 29

[0428] Table 30

[0429] Table 31

[0430] Table 32

[0431] Table 33

[0432] Table 34

[0433] Table 35

[0434] Table 36

[0435] Table 37

[0436] Table 38

[0437] Table 39

[0438] Table 40

[0439] Table 41

[0440] Table 42

[0441] Table 43

[0442] Table 44

[0443] Table 45

[0444] The foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. Other embodiments are within the scope of the claims.

Claims

1. A non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell containing at least one loss-of-function genetic modification in a lytic enzyme and at least one loss-of-function genetic modification in a protease.

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

3. 2. The ADAS of claim 1, wherein the loss-of-function genetic modification in a lytic enzyme results in increased stability of the ADAS compared to an ADAS derived from a parent bacterial cell that does not contain the modification.

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

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

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

7. 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 genetic modification is in lytC.

9. The ADAS of claim 1, wherein the parent bacterial cell further comprises a loss-of-function modification in a cell division topology specificity factor.

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

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

12. The ADAS of claim 1 , wherein the parent bacterial cell is gram-negative.

13. 2. The ADAS of claim 1, wherein the parent bacterial cell further comprises a loss-of-function genetic modification that disrupts sporulation.

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

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

16. The parent bacterium may be selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, and the like. hizobium), Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, winia), Francisella genus (Francisella), Fusobacterium genus (Fusobacterium), Gloeobacter genus (Gloeobacter), Gluconobacter genus (Gluconobacter), Helicobacter genus (Helicobacter), Legionella genus (Legionella), Magnetospirillum genus (Magnetospirillum), Mesorhizobium genus (Mesorhizobium), Methylobacterium genus (Methylococcus), Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychobacter, Ralstonia,Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, rmus), Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium ), Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc 2. The ADAS of claim 1, wherein the bacterium is a bacterium of the genus Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

17. 12. The ADAS of claim 11, wherein the parent bacterial cell is Bacillus subtilis.

18. The ADAS of claim 1, wherein the loss-of-function genetic modification in a 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. 2. The ADAS of claim 1, wherein the parent cell comprises loss-of-function genetic modifications 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. 20. The ADAS of claim 19, wherein the gene loss-of-function modifications are in sigF, divIVA, lytc, and the protease deletions are in aprE, ispA, wprA, nprB, Mpr, Vpr, Epr, Bpr, and nprE, or a combination thereof.

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

22. 22. The ADAS of claim 21, wherein the ADAS expresses a cytosolic cargo, a surface-displayed cargo, a secreted cargo, or a combination thereof.

23. 23. The ADAS of claim 22, wherein the cargo is an antigen, cytokine, mutein, protein, polypeptide, small molecule, large molecule, nucleic acid, polynucleotide, enzyme, or any combination thereof.

24. A non-chromosomal dynamic activity system (ADAS) derived from parental bacterial cells that contains engineered cell wall-embedded anchor structures for cargo presentation.

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

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

27. 26. The ADAS of 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. 26. The ADAS of claim 25, wherein the loss-of-function genetic modification in a lytic enzyme is in lytC.

29. 27. The ADAS of claim 26, wherein the parent bacterial cell further comprises a loss-of-function modification in a cell division topology specificity factor.

30. 30. The ADAS of claim 29, wherein the cell division topology specificity factor is a DivIVA, minC, minD, minE, minCD, or minCDE operon.

31. 25. The ADAS of claim 24, wherein the parent bacterial cell is Gram-positive.

32. 25. The ADAS of claim 24, wherein the parent bacterial cell is gram-negative.

33. 25. The ADAS of claim 24, wherein the parent bacterial cell further comprises a loss-of-function genetic modification that disrupts sporulation.

34. 34. The ADAS of claim 33, wherein the loss-of-function genetic modification that disrupts sporulation is a loss-of-function modification in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, or a combination thereof.

35. The ADAS of claim 33, wherein the loss-of-function modification in a sporulation gene is in SigF.

36. The parent bacterial cell may be selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, and the like. rhizobium), Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, rwinia), Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus , Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychobacter, Ralstonia,Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus mus), Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium , Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, 25. The ADAS of claim 24, which is a bacterium of the genus Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

37. 32. The ADAS of claim 31 , wherein the parent bacterial cell is Bacillus subtilis.

38. The loss-of-function genetic modification 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. The ADAS of any one of claims 26 to 30.

39. 27. The ADAS of claim 26, wherein the parent cell comprises loss-of-function genetic modifications 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. 40. The ADAS of claim 39, wherein the parent bacterial cell comprises a loss-of-function genetic modification in each of sigF, divIVA, and lytc and in one or more of the protease genes aprE, ispA, wprA, nprE, epr, vpr, bpr, mpr, nprB, or a combination thereof.

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

42. 42. The ADAS of claim 41, wherein the cargo is an antigen, a tumor antigen, a cytokine, a mutein, a protein, a polypeptide, a small molecule, a large molecule, a nucleic acid, a polynucleotide, an enzyme, IL-2, an IL-2 superkine, or any combination thereof.

43. 43. The ADAS of claim 42, wherein the ADAS expresses the cargo via an engineered cell wall-embedded anchor structure.

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

45. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor structure is an antiparallel coiled coil.

46. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor structure has a COOH terminal end embedded in the cell wall.

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

48. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor structure is a truncated antiparallel coiled-coil, a linker, a coiled linker, or a fusion construct.

