Stable non-chromosomal dynamic activity systems and uses thereof

Abstract

Disclosed herein is a non-chromosomal dynamic activity system (ADAS) derived from a parent bacterial cell comprising at least one loss of genetic function in a lyase to increase the stability of the ADAS. Also disclosed are methods of blocking sporogenesis in a parent bacterial cell in combination with a lyase deletion or other loss-of-function mutation.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing, which has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on November 14, 2023, is named 51296-057WO2_Sequence_Listing_11_14_23 and is 25,540 bytes in size. Technical Field

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

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

[0005] In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function alteration in a lyase. In some embodiments, relative to an ADAS derived from a parental bacterial cell that does not contain the alteration, the loss of function in the lyase results in increased stability of the ADAS.

[0006] In some aspects, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one genomic deletion in a lyase. In some embodiments, relative to an ADAS derived from a parental bacterial cell that does not contain the alteration, the genomic deletion in the lyase results in increased stability of the ADAS.

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

[0008] In some embodiments, the loss-of-function alteration in the parental bacterial cell decreases enzyme activity and / or lytic activity. In some embodiments, the genomic deletion in the parental bacterial cell decreases enzyme activity and / or lytic activity. In some embodiments, the parental bacterial cell has at least one loss-of-function alteration that decreases the activity of an endopeptidase, a cell wall lyase, and / or an autolysin. In some embodiments, the parental bacterial cell has at least one genomic deletion that decreases the activity of one or more endopeptidases, cell wall lyases, and / or autolysins.

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

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

[0011] In some embodiments, the parental bacterial cell is a Gram-positive bacterial cell.

[0012] In some embodiments, the parental bacterial cell is Bacillus subtilis or belongs to the genus Bacillus.

[0013] In some embodiments, the parental bacterial cell belongs to the genus Lactobacillus.

[0014] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0015] In some embodiments, the parental bacterial cell is from a genus selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, and Thermoanaerobacter.

[0016] In some embodiments, the parental cell comprises 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. [Molecular Biology and Evolution] 20(12):2076–2090, 2003).

[0017] In some embodiments, the parental bacterial cell contains a genomic deletion of a cell division topological specificity factor. In some embodiments, the genomic deletion belongs to the DivIVA, minC, minD, minE, minCD or minCDE operon.

[0018] In some embodiments, the ADAS further comprises at least one cargo.

[0019] In some embodiments, the cargo is a protein or polypeptide.

[0020] In some embodiments, the ADAS or the parental bacterial cell has been modified to increase the level of cargo in the ADAS.

[0021] In some embodiments, the cargo is an enzyme, a DNA modifier, a chromatin remodeler, a gene editing agent, a nuclear targeting agent, a binder, an immunogenic agent or a toxin. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editing agent is a component of the CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binder is an antibody or antibody fragment. In some embodiments, the binder is a VHH molecule. In some embodiments, the immunogenic agent is an immune stimulant. In some embodiments, the immunogenic agent is an immunosuppressant.

[0022] Other features and advantages of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a set of representative images showing overnight cultures of three different parental cell strains derived from Bacillus subtilis. These strains and their unique genomic deletions are listed in Table 1. M008: MACH008; m2347: MACH2347; m2403: MACH2403.

[0024] Figure 2 is a bar graph depicting the corresponding OD 600 measurements of each cultured strain at the following three different time points: t0 (after overnight culture and before enrichment of the chromosomally encoded dynamic activity system (ADAS)), t23 (after overnight culture, ADAS enrichment and incubation at 4°C for 23 hours), and t48 (after overnight culture, ADAS enrichment and incubation at 4°C for 48 hours).

[0025] Figure 3Aare a pair of representative micrographs showing ADAS and remaining parental cells from two different Bacillus subtilis strains after overnight culture. The left panel is strain MACH2347 (described in Table 1). The right panel is MACH2403 (described in Table 1), which was modified from MACH2347 to have an additional lytC genomic deletion. White arrows indicate examples of light-phase ADAS. Wedge symbols indicate examples of light-phase parental cells.

[0026] Figure 3B are a pair of representative micrographs showing ADAS and remaining parental cells from two different Bacillus subtilis strains after overnight culture and ADAS enrichment (t0). Left panel: MACH2347. Right panel: MACH2403. White arrows: Exemplary light-phase ADAS. Wedge symbols: Exemplary light-phase parental cells.

