Stabilized non-chromosomal dynamic activity systems and uses thereof

Abstract

Disclosed herein is a non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell, the ADAS comprising at least one genetic loss-of-function modification in a lytic enzyme to increase the stability of the ADAS. Also disclosed is a method for disrupting sporulation in the parent bacterial cell in combination with a lytic enzyme deletion or other loss-of-function mutation.

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 14, 2023, is entitled 51296-057WO2_Sequence_Listing_11_14_23, and is 25,540 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, 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. Specifically, there is a need for delivery vectors (e.g., non-chromosomal dynamic activation systems (ADAS)) with improved stability. Summary of the Invention

[0004] In some aspects, the disclosure features a non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell, the ADAS comprising at least one gene loss-of-function modification in a lytic enzyme. In some embodiments, the gene loss-of-function in the lytic enzyme results in increased stability of the ADAS compared to the ADAS derived from the parent bacterial cell that does not comprise the modification.

[0005] In some aspects, the present disclosure features a non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell, the non-chromosomal dynamic activation system (ADAS) comprising at least one genomic deletion in a lytic enzyme. In some embodiments, the genomic deletion 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.

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

[0007] In some embodiments, the genetic loss-of-function modification of the parent bacterial cell reduces enzymatic and / or lytic activity. In some embodiments, a genomic deletion in the parent bacterial cell reduces enzymatic and / or lytic activity. In some embodiments, the parent bacterial cell has at least one genetic loss-of-function modification that reduces the activity of an endopeptidase, a cell wall lytic enzyme, and / or an autolysin. In some embodiments, the parent bacterial cell has at least one genomic deletion that reduces the activity of one or more endopeptidases, cell wall lytic enzymes, and / or autolysins.

[0008] In some embodiments, the parent cell comprises a loss-of-function modification or 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.

[0009] In some embodiments, the parental cell comprises a loss-of-function modification or deletion of a gene that prevents sporulation 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 bacterial cell is a Gram-positive bacterial cell.

[0011] In some embodiments, the parent bacterial cell is of the genus Bacillus subtilis or Bacillus.

[0012] In some embodiments, the parent bacterial cells are of the genus Lactobacillus.

[0013] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0014] 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, Bacillus subtilis ... Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea talea), 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 otoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium dobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc The bacterium is derived from a genus selected from the group consisting of euconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, and Thermoanaerobacter.

[0015] In some embodiments, the parental cell comprises a gene deletion from the group consisting of SPβ, skin, PBSX, prophage 1, pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. 20(12):2076-2090, 2003).

[0016] In some embodiments, the parent bacterial cell comprises a genomic deletion of a cell division topology specificity factor, hi some embodiments, the genomic deletion is of the DivIVA, minC, minD, minE, minCD, or minCDE operon.

[0017] In some embodiments, the ADAS further comprises at least one cargo.

[0018] In some embodiments, the cargo is a protein or polypeptide.

[0019] In some embodiments, the ADAS or the parent bacterial cell has been modified to increase the level of cargo in the ADAS.

[0020] 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.

[0021] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0022] [Figure 1] A series of representative images showing overnight cultures of three different parental cell lines derived from Bacillus subtilis. The strains and their different genomic deletions are listed in Table 1: M008: MACH008; M2347: MACH2347; M2403: MACH2403. [Figure 2] 6 is a bar graph depicting the respective OD600 measurements of each culture at three different time points: t0 (after overnight culture and non-chromosomal dynamic activity system (ADAS) enrichment), t23 (after overnight culture, ADAS enrichment, and 23 hours of incubation at 4°C), and t48 (after overnight culture, ADAS enrichment, and 48 hours of incubation at 4°C). [Figure 3A] A pair of representative micrographs showing ADAS and residual parental cells from two different B. subtilis strains after overnight culture. The left panel is strain MACH2347 (listed in Table 1). The right panel is strain MACH2403 (listed in Table 1), which has been modified from MACH2347 by the addition of a genomic deletion of lytC. White arrows indicate examples of phase-light ADAS. Arrowheads indicate examples of phase-light parental cells. [Figure 3B] A pair of representative micrographs showing ADAS and residual parental cells from two different B. subtilis strains after overnight culture and DAS enrichment (t0). Left panel: MACH2347. Right panel: MACH2403. White arrow: exemplary phase-light ADAS. Arrowhead: exemplary phase-light parental cell. [Figure 3C] A pair of representative micrographs showing ADAS and residual parental cells from two different B. subtilis strains after overnight culture, ADAS enrichment, and 23 hours of incubation at 4°C (t23). Left panel: MACH2347. Right panel: MACH2403. White arrow: exemplary phase-light ADAS. Arrowhead: exemplary phase-light parental cell. DETAILED DESCRIPTION OF THE INVENTION

[0023] I. Definition As used herein, the term "non-chromosomal dynamic system" or "ADAS" refers to a genome-free, non-replicating, closed membrane system comprising at least one membrane and having 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 some embodiments, the ADAS is obtained from the parent bacterium using any suitable method, e.g., genetic manipulation of the parent cell or exposure to a culture medium or conditions that increase 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 surface of the parent cell, e.g., a cell wall, a cell wall modification, a flagellum, or a pilus, 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, the transcription machinery, or the translation machinery. In other embodiments, the ADAS may lack 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.

[0024] 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 apparatus (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, exhibiting increased ATP concentration, increased capacity for ATP generation, increased capacity for protein production, or increased protein production rate or amount, and / or increased responsiveness to a biological signal, e.g., promoter induction.

[0025] As used herein, the term "parent bacterial cell" refers to a cell (e.g., a Gram-negative bacterial cell or a 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 Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas chloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella ella), 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, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas as), Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus ctobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0026] 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 contains 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.

[0027] The term "cell division topology specificity factor" refers to a component of the cell division machinery in bacterial species that is involved in determining the septation site and functions by restricting the location of other components of the cell division machinery, e.g., 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 variety 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 cell poles. 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).

[0028] The term "Z-ring inhibitory protein" refers to a component of the cell division machinery in bacterial species that is involved in determining the septation site and functions by inhibiting the formation of a stable FtsZ ring or by anchoring such a component to the membrane. In some embodiments, the localization 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 Escherichia coli (E. coli) and have since been identified in a wide variety 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 occur at the same locus, which is sometimes referred to as the "min operon," the minCDE operon, or the min or minCDE locus.

[0029] As used herein, the term "reduction in the level or activity of a cell topology-specific factor" refers to an overall reduction of any 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 when compared to the level in a reference sample (e.g., an ADAS made from a wild-type cell or a cell harboring a wild-type minCDE operon or a wild-type divIVA gene), a reference cell (e.g., a wild-type cell or a cell harboring a wild-type minC, minD, minE, divIVA, or minCDE gene or operon), a control sample, or a control cell. In some embodiments, a decreased level or activity refers to a decrease in the level or activity in a sample that is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold the level or activity of the cell topology specific factor in a reference sample, reference cell, control sample, or control cell.

[0030] As used herein, the term "percent identity" refers to the percent (%) sequence identity 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, with, or compared 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 to, with, or compared 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 that are evaluated by a sequence alignment program (e.g., BLAST) as perfect matches 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, or 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.

[0031] 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., protein, transcript level or expression, or biological pathway activity). In some embodiments, increasing the state of a 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 the cellular parameter (e.g., protein, transcript level or expression, or biological pathway activity). 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).

[0032] 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 can be increased, for example, by adding a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by reducing or removing a negative regulator of T3SS expression.

[0033] 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.

[0034] 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 a cell from which the secretion apparatus (e.g., a T3SS) is derived (e.g., encoded by the wild-type version of that cell) and that is capable of being secreted by that secretion apparatus. The secretion apparatus and one or more of its endogenous effectors may be expressed in a 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.