49. The ADAS of any one of claims 1 to 48, wherein the parent bacterial cell further comprises at least one additional loss-of-function genetic modification in an enzyme.

50. 50. The ADAS of claim 49, wherein the parent bacterial cell comprises a loss-of-function genetic modification in one or more amylases.

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

52. 52. The ADAS of claim 51 , wherein the parent bacterial cell comprises a loss-of-function genetic modification in amyE, nprB, mpr, bpr, vpr, epr, wprA, ispA, nprE, aprE, sigF, lytC, divIVA, or any combination thereof.

53. 50. The ADAS of claim 49, wherein the parent bacterial cell further comprises a loss-of-function genetic modification in one or more lipases.

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

55. 50. The ADAS of claim 49, wherein the parent bacterial cell further comprises a loss of function of a gene in one or more cellulases.

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

57. 44. The ADAS of 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 greater than the stability of the cargo in a control ADAS.

58. 44. The ADAS of claim 43, wherein the ADAS is to be administered in an amount of more than 1, more than 10, more than 100, more than 1000, or more than 2000 ADAS per cell.

59. 44. The ADAS of claim 43, wherein the parent 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. 60. The ADAS of any one of claims 1 to 59, wherein the ADAS is to be delivered via subcutaneous, intraperitoneal, intravenous, intramuscular, oral, aerosol or nebulized administration.

61. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor 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 of any one of claims 43 to 67, wherein the cell wall-embedded anchor structure contains a cell wall binding domain.

69. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure contains a fusion promoter.

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

71. 71. The ADAS of 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. 44. The ADAS of claim 43, wherein the ADAS contains a proline-rich sequence that encodes the cell wall-embedded anchor structure.

73. 44. The ADAS of claim 43, wherein the ADAS contains a self-binding coil for the cell wall-embedded anchor structure.

74. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure is an unstructured, proline-rich construct.

75. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure is a structured construct.

76. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure contains a promoter.

77. 77. The ADAS of 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. 77. The ADAS of 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. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure sequence contains a ribosome binding site.

80. 80. The ADAS of claim 79, wherein the binding site is an 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. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure sequence contains a secretion signal.

82. 82. The ADAS of 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. 68. The ADAS of any one of claims 43 to 67, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is separated by a nucleotide sequence encoding the cargo.

84. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure uses a non-covalent cell-binding domain.

85. 85. The ADAS of 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. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure uses a covalent cell-binding domain.

87. 87. The ADAS of claim 86, wherein the binding domain is a 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. 44. The ADAS of claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure comprises a terminator sequence.

89. 44. The ADAS of claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is integrated into the deleted protease site.

90. 44. The ADAS of claim 43, wherein the nucleotide sequence encoding the cell wall-embedded anchor structure is integrated into the deleted amylase site.

91. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure is incorporated into a deleted phosphodiesterase site.

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

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

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

95. 95. The method of claim 94, wherein the ADAS is administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly, intrarectally, intraperitoneally, intratumorally, by aerosol and / or subcutaneous administration, or any combination thereof.

96. The ADAS is at least 1×10 ５ , 1×10 ６ , 1 x 10 ７ , 1×10 ８ , 5 x 10 ８ , 6 x 10 ８ , 8 x 10 ８ , 1×10 ９ , 2 × 10 ９ , 4 x 10 ９ , 6 x 10 ９ , 8 x 10 ９ or 1 x 10 １０ 96. The method of claim 95, wherein the ADAS is administered to the subject at a dose of 0.1 mg / kg or more.

97. 95. The method of claim 94, wherein 1, 2, 3, 4 or more than 4 doses of ADAS are administered to the subject.

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

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

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

101. The ADAS of claim 100, wherein the cytokine is IL-2 or an IL-2 mutein.

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

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

104. The ADAS of embodiment 102 or 103, wherein the toxin is listeriolysin O.

105. 44. The ADAS of claim 43, wherein the constructed cell wall-embedded anchor structure has an N-terminal portion embedded in the cell wall.

106. 44. The ADAS of claim 43, wherein the anchor structure comprises an internal cell wall-embedded anchor domain.

107. 44. The ADAS of claim 43, wherein the cell wall-embedded anchor structure incorporates a protease cleavage site.

108. 44. The ADAS of any one of claims 1 to 43, wherein the parent 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.

109. 1. A method for delivering a cancer therapeutic to a subject, comprising administering to the subject an ADAS derived from a parent bacterial cell that contains at least one loss-of-function genetic modification in a lytic enzyme and at least one loss-of-function genetic modification in a protease, or a composition comprising the same, wherein the ADAS has anti-cancer properties.

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

111. A method for delivering a cancer therapeutic agent to a subject, comprising administering to the subject an ADAS derived from a parent bacterial cell that contains an engineered cell wall-embedded anchor structure for cargo presentation, or a composition comprising the same, wherein the ADAS has anti-cancer properties.

112. 112. The method of claim 109 or 111, wherein the ADAS is administered by oral, intravenous, intradermal, intramuscular, intranasal, intraocular, intrarectal, intraperitoneal, intratumoral, aerosol and / or subcutaneous administration or any combination thereof.

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

114. 114. The method of claim 113, wherein 1, 2, 3, 4 or more than 4 doses of ADAS are administered to the subject.

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

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

117. 112. 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 mutein.

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

120. 112. 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 embodiment 119 or 120, wherein the toxin is listeriolysin O.

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

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

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

125. 125. The ADAS of any one of claims 109-124, wherein the parent 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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