[0027] Figure 3C are a pair of representative micrographs showing ADAS and remaining parental cells from two different Bacillus subtilis strains after overnight culture, ADAS enrichment, and incubation at 4 °C for 23 hours (t23). Left panel: MACH2347. Right panel: MACH2403. White arrows: Exemplary light-phase ADAS. Wedge symbols: Exemplary light-phase parental cells. Detailed Description

[0028] I. Definitions

[0029] As used herein, the term "achromosomal dynamic system" or "ADAS" refers to a genome-free non-replicating closed membrane system that includes at least one membrane and has an internal volume suitable for accommodating cargo (e.g., one or more of the following: nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs)). In some embodiments, the ADAS is a minicell or a modified minicell derived from a parental bacterial cell (e.g., a Gram-negative or Gram-positive bacterial cell). In other aspects, the ADAS is derived from a parental cell by modifying the parental cell to remove the genome and is substantially similar in size to the parental cell. In some embodiments, the ADAS is derived from a parental bacterium using any suitable method, such as genetically manipulating the parental cell or exposing it to cultures or conditions that increase the likelihood of bacterial minicell formation. An exemplary method for preparing the ADAS is a method that disrupts the cell division machinery of the parental cell. In some embodiments, the ADAS includes one or more endogenous or heterologous features on the surface of the parental cell, such as a cell wall, cell wall modification, flagella, or pili, and / or one or more endogenous or heterologous features in the internal volume of the parental cell, such as nucleic acids, plasmids, proteins, small molecules, transcriptional machinery, or translational machinery. In other embodiments, the ADAS may lack one or more features of the parental cell. In still other embodiments, the ADAS is loaded with or otherwise modified by features not contained in the parental cell.

[0030] As used herein, the term "highly active ADAS" refers to an ADAS that has high working potential, e.g., an ADAS that has the ability to perform a large amount of useful work. In some embodiments, the work is metabolic work, including chemical synthesis (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), chemical modification (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), or transport (e.g., import, export, or secretion, such as secretion by a bacterial secretion system (e.g., T3SS)) under suitable conditions. In certain embodiments, the highly active ADAS starts with a large amount of energy (e.g., energy in the form of ATP). In other embodiments, the ADAS has the ability to absorb or generate energy / ATP from another source. In some embodiments, highly active ADAS is identified by, e.g., an increase in ATP concentration, an increase in the ability to produce ATP, an increase in the ability to produce proteins, an increase in the rate or amount of protein production, and / or an increase in responsiveness to biological signals (e.g., induction of a promoter).

[0031] As used herein, the term "parental bacterial cell" refers to the cell (e.g., a Gram-negative or Gram-positive bacterial cell) from which the ADAS is derived. The parental bacterial cell is typically a live bacterial cell. The term "live bacterial cell" refers to a bacterial cell that contains a genome and is capable of cell division. Preferred parental bacterial cells are derived from any of the following strains: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phormidium, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacteria.

[0032] An ADAS composition or preparation that is "substantially free" of parental bacterial cells and / or live bacterial cells is defined herein as a composition having no more than 500, e.g., 400, 300, 200, 150, 100 or fewer colony forming units (CFU) / mL. In some embodiments, an ADAS composition that is substantially free of parental bacterial cells or live bacterial cells (including no bacterial cells) comprises less than 50, less than 25, less than 10, less than 5, less than 1, less than 0.1 or less than 0.001 CFU / mL.

[0033] The term "cell division topology-specific factor" refers to a component of the cell division machinery in a bacterial species that is involved in determining the site of the septum and acts by restricting the position of other components of the cell division machinery (e.g., restricting the position of one or more Z-ring inhibitory proteins). Exemplary cell division topology-specific factors include MinE, which was first discovered in Escherichia coli and has been identified in a large number of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). MinE acts by restricting the Z-ring inhibitory proteins MinC and MinD to the poles of the cell. A second exemplary cell division topology-specific factor is DivIVA, which was first discovered in Bacillus subtilis (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005).