[0035] 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 by the cell from which the secretion apparatus (e.g., T3SS) is derived (e.g., not encoded by the wild-type version of that cell) and that has the ability to be secreted by the secretion apparatus of the cell from which the heterologous effector is derived. In some embodiments, the effector has the ability to be secreted by the secretion apparatus to which it is heterologous, or has been modified to be secreted by the secretion apparatus to which it is heterologous. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0036] 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 the parent bacterium (e.g., a gram-negative or gram-positive bacterial cell) from which it is generated; for example, a cargo or payload (e.g., a nucleic acid such as an RNA or tRNA encoding a polypeptide, a protein, or a small molecule) or a structure (e.g., a plasmid or a gene editing system).

[0037] As used herein, the terms "phase-lite ADAS" and "phase-lite parent cell" refer to an ADAS or parent cell corpse (e.g., a dead ADAS or a dead parent cell) that appears paler or ghosted in an image (e.g., a photomicrograph taken using a light microscope), indicating disintegration and lysis compared to the darker ADAS or parent cell, referred to herein as an "intact ADAS" and an "intact parent cell," respectively.

[0038] As used herein, "increased stability" of an ADAS refers to an overall increase in ADAS integrity. ADAS stability (e.g., the stability of ADAS in a particular population, strain, or variety) can be measured as the ratio of "intact" ADAS to "phase-lite" ADAS (e.g., percent intact ADAS) in one or more representative images of multiple ADAS. For example, increased stability of an ADAS from a modified strain containing at least one lytic enzyme deletion can be defined as an increase in the percentage of "intact" ADAS in a representative image of the ADAS from the modified strain compared to ADAS 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 a modified ADAS has greater than 40% stability, greater than 40% of the visible ADAS is intact rather than phase-lite. In some embodiments, the stability of an ADAS is measured as a unitless ratio between the half-life of the ADAS from an unmodified strain (e.g., a strain not containing a lytic enzyme deletion) and the half-life of the 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 the ADAS from a modified strain compared to the ADAS from a control strain at one or more time points (e.g., 0 hours, 10 hours, 20 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, or more than 72 hours after ADAS enrichment) is another measure of the stability of the ADAS. The stability of the ADAS is modified (e.g., increased) by the genomic deletion of at least one lytic enzyme in the parental cell. In certain embodiments, an ADAS with one or more lytic enzyme deletions (e.g., derived from a parent cell containing one or more lytic enzyme deletions) has greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% stability, as measured by percent intact ADAS, compared to an ADAS without a lytic enzyme deletion (e.g., derived from a parent cell without a lytic enzyme deletion).

[0039] As used herein, "lytic enzyme" refers to an enzyme (e.g., an endopeptidase, cell wall lytic enzyme, or autolytic enzyme) that regulates the breakdown of the cell wall and membrane of a parent bacterium. Genomic deletion of one or more of these enzymes reduces lytic activity in the parent cell and, in some embodiments, can increase the stability and integrity of the ADAS structure, allowing for the production of greater numbers of intact ADAS and / or the survival of intact ADAS for longer periods of time. Specific genomic deletions that reduce lytic activity in 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.

[0040] As used herein, "sporulation" refers to the process of forming spores from cells under unfavorable conditions in a particular parent strain. Gene deletions that can inhibit sporulation include, but are not limited to, sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spoOA, and combinations thereof.

[0041] As used herein, "gene loss of function" refers to a substantial reduction or complete elimination of a protein (e.g., a substantial reduction in protein function, a complete loss of protein function, a substantial reduction in protein expression, or a complete loss of protein expression) caused by an alteration (e.g., a genetic mutation) in 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 for which gene loss of function can occur include, but are not limited to, enzymes such as lytic enzymes, proteases, amylases, lipases, or cellulases. In addition to gene loss of function due to mutation, promoter inactivation or chemical inhibition can affect (e.g., reduce or eliminate) expression of proteins (e.g., lytic enzymes, proteases, amylases, lipases, or cellulases).

[0042] 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 ADAS, that can provide a wide range of functions in numerous 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 interior 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)).

[0043] In some embodiments, the ADAS are minicells or modified minicells derived from parent bacterial cells (e.g., gram-negative or gram-positive bacterial cells). In some aspects, the ADAS are obtained from the parent bacteria using any suitable method, such as genetic manipulation of the parent cells or exposure to culture media or conditions that increase the likelihood of bacterial minicell formation.

[0044] In some embodiments, the ADAS has a major axis cross-section of about 100 nm to 500 μm (e.g., in certain embodiments, about: 100 to 600 nm, e.g., 100 to 400 nm; or about 0.5 to 10 μm, and 10 to 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 to 1 μm 3 , 0.3 to 5 μm 3 , 5 to 4000 μm 3 , or 4000 to 50 × 10 7 μm 3In 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.

[0045] In some embodiments, the present invention provides a highly active ADAS. A "highly active" ADAS is an ADAS with a high potential for activity, e.g., an ADAS capable of performing a large number of useful activities. In some embodiments, activity is defined as a metabolic activity, 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 translocation, 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.

[0046] The term "ADAS provided by the present invention" encompasses all embodiments of the ADAS described herein, including high-activity ADAS that are part of the ADAS provided by the present invention, a set of which in a particular embodiment can be referred to as "high-activity ADAS provided by the present invention."

[0047] In one aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic activation systems (ADAS), the ADAS having an initial ATP concentration of at least 1 mM, and the composition being substantially free of viable bacterial cells.

[0048] In another aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic activation systems (ADAS), the ADAS having an initial ATP concentration of at least 3 mM, and the composition being substantially free of viable bacterial cells.

[0049] 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.

[0050] In some embodiments, high activity is additionally or alternatively 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, e.g., 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.

[0051] 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).

[0052] 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, a highly active ADAS has 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 plasmid number in each ADAS, and the solution volume.

[0053] 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 protein expression 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, heterologous protein production 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, heterologous protein production 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 for each 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 for each ADAS. In some embodiments, high activity of an 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.

[0054] 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.

[0055] In some aspects, the present invention provides compositions comprising an ADAS and / or multiple ADASs derived from a parent bacterium that exhibits reduced levels, activity, or expression of a cell division topology-specific factor.

[0056] In some aspects, 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.

[0057] In some aspects, the invention provides a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells and produced by a method comprising: (a) producing, 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 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.

[0058] 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.

[0059] 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, Dechloro Dechloromonas, Desulfitobacterium, Desulfotalea, 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 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 The bacterium is a bacterium of the genus Lactobacillus, 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.

[0060] 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: 5), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the polypeptide encoded by SEQ ID NO: 5. In some embodiments, the cell division topology specificity factor comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 5. 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.

[0061] In some embodiments, the ADAS or parent bacterium that exhibits a reduced level or activity of a cell division topology specificity factor also exhibits a reduced level of one or more Z-ring inhibitory proteins.

[0062] 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., a polypeptide 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.

[0063] 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.

[0064] In some embodiments, the ADAS or parent bacterium exhibits reduced levels, activity, or expression of at least two Z-ring inhibitory proteins. In some embodiments, the ADAS or parent bacterium exhibits reduced expression of minC and minD polypeptides. In some embodiments, the ADAS or parent bacterium exhibits reduced expression of minC, minD, and minE polypeptides, e.g., a deletion of the minCDE operon (ΔminCDE).

[0065] In some embodiments, the reduction in the level, activity, or expression of a cell division topology specificity factor or Z-ring inhibitory protein, e.g., a reduction in ADAS or a 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., a gene deletion. In some embodiments, the loss-of-function mutation is an inducible loss-of-function mutation, and the loss of function is induced by exposing the 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.

[0066] In some embodiments, the parent cell has a deletion of the minCDE operon (ΔminCDE) or a deletion of a homologous operon.

[0067] 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 genetic loss-of-function modifications that stabilize (e.g., increase the stability of) the ADAS from the cell. In some embodiments, the parent bacterial cell has been modified to reduce enzyme activity. In certain embodiments, ADAS stability is improved by genetic deletion of one or more enzymes.

[0068] In some embodiments, the parent cell comprises one or more gene loss-of-function modifications that reduce or eliminate enzymatic and / or lytic activity (e.g., reduce or eliminate enzymatic or lytic activity carried out by a protein encoded by a gene comprising the gene loss-of-function modification). In some embodiments, the parent cell comprises a genomic deletion that reduces or eliminates enzymatic and / or lytic activity (e.g., reduce or eliminate 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 gene loss-of-function (e.g., genomic deletion) from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0069] In some embodiments, the parent cell comprises a gene loss-of-function in lytC. For example, in cases where a wild-type parent cell comprises a lytC sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, a parent cell of the invention may comprise a deletion as indicated by a comparison of SEQ ID NOs: 8 and 9. For example, the parent cell may comprise a genomic sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. The loss-of-function modification to lytC may comprise a deletion of all or part of the coding region of the gene.

[0070] In some embodiments, the parent cell comprises a gene loss-of-function in sigF. For example, in cases where a wild-type parent cell comprises a sigF sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, the parent cell of the invention may comprise a deletion as shown by a comparison of SEQ ID NOs: 4 and 5. For example, the parent cell may comprise a genomic sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. The loss-of-function modification to sigF may comprise a deletion of all or part of the coding region of the gene.

[0071] One of skill in the art would be readily able to identify modifications to a nucleotide sequence encoding a protein (eg, a protein having enzymatic and / or lytic activity, such as a lytic enzyme) that result in a loss of function of the protein.

[0072] In some embodiments, the parent cell comprises a gene loss of function in lytC.

[0073] In some embodiments, the parent cell comprises a genomic deletion in lytC.

[0074] In some embodiments, the parent bacterial cell comprises a gene loss-of-function (e.g., genomic deletion) of at least one of a gene encoding a lytic enzyme, a gene affecting the sporulation mechanism, or a cell division topology factor, and combinations thereof (e.g., comprising two or more gene loss-of-function modifications affecting one or more lytic enzymes, one or more genes affecting the sporulation mechanism, and / or one or more cell division topology factors).

[0075] In some embodiments, the parent bacterial cell comprises a gene loss of function (e.g., a genomic deletion) of lytC, sigF, and divIVa. In some embodiments, the parent bacterial cell comprises a genomic deletion of lytC, sigF, and divIVa.

[0076] In some embodiments, the parent bacterial cell is a Gram-positive bacterial cell.

[0077] In some embodiments, the parent bacterial cell is Bacillus subtilis or is of the genus Bacillus (eg, a Bacillus species).

[0078] In some embodiments, the parent bacterial cell is of the genus Lactobacillus (eg, a Lactobacillus species).

[0079] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0080] 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, Bacillus subtilis ... Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea talea), 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 Fidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc The bacterium is a bacterium belonging to the genus Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0081] 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). Further deletion of portions of the parental bacterial cell's genome can result in the reduction of many lytic elements through the removal of prophage and prophage-like segments. These deletions are not essential for the formation of an intact and stable ADAS, as are deletions or loss-of-function mutations of certain lytic enzymes (e.g., lytC).

[0082] D. ADAS derived from parent cells in which sporulation was disrupted In some embodiments, the parental cells comprise a gene deletion or loss-of-function modification (e.g., a loss-of-function mutation) that disrupts sporulation (e.g., reduces or abolishes sporulation). In some embodiments, the loss-of-function mutation 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 in an ADAS and the genomic deletions used to reduce enzyme activity are not necessary or dependent on each other, although each provides unique advantages for the generation and use of an ADAS. Disrupting sporulation is beneficial for the generation of ADAS because it maintains the parental cell population while eliminating spores that may be difficult to distinguish from ADAS. Thus, in some embodiments, the parent cells provided herein contain both one or more modifications that reduce enzymatic activity (e.g., loss-of-function mutations) and one or more modifications that prevent sporulation (e.g., loss-of-function mutations).

[0083] E. ADAS including cargo In some embodiments, the ADAS provided by the present invention comprises a cargo housed within the ADAS. In some embodiments, the cargo is any moiety disposed within the ADAS (e.g., encapsulated in the ADAS) or conjugated to the surface of the ADAS. 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 agent, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP), or a combination of the foregoing. 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.

[0084] 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).

[0085] 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.

[0086] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme modifies a substrate such that a target product is produced. 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.

[0087] In certain embodiments, the cargo is modified to enhance stability compared to the unmodified version of the cargo. The "stability" of a cargo is the unitless ratio of the half-life of the unmodified cargo to the half-life of the modified cargo when 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 "ADAS plasm" (i.e., the contents of the internal volume of the ADAS, e.g., after dissolution). In some applications, it is an agricultural environment, such as various field soils, river water, or ocean water. In other embodiments, the environment is real or simulated: an animal's intestine, animal skin, animal reproductive system, animal respiratory tract, animal bloodstream, or animal extracellular space. In certain embodiments, the ADAS does not substantially degrade the cargo.

[0088] In certain embodiments, the cargo comprises a protein. In certain embodiments, the protein has a stability in the cytoplasm or other environment greater than about: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 10000000. The protein may 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.

[0089] In some embodiments, the cargo comprises a plant hormone such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.

[0090] In some embodiments, the cargo is an anti-inflammatory agent, e.g., a cytokine (e.g., a heterologously expressed anti-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).

[0091] 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)). 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., a molecule that alters the compartmentalization, presentation, or profile of an immunomodulatory molecule or one or more costimulatory molecules associated with a vaccine antigen. In some instances, the adjuvant is an activator of an immune pathway upstream of the desired immune response (e.g., an activator of 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 embodiments, the ADAS comprises an antigen and one or more adjuvants.

[0092] In some embodiments, the cargo is a cancer therapeutic or prophylactic agent, 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 individuals with cancer).

[0093] Agents for preventing cancer include, but are not limited to, anti-inflammatory agents and growth inhibitors. Agents for treating cancer (e.g., solid tumor cancer) include, but are not limited to, anti-inflammatory agents, growth inhibitors, chemotherapeutic agents, immunotherapeutic agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as 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.

[0094] In some aspects, the ADAS therapeutic composition is administered orally, intravenously, intradermally, intramuscularly, intraperitoneally, intratumorally, intranasally, intraocularly, or intrarectally, and / or subcutaneously. In certain embodiments, the ADAS is administered orally, intravenously, intramuscularly, and / or subcutaneously. In some embodiments, the ADAS is administered to a subject once, twice, three times, four times, or more times. In some embodiments, the dose of the ADAS is at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 6×10 8 , 8×10 8 , 1×10 9 , 2 × 10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 For example, the administration is at least 1 x 10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×108 , 6×10 8 , 8×10 8 , 1×10 9 , 2 × 10 9 , 4×10 9 , 6×10 9 , 8×10 9 , or 1×10 10 The method includes administering an ADAS to a subject.

[0095] 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, an oxidoreductase, or a combination thereof.

[0096] 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.

[0097] In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally via a linker to the outer membrane.

[0098] Alternatively, in some embodiments, the cargo is a nucleic acid encoding any of the enzymes described herein.

[0099] In some embodiments, the cargo is an agent that activates or inhibits the autophagy process (e.g., an activator such as listerialysin o or an inhibitor such as IcsB).

[0100] 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.

[0101] 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.

[0102] For cargo-containing ADASs, 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, e.g., a stem-loop structure. For example, non-human tRNALys3 and E. coli tRNAMet (Nat. Methods, Ponchon 2007). Both are well characterized and recombinantly expressed. However, various other types, such as aptamers, lncRNAs, and ribozymes, may also be used. 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.

[0103] 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 an 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.