[0034] The term "Z-ring inhibitory protein" refers to a component of the cell division machinery in a bacterial species that is involved in determining the site of the septum and acts by inhibiting the formation of a stable FtsZ ring or anchoring such a component to the membrane. In some embodiments, the localization of the Z-ring inhibitory protein is regulated by cell division topology-specific factors (e.g., MinE and DivIVA). Exemplary Z-ring inhibitory proteins include MinC and MinD, which were first discovered in Escherichia coli and have been identified in a large number of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). In Escherichia coli and in other species, MinC, MinD, and MinE are present at the same genetic locus, which may be referred to as the "min operon", the MinCDE operon, or the Min or MinCDE genetic locus.

[0035] As used herein, the term "reduced level or activity of a cellular topology-specific factor" means a general reduction of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level or activity of a cellular topology-specific factor (e.g., a protein or a nucleic acid (e.g., a gene or an mRNA)) as detected by standard methods, compared to the level in a reference sample (e.g., an ADAS produced by wild-type cells or cells having a wild-type minCDE operon or a wild-type divIVA gene), a reference cell (e.g., a wild-type cell or a cell having a wild-type minC, minD, minE, divIVA or minCDE gene or operon), a control sample or a control cell. In some embodiments, the reduced level or activity means a reduction in the level or activity in a sample that is at least about 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x or 0.01x the level or activity of the cellular topology-specific factor in a reference sample, reference cell, control sample or control cell.

[0036] As used herein, the term "percent identity" means the percent (%) sequence identity relative to a reference polynucleotide or polypeptide sequence as determined by alignment using standard techniques. The alignment for purposes of determining the percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of those of skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST or Megalign software. Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. For example, in some embodiments, the sequence comparison computer program BLAST is used to generate the percent sequence identity values. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A with, compared to, or relative to a given nucleic acid or amino acid sequence B (which can alternatively be expressed as the phrase: a given nucleic acid or amino acid sequence A having a certain percent sequence identity with, compared to, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows:

[0037] 100×(fraction X / Y)

[0038] Where X is the number of nucleotides or amino acids scored as identical matches by the program in the alignment of A and B by a sequence alignment program (e.g., BLAST), and where Y is the total number of nucleotides or amino acids in B. In some embodiments, for example, in homologs of the MinE or DivIVA proteins, the sequence identity to the native sequence MinE (or minE) or DivIVA (or divIVA) sequences disclosed herein will have at least about 40%, 50%, 60%, 70%, 80%, 85%, 90% or even 95% or greater amino acid or nucleic acid sequence identity, alternatively, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence or nucleic acid identity.

[0039] As used herein, the phrase "modulating the state of a cell" refers to an observable change in the state of a cell (e.g., a transcriptome, proteome, epigenome, biological effect, or health or disease state) 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 proteins, transcripts, epigenetic markers, or for measuring an increase or decrease in biological pathway activity). In some embodiments, modulating the state of a cell results in a change of at least 1% relative to before administration (e.g., a change of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., a change of up to 100% relative to before administration). In some embodiments, modulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In some embodiments, increasing the state of a cell results in an increase in the parameter of at least 1% relative to before administration (e.g., an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., an increase of up to 100% relative to before administration). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In some embodiments, decreasing the state of a cell results in a decrease in the parameter of at least 1% relative to before administration (e.g., a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% or more relative to before administration; e.g., a decrease of up to 100% relative to before administration).

[0040] As used herein, the term "endogenous type III secretion system" or "endogenous T3SS" refers to a T3SS that is present on a cell (e.g., a parental cell) or an ADAS derived therefrom and that is naturally encoded by the cell (e.g., encoded by the wild-type version of the cell). The T3SS can be expressed from endogenous genes of the cell and / or can be encoded and expressed by a synthetic construct in the cell. The expression or abundance of the endogenous T3SS can be increased, for example, by adding a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by reducing or removing a negative regulator of T3SS expression.

[0041] As used herein, the term "heterologous type III secretion system" or "heterologous T3SS" refers to a T3SS that is present on or derived from a cell (e.g., a parental cell) and is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell). The cell may encode another T3SS or may not encode any T3SS. In some embodiments, the T3SS is expressed by a synthetic construct in the cell.

[0042] As used herein, an "endogenous effector" of a secretion system (e.g., T3SS, T4SS, or T6SS) is a moiety (e.g., a protein or polypeptide) that is naturally encoded by the cell (e.g., encoded by the wild-type version of the cell) from which the secretion system (e.g., T3SS) is derived and is capable of being secreted by the secretion system. One or more of the secretion system and its endogenous effector(s) may be expressed in the cell in which they naturally occur or may be heterologously expressed, e.g., by a cell that does not naturally encode the endogenous effector or the secretion system.