[0104] 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) 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, and a nucleic acid associated with the function of such a protein, such as a guide RNA. 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.

[0105] 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 the 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. Next, a plasmid containing the IPTG-inducible promoter PLac and the heat-inducible promoter pL, both of which drive the expression of RAJ11 sRNA, can 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.

[0106] 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.

[0107] 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.

[0108] F. ADAS containing secretion 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 capable of exporting cargo from the cytoplasm of a bacterial cell (or, e.g., an ADAS derived therefrom) to the extracellular space, the periplasm of Gram-negative bacteria, or the intracellular space of another cell. In some embodiments, the bacterial secretion apparatus functions 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 the protein tube, and the T6SS, which carries cargo at the end of a spike. Other exemplary bacterial secretion apparatuses include the T1SS, T2SS, T5SS, T7SS, Sec, and Tat, which are transmembrane.

[0109] 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) heterologous to the parent bacterial cell.

[0110] In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.

[0111] In some embodiments, the parent bacterial cell does not contain an endogenous T3SS.

[0112] 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.

[0113] In some embodiments, the parent bacterial cells are probiotic cells.

[0114] 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.

[0115] 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 the ADAS. In some embodiments, the moiety is a transcriptional activator of the one or more heterologous nucleotide sequences encoding components of a T3SS.

[0116] In another aspect, 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) endogenous to the parent bacterial cell, wherein the parent bacterial cell has been modified to reduce the levels of an endogenous protein or polypeptide capable of being secreted by the T3SS.

[0117] In some embodiments, the parental bacterium has been modified by deleting a transcriptional activator of an endogenous protein or polypeptide that has the ability to be secreted by a T3SS.

[0118] In some embodiments, the parent bacterium cell is a gram-negative bacterial cell.

[0119] 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.

[0120] 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.

[0121] In some embodiments, the parent bacterial cell has been modified to reduce 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.

[0122] In some embodiments, the parent bacterial cell has been modified to reduce levels of one or more of: LPS; metabolically non-essential proteins; non-T3SS-associated toxins; endotoxins; flagella; and pillus.

[0123] In some embodiments, the ADAS further comprises at least one cargo, wherein the T3SS is capable of delivering the cargo to a target cell, hi some embodiments, the delivery is to the cytoplasm of the target cell.

[0124] In some embodiments, the cargo is a protein or polypeptide.

[0125] In some embodiments, the cargo is endogenously secreted by a T3SS.

[0126] In some embodiments, the ADAS or the parent bacterial cell is modified to increase the level of cargo in the ADAS.

[0127] In some embodiments, the cargo is not endogenously secreted by a T3SS.

[0128] In some embodiments, the cargo is endogenously secreted by a T3SS of a species other than the ADAS T3SS species.

[0129] In some embodiments, the cargo is endogenously secreted by a type 4 secretion system (T4SS) or a type 6 secretion system (T6SS).

[0130] In some embodiments, the cargo is modified for delivery by the T3SS.

[0131] 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.

[0132] In some embodiments, the cargo is modified by the addition of a secretion signal.

[0133] In another aspect, the disclosure features a method of delivering a 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.

[0134] In some embodiments, the ADAS comprises a cargo, wherein the cargo comprises a moiety that directs export by the bacterial secretion apparatus, e.g., in some embodiments, the moiety is Pho / D, Tat, or a synthetic peptide signal.

[0135] 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.

[0136] The ADAS provided by the present invention, in some embodiments, comprises a bacterial secretion apparatus.

[0137] In some embodiments, the bacterial secretion apparatus has the ability to export cargo across the ADAS outer membrane, such as a T3SS, a T4SS, a T3 / T4SS, or a T6SS, into a target cell, such as an animal cell, a fungal cell, a bacterial cell, or a plant cell.

[0138] 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 apparatuses as well as unmodified versions, that links the two and delivers one or more effectors between a bacterium (or ADAS) and a target cell. The target cell may 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 apparatuses 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 apparatuses include the Agrobacterium Ti plasmid apparatus and the Helicobacter pylori T4SS. In certain 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 specific embodiments, the modified effector function is for intracellular targeting, such as translocation into the nucleus, Golgi, mitochondria, actin, microvilli, ZO-1, microtubules, or cytoplasm. In even more specific embodiments, the modified effector function is nuclear targeting based on NleE2 from Escherichia coli (E. coli). In other specific embodiments, the modified effector function is for filopodia formation, disruption of tight junctions, loss of microvilli, or inactivation of SGLT-1.

[0139] In other embodiments, the ADAS provided by the present invention, which 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).

[0140] 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.

[0141] G. ADAS lacking proteases, RNases, and / or LPS In another aspect, the invention provides a composition further comprising a plurality of ADAS (e.g., a hyperactive ADAS), which has reduced protease levels or activity compared to an ADAS produced from a wild-type parent bacterium. In some aspects, the ADAS is produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one protease.

[0142] In another aspect, the invention provides compositions comprising multiple ADAS (e.g., hyperactive ADAS) that have reduced RNase levels or activity compared to 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 RNase. In some embodiments, the RNase is an endoribonuclease or exoribonuclease.

[0143] 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). In some embodiments, the modification is a mutation in lipid A biosynthesis myristoyltransferase (msbB).

[0144] 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, fimbriae, 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, 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.

[0145] H. ADAS Containing a Targeting Moiety In another embodiment, the present invention provides a composition comprising a plurality of ADASs, each of which 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.

[0146] 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.

[0147] I. 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), the ADAS being derived from a parent bacterium that is a mammalian pathogen or a mammalian commensal. In some examples, 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.

[0148] In another embodiment, the invention provides a composition comprising multiple ADAS (e.g., highly active ADAS) derived from a parent bacterium that is a plant pathogen or a plant commensal bacterium. In some examples, the plant commensal bacterium is Bacillus subtilis or Pseudomonas putida, or the plant pathogenic bacterium is Xanthomonas species or Pseudomonas syringae.

[0149] J. ADAS derived from auxotrophic parent strains In another embodiment, the present invention provides a composition comprising multiple ADAS (e.g., highly active ADAS) that are derived from an auxotrophic parent bacterium, i.e., a parent bacterium that lacks the ability to synthesize an organic compound required for growth. Such bacteria can grow only when the organic compound is provided.

[0150] K. ADAS with additional components 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 induced 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 synthesis, optionally including fractions of bacterial cell preparations. In some embodiments, a highly active ADAS has a saturation of at least: 10,000 nm 2 1 per piece, 5000nm 2 1 per piece, 3500nm 2 1 per 1000nm 2 The concentration of ATP synthase is one per unit volume.

[0151] 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 apparatus, and transporters, including combinations of the foregoing, including the specific detailed embodiments described below. In certain embodiments, the ADAS lacks metabolically non-essential genes and / or other elements, such as certain enzymes, nucleases, or proteases.

[0152] In certain embodiments, the ADAS provided by the present invention comprises an ATP synthase that optionally lacks a regulatory domain, such as lacking the ε 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 may be achieved by flanking LoxP sites and inducible Cre expression or CRISPR knockout, or may be inducible (placed on a plasmid under the tTa tet transactivator in an ATP synthase knockout strain).

[0153] 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.

[0154] 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.

[0155] 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.

[0156] L. ADAS Compositions and Formulations The present invention provides compositions or preparations containing the ADAS provided by the present invention, including, inter alia, highly active ADAS preparations provided by the present invention or ADAS preparations in which multiple individual ADAS lack a cell division topology specificity factor, e.g., lack the minE gene product, and optionally are substantially free of viable cells. Collectively, these may be "compositions provided by the present invention" or "a composition 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.

[0157] 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 an ADAS comprising a bacterial secretion apparatus. In particular embodiments, the bacterial secretion apparatus is one of a T3SS, a T4SS, a T3 / 4SS, or a T6SS.

[0158] In some embodiments, the compositions provided herein contain an ADAS that comprises a T3SS, the ADAS having a T3SS activity of about: 40000, 35000, 30000, 25000, 19600, 15000, 10000, or 5000 nm 2 In certain detailed embodiments, the ADAS is derived from a S. typhimurium or E. coli parent strain.

[0159] Certain embodiments of the compositions provided by the present invention include an ADAS containing a T3SS, the ADAS having a molecular weight of about: 300,000, 250,000, 200,000, 150,000, 100,000, 50,000, 20,000, 10,000, 5,000 nm 2In certain detailed embodiments, the ADAS comprises an average T3SS membrane density of greater than 1 per 1000 cells / ml. In certain detailed embodiments, the ADAS is derived from an Agrobacterium tumefaciens parent strain.

[0160] In another aspect, the present invention provides an ADAS composition, 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 comprises a bacterial secretion apparatus including a T3, T4, T3 / 4SS, T6SS, and optionally one or more of an exogenous carbohydrate, a phosphate-producing synthase, a light-responsive protein, an intracellular internalization protein, an enzyme, a functional cargo, an organism-specific effector, or a fusion protein.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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%, e.g., at least 95%, 98%, or at least equal to the ATP concentration of the non-lyophilized ADAS.

[0165] 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.

[0166] In some embodiments, the ADAS is maintained or in some "resting" state and then rapidly activated.

[0167] 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.

[0168] 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 + nitrogen source, an organosilicone surfactant, or a high surfactant oil concentrate), a crop oil concentrate, a vegetable oil concentrate, a modified vegetable oil, a nitrogen source, an adhesion agent (drift control agent) and / or a retention agent (with or without ammonium sulfate and / or an antifoam agent), a compatibilizer, a buffer and / or an acidifier, a water conditioner, a base blend, an extension agent-spreading agent and / or a bulking agent, an adjuvant + foliar fertilizer, an antifoam agent, a foam marker, a fragrance, or a tank cleaner and / or a neutralizer. In some embodiments, the adjuvant is selected from the group consisting of herbicide adjuvants listed in the Compendium of Herbicide Adjuvants (Young et al. (2016)). Compendium of Herbicide Adjuvants (13 th It is an adjuvant drug described in "The Journal of Clinical Nutrition and Medicine, Vol. 1, No. 1, pp. 111-115, 2002," ed., Purdue University.

[0169] 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.

[0170] In some embodiments, the composition is formulated as a liquid, solid, aerosol, paste, gel, or gas composition.

[0171] M. ADAS containing enzymes In one aspect, the invention features a composition including multiple ADASs, where the ADASs include an enzyme that modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS, and the target product is produced in the ADAS. In other embodiments, the substrate is present in 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.

[0172] III. ADAS manufacturing method A. Creation of ADAS and highly active ADAS In some embodiments, production of an ADAS features a method for making a composition comprising a plurality of ADASs, the composition being substantially free of viable bacterial cells, the method comprising: (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 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.

[0173] Parental bacteria include any suitable bacterial species from which an ADAS can be produced (e.g., a species that may 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 may be derived: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella (B artonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas ), Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum gnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium,Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus cus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella lla), Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus llus), Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0174] In some embodiments, any of the methods for producing an ADAS composition, e.g., a highly active ADAS composition, described in Section I herein are used. For example, provided herein are methods for producing a highly active ADAS; methods for producing an ADAS that lacks a cell division topology specificity factor, and optionally lacks a Z-ring inhibitory protein (e.g., methods for producing an ADAS from a ΔminCDE parent bacterium); and methods for producing any of the ADASs mentioned herein, wherein the ADAS comprises a cargo.

[0175] In some embodiments, the ADAS (e.g., a highly active ADAS) is generated from a parent strain that is a plant bacterium, such as a plant-symbiotic bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogenic bacterium (e.g., Xanthomonas sp. or Pseudomonas syringae), or a bacterium capable of colonizing the plant rhizosphere and / or forming root nodules, e.g., a rhizobia.

[0176] In some embodiments, the ADAS (e.g., a highly active ADAS) is generated from a parent strain that is a symbiont of an invertebrate, such as an arthropod (e.g., an insect or arachnid), a nematode, a protozoan, or an annelid. In embodiments, the invertebrate is a pest or pathogen of a plant or animal.

[0177] In some embodiments, an ADAS (eg, a highly active ADAS) is generated from a parent strain capable of genetic transformation, such as an Agrobacterium.

[0178] In some embodiments, the ADAS (e.g., a highly active ADAS) is generated from a parent strain that is a human bacterium, such as a commensal human bacterium (e.g., E. coli, Staphylococcus sp., Bifidobacterium sp., Micrococcus sp., Lactobacillus sp., or Actinomyces sp.) or a pathogenic human bacterium (e.g., Escherichia coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori), or an extremophile.

[0179] 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.

[0180] 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 insecticides, survive extreme environments, make nanoparticles, integrate into other organisms, respond to the environment, and produce reporter signals.

[0181] 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 oIt is modified to express F1ATP synthase.

[0182] 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.

[0183] The highly active ADAS of any one of the preceding claims, which is made from an extremophile microorganism, including a functionalized derivative of any of the foregoing, including a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial, digest plastic, secrete a pesticide, survive in an extreme environment, make nanoparticles, integrate into other organisms, respond to the environment, and produce a reporter signal.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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).

[0190] 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.

[0191] In some embodiments of the methods provided by the 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.

[0192] 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.

[0193] 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.

[0194] Purification separates the ADAS from larger, genome-containing, viable parent bacterial cells. 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.

[0195] 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.

[0196] 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).

[0197] 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.

[0198] 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 a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a), wherein the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0199] In another aspect, the invention features a method for delivering an ADAS to a target cell, the method including: (a) providing a composition including a plurality of ADAS; and (b) contacting the target cell with the composition of step (a), wherein the ADAS is derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0200] In some embodiments, the target cell is, for example, an animal cell, a plant cell, or a fungal cell.

[0201] 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 including a plurality of ADASs, wherein the ADASs comprise the cargo, and wherein the composition is substantially free of viable bacterial cells; and (b) contacting a target cell with the composition of step (a), wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0202] 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 including a plurality of ADAS; and (b) contacting a target cell with the composition of step (a), wherein the ADAS is derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0203] 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 including a plurality of ADASs, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme), wherein the ADASs include cargo, and wherein the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a). In some aspects, the ADASs are derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0204] 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.

[0205] C. Methods for Regulating Cellular Conditions In one aspect, the invention features a method of modulating the state of an animal cell, the method including: (a) providing a composition comprising a plurality of ADASs, wherein 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, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the plurality of ADASs are a plurality of high-activity ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0206] In another aspect, the invention features a method for modulating the state of a plant cell, the method including: (a) providing a composition including a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting a plant cell with the composition of step (a), thereby modulating the state of an animal cell, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the plurality of ADASs are a plurality of high-activity ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0207] In another aspect, the invention features a method of modulating the state of an insect cell, the method including: (a) providing a composition comprising a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby modulating the state of the insect cell, wherein the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the plurality of ADASs are a plurality of high-activity ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0208] In another aspect, the invention features a method of modulating the state of an animal cell, the method including: (a) providing a composition including a plurality of ADASs, wherein 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, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADASs are derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0209] In another aspect, the invention features a method of modulating the state of a plant cell, the method including: (a) providing a composition including a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting a plant cell with the composition of step (a), thereby modulating the state of the plant cell, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADASs are derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0210] In another aspect, the invention features a method of modulating the state of an insect cell, the method including: (a) providing a composition including a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), thereby modulating the state of the insect cell, wherein the ADASs are derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADASs are derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0211] In one aspect, the invention features a method of modulating the state of an animal cell, the method including: (a) providing a composition comprising a plurality of ADASs, wherein 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. In some embodiments, the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0212] In another aspect, the invention features a method of modulating the state of a plant cell, the method including: (a) providing a composition comprising a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting a plant cell with the composition of step (a), thereby modulating the state of the plant cell. In some embodiments, the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0213] In another aspect, the invention features a method of modulating the state of an insect cell, the method including: (a) providing a composition comprising a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting an insect cell with the composition of step (a), thereby modulating the state of the insect cell. In some embodiments, the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0214] In some embodiments, modulation 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 measurements, e.g., methods for measuring the level or expression of a protein, transcript, epigenetic mark, or methods for measuring 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).