[0043] As used herein, an effector that is heterologous relative to a secretion system ("heterologous effector") is a moiety (e.g., a protein or polypeptide) that is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell) from which the secretion system (e.g., T3SS) is derived and is capable of being secreted by the secretion system of the cell from which the heterologous effector is derived. In some embodiments, the effector is capable of being secreted by a secretion system to which it is heterologous or is modified to be secreted by a secretion system to which it is heterologous. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0044] As used herein, the term "heterologous" means not inherent to a cell or composition in its natural state. In some embodiments, "heterologous" refers to a molecule; e.g., a cargo or payload (e.g., a polypeptide, a nucleic acid such as an RNA encoding a protein or a tRNA, or a small molecule) or a structure (e.g., a plasmid or a gene editing system) that is not naturally present in an ADAS or the parental bacterium (e.g., a Gram-negative or Gram-positive bacterial cell) from which the ADAS is produced.

[0045] As used herein, the terms "phase-light ADAS" and "phase-light parental cell" refer to an ADAS or parental cell body (e.g., a dead ADAS or dead parental cell) that appears brighter or has a ghostly image (indicating rupture and lysis) in an image (e.g., a micrograph taken using an optical microscope) compared to a dark ADAS or parental cell, which are described herein as "intact ADAS" and "intact parental cell," respectively.

[0046] As used herein, "increased stability" of an ADAS refers to an overall enhancement of ADAS integrity. The stability of an ADAS (e.g., the stability of an ADAS of a particular population, strain, or variety) can be measured as the ratio (e.g., percentage) of "intact" ADAS to "attenuated phase" ADAS in one or more representative images of a plurality of ADAS. For example, an increase in the stability of an ADAS derived from a modified strain containing at least one lyase deletion can be defined as an increase in the percentage of "intact" ADAS in the representative images of the ADAS derived from the modified strain compared to the ADAS derived from a control strain (e.g., a strain not containing a lyase deletion), where measurements (e.g., scoring) are made under the same conditions and time points. For example, if the modified ADAS has a stability greater than 40%, more than 40% of the visible ADAS is intact rather than in the attenuated phase. In some embodiments, the stability of an ADAS is measured as the unitless ratio of the half-life of an ADAS derived from an unmodified strain (e.g., a strain not containing a lyase deletion) to the half-life of an ADAS derived from a modified strain (e.g., a strain containing a lyase deletion), where measurements are made under the same environmental conditions. In some embodiments, at one or more time points (e.g., 0 hours, 10 hours, 20 hours, 23 hours, 24 hours, 48 hours, 72 hours, or more than 72 hours after ADAS enrichment), the fold change in the optical density at wavelength 600 (OD600) of an ADAS derived from a modified strain compared to an ADAS derived from a control strain is another measure of ADAS stability. The stability of an ADAS is improved (e.g., increased) by at least one genomic deletion of a lyase in the parental cell. In certain embodiments, an ADAS having one or more lyase deletions (e.g., derived from a parental cell containing one or more lyase deletions) has a stability greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (as measured by the percentage of intact ADAS) compared to an ADAS not having a lyase deletion (e.g., derived from a parental cell not containing a lyase deletion).

[0047] As used herein, "lyase" refers to an enzyme that regulates the rupture of the parental bacterial cell wall and membrane (e.g., endopeptidase, cell wall lyase, or autolysin). Genomic deletion of one or more of these enzymes reduces the lytic activity in the parental cell and, in some embodiments, can increase the stability and integrity of the ADAS structure, thereby allowing for the production of a greater number of intact ADAS and / or increasing the survival period of intact ADAS. Specific genomic deletions that reduce the lytic activity in the parental cell include, but are not limited to, lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, and combinations thereof.

[0048] As used herein, "sporulation" refers to the process by which certain parental bacterial strains form spores from cells during adverse conditions. Genetic deletions that may disrupt sporulation include, but are not limited to, sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof.