[0215] D. Methods for Treating Animals, Plants, Insects, or Fungi In some aspects, the invention features a method of treating an animal in need thereof, the method including: (a) providing a composition comprising a plurality of ADASs, 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, wherein the ADASs are derived from parent cells that contain one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in lytic enzymes). In some embodiments, the plurality of ADASs are a plurality of high-activity ADASs, wherein the ADASs have an initial ATP concentration of at least 1.25 mM.

[0216] In some embodiments, the invention features the use of an ADAS in the manufacture of a medicament for the treatment of an animal, a plant, an insect, or a fungus.

[0217] In some embodiments, the invention features the use of an ADAS in the manufacture of a medicament for the treatment of an animal, wherein the ADAS has an initial ATP concentration of at least 1.25 mM, and wherein the composition is substantially free of viable bacterial cells.

[0218] In some embodiments, the invention features the use of an ADAS in the manufacture of a medicament for the treatment of an animal, wherein the ADAS is derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme), and wherein the composition is substantially free of viable bacterial cells.

[0219] In another aspect, the invention features a method of treating an animal in need thereof, the method including: (a) providing a composition comprising a plurality of ADAS, 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, wherein the ADAS is derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADAS is derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0220] In another aspect, the invention features a method of treating an animal in need thereof, the method including: (a) providing a composition comprising a plurality of non-chromosomal dynamic activation systems (ADAS); and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal. In some embodiments, the ADAS is derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0221] In some embodiments, the animal in need of treatment has a disease, for example, cancer, hi some embodiments, the ADAS carries a chemotherapeutic or immunotherapeutic cargo.

[0222] In some aspects, the invention features a method of treating a plant in need thereof, the method including: (a) providing a composition comprising a plurality of ADASs, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant or a pest thereof (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant, wherein the ADASs are derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADASs are derived from a parent bacterium that has a reduced level or activity of a cell division topology specificity factor.

[0223] In another aspect, the invention features a method of treating a plant in need thereof, the method including: (a) providing a composition including a plurality of ADASs, wherein the ADASs are derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant or a pest thereof (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant, wherein the ADASs are derived from a parent bacterium having one or more genetic loss-of-function modifications that stabilize the ADASs (e.g., one or more loss-of-function modifications in a lytic enzyme). In some embodiments, the ADASs are derived from a parent bacterium having a reduced level or activity of a cell division topology specificity factor.

[0224] In some aspects, the invention features a method of treating a plant in need thereof, the method including: (a) providing a composition including a plurality of non-chromosomal dynamic activation systems (ADAS); and (b) contacting the plant or a pest thereof (e.g., an insect pest) with an effective amount of the composition of step (a), thereby treating the plant. In some embodiments, the ADAS is derived from a parent cell that includes one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0225] In a further aspect, the present invention provides methods for modulating a target cell. The target cell can be any cell, including an animal cell (e.g., a human and a non-human animal, including a farm animal or livestock animal, including a pest), a plant cell (including from a crop or a pest), a fungal cell, or a bacterial cell. In some embodiments, the cell is isolated, e.g., in vitro, or in other embodiments, in vivo within an organism. These methods involve providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention to the target cell. In some embodiments, the access to the target cell is either direct (e.g., the target cell is directly modulated by the ADAS, such as by secretion of an agent proximate 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., modulating a neighboring cell that may be commensal or pathogenic to the target cell. The neighboring cells, like the target cells, may be either in vitro or in vivo - i.e., in an organism, which 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.

[0226] For example, in some embodiments, the present invention provides methods for modulating the condition of an animal cell by providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention with access to the animal cell. In certain embodiments, the ADAS or composition is provided with access to the animal cell in vivo in an animal, such as a mammal, such as 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 the bacterial secretion apparatus and a cargo comprising an anti-inflammatory agent, for example, 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).

[0227] 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, an ulcer, a tumor, or an inflammatory disorder.

[0228] 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.

[0229] In certain detailed embodiments, the animal cell is contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and a cargo, wherein the cargo is delivered to the animal cell. In some detailed embodiments, the animal cell is provided with access to an effective amount of an ADAS comprising a cargo and a secretion apparatus, wherein the cargo is secreted extracellularly and contacts the animal cell.

[0230] 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.

[0231] 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 / 4SS or T6SS and a cargo, where 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 provided with access to an effective amount of an ADAS that extracellularly secretes the cargo that contacts the bacterial or fungal cells.

[0232] 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.

[0233] As will be appreciated, 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.

[0234] Accordingly, in a related aspect, the present invention provides a method of modulating the condition of a plant or fungal cell by providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention with access to: a) the plant or fungal cell, b) a neighboring bacterial or 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.

[0235] In certain embodiments, the ADAS is provided with access to plant cells in planta within crop plants, such as vegetable crops including row crops including corn, wheat, soybean, and rice, as well as solanaceous plants such as tomato and pepper; cucurbits such as melon and cucumber; cruciferous plants such as cabbage and broccoli; leafy vegetables such as kale and lettuce; tuberous roots and roots such as potato and carrot; large seed vegetables such as beans and corn; and mushrooms. In some embodiments, the plant or fungal cells are exposed to the bacteria within a healthy plant or fungus. In other embodiments, the plant or fungal cells are exposed to the bacteria in a diseased state.

[0236] In certain embodiments, the plant or fungal cell is a dividing cell, such as a meristematic cell, or a pathogenic cell, such as a tumor. In some embodiments, the plant or fungal cell is exposed to bacteria in a diseased state, such as a wound, or the plant or fungal cell is not part of the human diet.

[0237] 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.

[0238] In some embodiments, the ADAS comprises a T3 / 4SS or T6SS 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, where the bacterial secretion apparatus secretes the cargo extracellularly, thereby contacting the cargo with the plant or fungal cell.

[0239] In some embodiments, the methods involve providing an effective amount of the ADAS or composition with access to neighboring bacteria or fungal cells in the vicinity of the plant or fungal cell. In more particular 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 particular 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 particular embodiments, the fitness is increased by the reduction of competing bacteria or fungi, which may be neutral, commensal, or pathogenic.

[0240] 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, wherein the cargo is delivered to the adjacent bacterial or fungal cell.

[0241] 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, wherein the bacterial secretion apparatus secretes the cargo extracellularly, thereby contacting the cargo with adjacent bacterial or fungal cells.

[0242] 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.

[0243] In particular embodiments, the methods involve providing an effective amount of the ADAS or composition to access invertebrate (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cells 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 cells is reduced. In even more particular embodiments, the fitness of the pathogenic invertebrate cells is reduced by modulation of symbionts in the invertebrate cells. In other particular embodiments, the invertebrate is commensal. In more particular embodiments, the fitness of the commensal invertebrate cells is increased. In even more particular embodiments, the fitness is increased by a reduction in competitor bacteria or fungi, which may be neutral, commensal, or pathogenic.

[0244] In yet another aspect, the present invention provides a method for removing one or more undesirable materials 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 (such as proteins, polymers, nanoparticles, binders, or combinations thereof) that capture, chelate, or degrade the one or more undesirable materials. "Environment" is defined as non-cellular targets such as the ocean, soil, Superfund sites, skin, ponds, the intestinal lumen, and food in containers.

[0245] In certain embodiments, the undesirable material includes a heavy metal such as mercury, and the ADAS includes one or more molecules (e.g., proteins, polymers, nanoparticles, binders, or combinations thereof) that bind to the heavy metal, such as MerR for mercury. In some embodiments, the undesirable material includes a plastic such as PET, and the ADAS includes one or more plastic-degrading enzymes, such as PETase. In certain embodiments, the undesirable material includes one or more small organic molecules, and the ADAS includes one or more enzymes capable of metabolizing the one or more small organic molecules.

[0246] E. RNA delivery method In another aspect, the invention provides a composition comprising a bacterium or ADAS provided by the invention, the bacterium or ADAS comprising a T4SS, an RNA-binding protein cargo, and an RNA cargo to which the RNA-binding protein binds and 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 an Agrobacterium Ti apparatus. In other embodiments, the RNA-binding protein is Carnation Italian ringspot virus p19 fused to VirE2 or VirF, the RNA cargo is an siRNA, and optionally, the T4SS is an Agrobacterium Ti apparatus.

[0247] In related aspects, the invention provides methods for making these particular compositions, which involve transfecting Agrobacterium cells with a plasmid containing Cas9 fused to VirE2 and VirF and an RNA cargo.

[0248] 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, 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. In some embodiments, the ADAS is derived from a parent cell that contains one or more genetic loss-of-function modifications that stabilize the ADAS (e.g., one or more loss-of-function modifications in a lytic enzyme).

[0249] VI. Other Embodiments Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0250] Embodiment 1. A non-chromosomal dynamic activation system (ADAS) derived from a parent bacterial cell, comprising at least one gene loss-of-function modification in a lytic enzyme.

[0251] Embodiment 2. The ADAS of embodiment 1, wherein the genetic loss-of-function modification results in increased stability of the ADAS compared to an ADAS from a parent bacterial cell that does not contain the modification.

[0252] Embodiment 3. The ADAS of embodiment 1, wherein the gene loss-of-function modification is a non-silent codon change, a deletion, an insertion, a mutation, or any combination thereof.

[0253] Embodiment 4 The ADAS of embodiment 1, wherein the gene loss-of-function alteration is a deletion.

[0254] Embodiment 5. The ADAS of embodiment 1, wherein the lytic enzyme is an endopeptidase, a cell wall lytic enzyme, and / or an autolytic enzyme.

[0255] Embodiment 6. The ADAS of embodiment 2, 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.

[0256] Embodiment 7 The ADAS of embodiment 5, wherein the gene loss-of-function alteration is in lytC.

[0257] Embodiment 8 The ADAS of embodiment 1, wherein the parent bacterial cell further comprises a loss-of-function modification in a cell division topology specificity factor.

[0258] Embodiment 9 The ADAS of embodiment 8, wherein the cell division topology specificity factor is a DivIVA, minC, minD, minE, minCD, or minCDE operon.

[0259] Embodiment 10 The ADAS of embodiment 1, wherein the parent bacterial cell is Gram-positive.

[0260] Embodiment 11 The ADAS of embodiment 1, wherein the parent bacterial cell is Gram-negative.

[0261] Embodiment 12 The ADAS of embodiment 1, wherein the parent bacterial cell further comprises a gene loss-of-function modification that prevents sporulation.

[0262] Embodiment 13. The ADA of embodiment 12, wherein the gene loss-of-function modification that disrupts sporulation is a loss-of-function modification of 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.

[0263] Embodiment 14 The ADAS of embodiment 13, wherein the loss-of-function modification of a sporulation gene is in SigF.

[0264] Embodiment 15. 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. Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea ea), 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 toga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium bacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc The ADAS of embodiment 1, wherein the bacterium is selected from the group consisting of: Ostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, and Thermoanaerobacter.

[0265] Embodiment 16 The ADAS of embodiment 15, wherein the parent bacterial cell is Bacillus subtilis.

[0266] Embodiment 17. The ADAS of embodiment 2, wherein the stability of the ADAS is greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%, as measured by the percentage of intact ADAS.

[0267] Embodiment 18. The ADAS of any one of embodiments 1 to 17, 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 both of SEQ ID NO:5 and SEQ ID NO:9. [Example]

[0268] The present invention will now be generally described, but will be more readily understood by reference to the following examples. These examples are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention. It will be understood that various other embodiments may be practiced from the general description provided above.

[0269] Example 1. ADAS manufacturing 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.

[0270] In this example, ADAS is generated by disruption of one or more genes involved in regulating parental cell division function, i.e., ΔminC, ΔminD, ΔminCDE, ΔminCdivIVA, or ΔdivIVA. This example details genetic means to generate ADAS-producing strains by disruption of the min operon or overexpression of the septation machinery component FtsZ.

[0271] A. Production of ADAS by min mutation To disrupt the min operon, we employed the λ-RED recombineering method according to the protocol provided by Datsenko and Wanner, PNAS, 97(12):6640-6645, 2000. Strains containing the plasmids for the λ-RED system were obtained from the Coli Genetic Stock Center (CGSC) at Yale University. Briefly, primers were designed to non-polarly 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 were homologous to the λ-RED system plasmids pKD3 and pKD4, which provided antibiotic markers that were used to select parental bacterial strains that inherited the targeted mutation. The primer sequences used for deletion are listed in Table 2. After standard PCR using primers with pKD3 as the DNA template, the purified amplicon was transformed by electroporation into bacteria prepared with pKD46, a plasmid containing the phage-derived λ-RED homologous recombination system, according to the method described by Datsenko and Wanner, PNAS, 97(12):6640-6645, 2000. Transformants were selected on LB agar medium supplemented with 35 pg / mL chloramphenicol. Standard allele-specific PCR was used to confirm that the resulting colonies contained the gene disruption (i.e., AminC, AminD, or AminCDE).

[0272] ADAS production by B. divIVA mutations Briefly, primers were designed to delete the coding sequences of B. subtilis divIVA or divIVA and minC. The primer sequences used for the deletions, including both the WT and divIVA deletions, are listed in Table 2. Deletion of divIVA, along with deletion of sigF (sequence in Table 2) to prevent sporulation, produced the parent strain MACH2347. The strain genotype is listed in Table 1. An erm (erythromycin) cassette was introduced into growing bacteria along with the selected deletion primers to allow selection of transformed colonies by growth on LB-erm5 plates. Once transformed bacteria were plated, the colonies were cured of the erm cassette using a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. Transformed bacteria were selected by plating on LB-Spec plates and incubated at 30°C. Selected bacteria were then streaked onto LB plates without antibiotics and incubated at a higher temperature of 42°C to eliminate the temperature-sensitive plasmid. Isolated colonies were confirmed to contain the intended deletion.

[0273] [Table 1]

[0274] Production of ADAS by overexpression of C. ftsZ To generate an ADAS from overexpression of the septation mechanism, we constructed a plasmid directing expression of the FtsZ Z-ring protein from wild-type E. coli (E. coli). Briefly, a strong ribosome binding site and the coding sequence of the E. coli FtsZ protein were optimized de novo using computational tools from De Novo DNA. 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 compatible bacteria, pFtsz can be induced to overproduce the FtsZ protein by adding anhydrotetracycline to the culture. This protein can spontaneously form protofilaments, which trigger asymmetric cell division of the parent cell, thereby leading to ADAS production.

[0275] 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.

[0276] 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 Table 1. Purification separates ADAS from larger, viable, genome-containing parent bacterial cells. ADAS was purified from high-density cell cultures of ADAS-producing strains by a combination of 1) high- or low-speed centrifugation, 2) selective growth, and 3) buffer exchange / concentration. Centrifugation was used to selectively remove viable parent bacterial cells and large cellular debris while simultaneously concentrating ADAS in the mixed suspension. The selective growth procedure was used to reduce the number of viable parent bacterial cells present in the sample by adding compounds that are directly antimicrobial (i.e., toxic to cells carrying the microbial genome) and / or that promote the sedimentation of viable cells by centrifugation. The buffer exchange / concentration procedure was used to transfer ADAS from a large volume of bacterial culture medium to a small volume of 1x PBS while removing culture additives and cellular debris.