[0049] As used herein, "loss of genetic function" refers to a significant reduction or complete elimination of a protein (e.g., a significant decrease in the function of the protein, a complete loss of the function of the protein, a significant decrease in the expression of the protein, or a complete loss of the expression of the protein) caused by a change (e.g., gene mutation) in the gene encoding the protein. Mutations include, but are not limited to, insertions or deletions of one or more nucleotides, non-silent codon changes, and duplications. Proteins in which loss of genetic function may occur include, but are not limited to, enzymes such as lyase, protease, amylase, lipase, or cellulase. In addition to loss of genetic function by mutation, promoter inactivation or chemical inhibition can also affect (e.g., reduce or eliminate) the expression of a protein (e.g., lyase, protease, amylase, lipase, or cellulase).

[0050] II. Composition

[0051] A. ADAS and highly active ADAS

[0052] The present invention is based at least in part on the applicant's discovery of non-chromosomal dynamic active systems (ADAS) (including highly active ADAS), which are capable of providing multiple functions in a large number of environments. "ADAS" is a genome-free non-replicating closed membrane system comprising at least one membrane (in some embodiments, two membranes, wherein the two membranes are disjoint) and having an internal volume suitable for accommodating cargo (e.g., nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles or ribonucleoprotein complexes (RNPs)).

[0053] In some embodiments, ADAS is a minicell or modified minicell derived from a parent bacterial cell (e.g., a Gram-negative or Gram-positive bacterial cell). In some aspects, ADAS is derived from a parent bacterium using any suitable method, such as genetic manipulation of the parent cell or exposure to a culture or condition that increases the likelihood of bacterial minicell formation.

[0054] In some embodiments, ADAS has a long axis cross section between about 100nm-500μm (e.g., in some embodiments, about: 100-600nm, such as 100-400nm; or between about 0.5-10μm and 10-500μm). In some embodiments, the short axis cross section of ADAS is between about: 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100% of the long axis. In some embodiments, ADAS has about: 0.001-1μm 3 , 0.3-5μm 3 、5-4000μm 3 or 4000-50×10 7 μm 3 In some embodiments, the size of the ADAS is substantially similar to that of the parent cell, for example, the size (e.g., internal volume, major axis cross-section, and / or minor axis cross-section) is about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the size of the parent cell, the size is the same as the size of the parent cell, or the size is about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of the size of the parent cell.

[0055] In some embodiments, the present invention provides highly active ADAS. "Highly active ADAS" is an ADAS having high working potential, e.g., an ADAS having the ability to perform a large amount of useful work. In some embodiments, work is defined as, for example, metabolic work, including chemical synthesis (e.g., synthesis of proteins, nucleic acids, lipids, carbohydrates, polymers or small molecules), chemical modification (e.g., modification of proteins, nucleic acids, lipids, carbohydrates, polymers or small molecules) or transport (e.g., import, export or secretion) under suitable conditions. In some embodiments, the highly active ADAS starts with a large amount of energy (e.g., energy in the form of adenosine triphosphate (ATP)). In other embodiments, the ADAS has the ability to absorb or generate energy (e.g., ATP) from another source.

[0056] The term "ADAS provided by the present invention" encompasses all embodiments of the ADAS described herein, including the highly active ADAS in specific embodiments, and this group can be referred to as "highly active ADAS provided by the present invention", which is a subgroup of the ADAS provided by the present invention.

[0057] In one aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic active systems (ADAS), wherein the initial ATP concentration of the ADAS is at least 1 mM, and wherein the composition is substantially free of live bacterial cells.

[0058] In another aspect, the present invention provides a composition comprising a plurality of highly active non-chromosomal dynamic active systems (ADAS), wherein the initial ATP concentration of the ADAS is at least 3 mM, and wherein the composition is substantially free of live bacterial cells.

[0059] In some embodiments, the initial ATP concentration of the highly active ADAS is at least 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, 2.5 mM, 3 nM, 3.5 nM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM or 50 mM. The ATP concentration can be evaluated by various means, including the BacTiter-Glo TM assay (Promega) for lysed ADAS in certain embodiments.

[0060] In some embodiments, high activity is additionally or alternatively assessed by the rate or amount of increase in ATP concentration over time in the ADAS. In some embodiments, after incubation under suitable conditions (e.g., incubation at 37 °C for 12 hours), the ATP concentration in the ADAS increases by at least 50%, at least 60%, at least 75%, at least 100%, at least 150%, at least 200%, or more than 200%. In certain embodiments, the ATP production rate of the high activity ADAS is greater than approximately: 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2, 3, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 500, 1000, 10000 ATP / sec / nm 2 , for at least approximately: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, or two weeks.

[0061] In other aspects, high activity is assessed by the rate of decrease in ATP concentration over time. In some embodiments, the ATP concentration in the high activity ADAS decreases more slowly than in the non-high activity ADAS. In some embodiments, for example, as measured using BacTiter-Glo TM assay (Promega Corporation), the ATP concentration in the ADAS or ADAS composition 24 hours after preparation decreases by less than approximately 50% (e.g., less than approximately: 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) compared to the initial ATP concentration (e.g., ATP per cell volume).

[0062] In some embodiments, high activity is additionally or alternatively assessed by the longevity index of the ADAS. The longevity index is calculated as the ratio of the GFP production rate at 24 hours to that at 30 minutes. In some embodiments, the longevity index of the high activity ADAS is greater than approximately: 0.13, 0.14, 0.15, 0.16, 0.18, 0.2, 0.25, 0.3, 0.35, 0.45, 0.5, 0.60, 0.70, 0.80, 0.90, 1.0, or greater. In more specific embodiments, the longevity index is measured in an ADAS containing a functional GFP plasmid with a species-appropriate promoter, where at 30 minutes and 24 hours, the GFP concentration is measured relative to the number of ADAS, the average number of plasmids per ADAS, and the solution volume using a plate reader.

[0063] In some aspects, the ADAS produces proteins, such as heterologous proteins. In some aspects, high activity is assessed by the rate, amount, or duration of protein production or the rate of inducing protein expression (e.g., the response of the ADAS to a signal). For example, in some embodiments, the ADAS comprises a plasmid that comprises an inducible promoter and a nucleotide sequence encoding the heterologous protein, wherein contacting the ADAS with an inducer of the inducible promoter under appropriate conditions causes production of the heterologous protein. In some aspects, in an ADAS that has been contacted with the inducer (e.g., a high-activity ADAS), the production of the heterologous protein is increased by at least 1.6-fold relative to an ADAS that has not been contacted with the inducer. For example, in some embodiments, in an ADAS that has been contacted with the inducer (e.g., a high-activity ADAS), the production of the heterologous protein is increased by at least 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more than 10-fold. In some embodiments, the rate of production of the heterologous protein by the high-activity ADAS reaches a target level within a specific duration after the ADAS is contacted with the inducer, e.g., within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or more than 3 hours. In some embodiments, the protein (e.g., heterologous protein) is produced at a rate of at least 0.1 femtograms per hour per high-activity ADAS, e.g., at least 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10, 25, 50, 100, 250, 500, 1000, 2000, 3000, or 3500 fg / hour / ADAS. In some embodiments, the high activity of the ADAS is assessed by the duration of protein production. In some embodiments, the high-activity ADAS produces protein (e.g., heterologous protein) for a duration of at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or more than 48 hours.

[0064] B. ADAS and high-activity ADAS derived from a parental bacterium defective in a cell division topology-specific factor

[0065] In some embodiments, as described herein, the ADAS is derived from a bacterial parental cell.

[0066] In some aspects, the present invention provides an ADAS and / or a composition comprising a plurality of ADASs, wherein the ADAS and / or the composition is derived from a parental bacterium having a reduced level, activity, or expression of a cell division topology-specific factor.

[0067] In some aspects, the present invention provides a composition comprising a plurality of ADASs, wherein the ADASs do not comprise a cell division topology-specific factor and wherein the composition is substantially free of live bacterial cells.

[0068] In some aspects, the present invention provides a composition comprising multiple ADASs, the composition being substantially free of live bacterial cells and produced by a method comprising: (a) preparing, providing, or obtaining multiple parental bacteria with a reduced level or activity of a cell division topological specificity factor; (b) exposing the parental bacteria to conditions permitting the formation of minicells, thereby producing the highly active ADASs; and (c) separating the ADASs from the parental bacteria, thereby producing a composition substantially free of live bacterial cells.

[0069] In some embodiments of the above aspects, the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 20% identity to an Escherichia coli minE polypeptide (e.g., as encoded by SEQ ID NO:1), such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to the polypeptide encoded by SEQ ID NO:1. In some embodiments, the cell division topological specificity factor comprises the amino acid sequence encoded by SEQ ID NO:1. In some embodiments, the cell division topological specificity factor is a minE polypeptide. Exemplary species having a minE polypeptide are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005.