[0277] A. Purification of ADAS Using the molecular cloning procedures described in Example 1, ADAS-producing strains were generated and then grown to high cell density in culture medium. Cultures can also be scaled up, for example, from 1 mL to 1000 mL or more of culture medium.

[0278] The culture was transferred to a centrifuge tube and subjected to a high- or low-speed centrifugation procedure to pellet intact cells and large cellular debris while preserving the ADAS in the supernatant. Centrifugation was performed at either 4°C or room temperature. In some cases, a low-speed centrifugation procedure was used, involving a series of consecutive spins at 1,000 x g, 2,000 x g, 3,000 x g, and 4,000 x g for 10 minutes using an Allegra® x14R tabletop centrifuge (Beckman Coulter) or an Eppendorf™ 5424R tabletop centrifuge (Fisher Scientific). In some cases, the low-speed centrifugation procedure consisted of consecutive spins at 2,000 x g for 20 minutes at 4°C, with the supernatant from the first spin being decanted into a sterile centrifuge bottle before the second spin. In some cases, the low-speed centrifugation procedure was a single 40-minute spin at 4,000 × g using a Sorvall™ Lynx 6000 Superspeed Centrifuge (Thermo Scientific™), with the rotor acceleration set as low as possible. In some cases, a high-speed centrifugation procedure was used, involving successive pulses at 20,000 × g, with the spin stopped as soon as the required speed was reached and the supernatant transferred to a new high-speed bottle before the next spin. In some cases, the high-speed centrifugation consisted of a 30-minute spin at 4°C and 17,000 × g, after which the pellet was resuspended in growth medium.

[0279] After low-speed centrifugation, the culture supernatant was decanted into sterile culture tubes and subjected to a selective growth process. After high-speed centrifugation, the culture supernatant was decanted, 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 drug 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 embodiments, the culture supernatant was pelleted by high-speed centrifugation at 10,000 × g to 20,000 × g for 5 to 60 minutes, and the pellet was resuspended in fresh medium containing a concentration of antibiotic or other drug solution that is inhibitory to viable cells. Selective growth was performed by incubating ADAS at 4°C to 42°C for 1 to 3 hours with agitation at 250 rpm. The ADAS were then transferred to a sterile centrifuge tube and subjected to an additional round of centrifugation.

[0280] 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 by centrifugation at 10,000–20,000×g for 5–60 minutes, washed with 1–9 volumes of 1x PBS, pelleted again, and resuspended in 1x PBS to a concentration of 1–100,000-fold from the starting culture volume. In other embodiments, ADAS were pelleted with successive high-speed pulses at 16,000×g spaced 1 min apart, followed by an extended high-speed spin at 20,000×g for 20 minutes at 4°C, after which the pellet was resuspended and washed several times. In some cases, washing consisted of a 5-minute spin at 15,000×g at 4°C.

[0281] B. Purification of ADAS from the Auxotrophic ADAS-Producing Parent Strain ADAS-producing parent strains that are auxotrophic, i.e., incapable of synthesizing an organic compound required for growth, are useful for producing ADAS. Such strains can grow only when the organic compound is supplied. Therefore, storing or incubating the ADAS preparation in a medium lacking the organic compound can counterselect for auxotrophic parent strains, thus providing a further method for reducing the parental load in an ADAS preparation.

[0282] Example 3: Deletion of lytic enzymes in ADAS Primers were designed to delete coding sequences of interest (e.g., deletion of genomic sequences encoding lytic enzymes) in a manner similar to that described in Example 1B. The sequences targeted for deletion are shown in Table 2, including 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 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 loss-of-function deletion.

[0283] The genotype of the strain is shown in Table 1. An erm (erythromycin) cassette was introduced into growing bacteria along with selected deletion primers to allow selection of transformed colonies by growth on LB-erm5 plates. Once the transformed bacteria were cultured, the colonies restored the erm cassette using a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. Transformed bacteria were selected by plating onto LB-Spec plates and incubated at 30°C. Selected bacteria were then streaked onto antibiotic-free LB plates and incubated at a higher temperature of 42°C to eliminate the temperature-sensitive plasmid. Isolated colonies were confirmed to contain the intended deletion (e.g., lytC). If additional genomic deletions (e.g., sigF) were required, a similar process was used.

[0284] [Table 2]

[0285] [Table 3]

[0286] [Table 4]

[0287] [Table 5]

[0288] [Table 6]

[0289] Example 4: Stability of ADAS The ADAS-producing strains were produced and purified using the procedures described in Examples 1-3 and are shown in Table 1. Bacillus subtilis strains were pre-cultured in LB broth and incubated at 37°C and 250 rpm for 6 hours. The culture was then diluted and grown overnight at 30°C and 250 RPM.

[0290] A. Imaging and absorbance As shown in Figure 2, the absorbance at optical density 600 (OD600) of the various cultures was measured at three different time points: (1) after overnight culture and ADAS enrichment (t0); (2) after overnight culture, ADAS enrichment, and 23 hours of incubation at 4°C (t23); and (3) after overnight culture, ADAS enrichment, and 48 hours of incubation at 4°C (t48). Generally, a higher OD600 at later time points indicates a more stable ADAS chassis compared to the control strain.

[0291] Representative images of overnight cultures clearly demonstrate differences in parental cell growth before ADAS enrichment; parental bacterial cells containing the ΔlytC mutation (MACH2403; Figure 1) exhibit fewer breakpoints in rod length. To visually measure ADAS stability, representative images of two different cultures were also taken at three different time points. ADAS and residual parental cells are shown from two different B. subtilis strains after overnight culture (Figures 1 and 3A), after overnight culture and ADAS enrichment (t0, Figure 3B), or after 23 hours of overnight culture, ADAS enrichment, and incubation at 4°C (t23, Figure 3C). The left panel is strain MACH2347 (listed in Table 1). The right panel is strain MACH2403 (listed in Table 1), which was modified from strain MACH2347 by the addition of a genomic deletion of lytC. White arrows indicate examples of phase-light ADAS (fainter or ghost-appearing ADAS, indicating collapse and dissolution). Arrowheads indicate examples of phase-light parental cells. ADAS populations derived from parental cell lines containing a deletion of lytC show visual evidence of increased ADAS stability, as evidenced by an increased number of intact (non-phase-light) ADAS. The strains listed and images shown should not be considered limiting.

[0292] The present invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding; however, these 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 following claims.

Claims

1. A non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell, which contains at least one gene loss-of-function modification in a lytic enzyme.

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

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

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

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

6. 3. The ADAS of claim 2, 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.

7. The ADAS of claim 5, wherein the gene loss-of-function alteration is in lytC.

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

9. The ADAS of claim 8, wherein the cell division topology specificity factor is a DivIVA, minC, minD, minE, minCD, or minCDE operon.

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

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

12. The ADAS of claim 1 , wherein the parent bacterial cell further comprises a gene loss-of-function modification that prevents sporulation.

13. 13. The ADAS of claim 12, wherein the gene loss-of-function modification that disrupts sporulation is a loss-of-function modification of 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.

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

15. The parent bacterial cell 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, 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 oc), Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter bacteria.

16. 16. The ADAS of claim 15, wherein the parent bacterial cell is Bacillus subtilis.

17. 3. The ADAS of claim 2, wherein the stability of the ADAS, as measured by the percent intact ADAS, is greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%.

18. 18. The ADAS of any one of claims 1 to 17, 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 both of SEQ ID NO:5 and SEQ ID NO:9.

Images