[0070] In some embodiments, the parental bacterium is Escherichia coli and the MinE polypeptide is E. coli MinE. In other embodiments, the parental bacterium is Salmonella typhimurium and the MinE polypeptide is S. typhimurium MinE. In yet other embodiments, the parental bacterium is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phormidium, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter, and the cell division topology-specific factor is the endogenous MinE or DivIVA of the parental bacterium.

[0071] In some embodiments of the above aspects, the cell division topology-specific factor is a polypeptide having an amino acid sequence that is at least 20% identical to a Bacillus subtilis DivIVA polypeptide (e.g., as encoded by SEQ ID NO:5), such as 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-specific factor comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO:5. In some embodiments, the cell division topology-specific factor is a DivIVA polypeptide. Exemplary species having a DivIVA polypeptide are provided in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005. In some embodiments, the parental bacterium is Bacillus subtilis, and the cell division topology-specific factor is Bacillus subtilis DivIVA.

[0072] In some embodiments, the level of one or more Z-ring inhibitory proteins of the ADAS or parental bacterium in which the level or activity of the cell division topology-specific factor is reduced is also reduced.

[0073] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence that is at least 20% identical to an Escherichia coli minC polypeptide (e.g., as encoded by SEQ ID NO:2), such as 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.

[0074] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence that is at least 20% identical to an Escherichia coli minD polypeptide (e.g., as encoded by SEQ ID NO:3), such as 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.

[0075] In some embodiments, the levels, activities, or expression of at least two Z-ring inhibitory proteins of the ADAS or the parental bacterium are reduced. In some embodiments, the expression of the MinC polypeptide and the MinD polypeptide of the ADAS or the parental bacterium is reduced. In some embodiments, the expression of the MinC polypeptide, the MinD polypeptide, and the MinE polypeptide of the ADAS or the parental bacterium is reduced, such as a deletion of the minCDE operon (ΔminCDE).

[0076] In some embodiments, any suitable method is used to achieve a reduction in the level, activity, or expression of a cell division topology-specific factor or a Z-ring inhibitory protein, such as a reduction in the ADAS or in the parental bacterial cell. For example, in some embodiments, the reduction in the level or activity is caused by a loss-of-function mutation (such as a gene deletion). In some embodiments, the loss-of-function mutation is an inducible loss-of-function mutation, and the loss of function is induced by exposing the parental cell to an inducing condition. For example, the inducible loss-of-function mutation is a temperature-sensitive mutation, and the inducing condition is a temperature condition.

[0077] In some embodiments, the parental cell has a deletion of the minCDE operon (ΔminCDE) or a homologous operon deletion.

[0078] C. ADAS with increased stability due to a genetic loss of function of a lyase

[0079] In some embodiments, the parental bacterial cell has one or more genetic loss-of-function alterations that stabilize the ADAS derived from the cell (e.g., resulting in an increase in stability in the ADAS derived from the cell). In some embodiments, the parental bacterial cell has been modified to reduce enzyme activity. In certain embodiments, the ADAS stability is improved by a genetic deletion of one or more enzymes.

[0080] In some embodiments, the parental cell comprises one or more loss-of-function alterations that reduce or eliminate enzymatic activity and / or lytic activity (e.g., reduce or eliminate the enzymatic activity or lytic activity performed by a protein encoded by a gene comprising a loss-of-function alteration). In some embodiments, the parental cell comprises a genomic deletion that reduces or eliminates enzymatic activity and / or lytic activity (e.g., reduces or eliminates the enzymatic activity or lytic activity performed by a protein encoded by a deleted genomic region). In some embodiments, the parental bacterial cell comprises one or more genomic deletions that reduce the activity of one or more endopeptidases, cell wall-lytic enzymes, and / or autolysins. In some embodiments, the parental cell comprises a loss-of-function (e.g., genomic deletion) from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwlK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ, and combinations thereof.

[0081] In some embodiments, the parental cell comprises a loss-of-function in lytC. For example, in the case where a wild-type parental cell comprises a lytC sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:8, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:8 and 9. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:9. The loss-of-function alteration of lytC may comprise a deletion of all or a portion of the coding region of the gene.

[0082] In some embodiments, the parental cell comprises a loss-of-function in sigF. For example, in the case where a wild-type parental cell comprises a sigF sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4, the parental cell of the present invention may comprise a deletion as shown by comparison of SEQ ID NO:4 and 5. For example, the parental cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5. The loss-of-function alteration of sigF may comprise a deletion of all or a portion of the coding region of the gene.

[0083] One of ordinary skill in the art can readily identify alterations in nucleotide sequences encoding proteins (e.g., proteins having enzymatic activity and / or lytic activity, such as lytic enzymes) that result in a loss of protein function.

[0084] In some embodiments, the parental cell contains a loss-of-function in lytC.

[0085] In some embodiments, the parental cell contains a genomic deletion of lytC.

[0086] In some embodiments, the parental bacterial cell contains at least one loss of genetic function (e.g., genomic deletion) of a gene encoding a lytic enzyme, a gene affecting the sporulation mechanism, or a cell division topological factor, or a combination thereof (e.g., comprising two or more loss-of-function alterations affecting one or more lytic enzymes, one or more genes affecting the sporulation mechanism, and / or one or more cell division topological factors).

[0087] In some embodiments, the parental bacterial cell contains a loss of genetic function (e.g., genomic deletion) of lytC, sigF, and divIVa. In some embodiments, the parental bacterial cell contains genomic deletions of lytC, sigF, and divIVa.

[0088] In some embodiments, the parental bacterial cell is a Gram-positive bacterial cell.

[0089] In some embodiments, the parental bacterial cell is Bacillus subtilis or belongs to the genus Bacillus (e.g., is a Bacillus species).

[0090] In some embodiments, the parental bacterial cell belongs to the genus Lactobacillus (e.g., is a Lactobacillus species).

[0091] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0092] In some embodiments, the parental bacterial cell is a bacterium of the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter.

[0093] In some embodiments, the parental cell comprises a gene deletion from the group consisting of SPβ, skin, PBSX, prophage 1, pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. [Molecular Biology and Evolution] 20(12):2076 - 2090, 2003). Additional deletions of genomic segments in the parental bacterial cell can lead to a reduction of multiple lysis elements through the removal of prophages and prophage-like segments. These deletions, like certain lytic enzyme deletions or loss-of-function mutations (e.g., lytC), are not necessary for establishing a complete and stable ADAS.

[0094] D. ADAS Derived from Parental Cells with Sporulation Blocked

[0095] In some embodiments, the parental cell comprises a gene deletion or loss-of-function alteration (e.g., loss-of-function mutation) that blocks sporulation (e.g., reduces or eliminates sporulation). In some embodiments, the loss-of-function alteration is selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, and combinations thereof. Although the genomic deletion for blocking sporulation and the genomic deletion for reducing enzyme activity in the ADAS are not required or dependent on each other, each genomic deletion provides unique benefits for ADAS formation and use. Disrupting sporulation is beneficial for ADAS formation because it maintains the parental cell population and removes spores that may be difficult to distinguish from the ADAS. Thus, in some aspects, the parental cells provided herein simultaneously comprise one or more alterations (e.g., loss-of-function mutations) that reduce enzyme activity and one or more alterations (e.g., loss-of-function mutations) that block sporulation.

[0096] E. ADAS containing cargo

[0097] In some embodiments, the ADAS provided by the present invention includes cargo contained within the ADAS. In some embodiments, the cargo is any moiety disposed within the ADAS (e.g., encapsulated by the ADAS) or conjugated to the surface of the ADAS. In some embodiments, the cargo comprises nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulators, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs), or combinations of the foregoing. In some aspects, the cargo is delivered by a secretion system (e.g., T3SS). In other aspects, the cargo is not delivered by the T3SS.

[0098] In some embodiments, the nucleic acid is DNA, RNA, or a plasmid. In some embodiments, the nucleic acid (e.g., DNA, RNA (e.g., mRNA, ASO, circular RNA, siRNA, shRNA, tRNA, dsRNA, or combinations thereof), or plasmid) encodes a protein. In some embodiments, the protein is transcribed and / or translated in the ADAS. In some embodiments, the nucleic acid inhibits the translation of a protein or polypeptide, e.g., is siRNA or an antisense oligonucleotide (ASO).

[0099] In some embodiments, the cargo is an agent that can modulate the microbiome of a target organism (e.g., a human, animal, plant, or fungal microbiome), such as a polysaccharide, an amino acid, an antimicrobial agent (e.g., an anti-infective or antimicrobial peptide, protein, and / or natural product), a short-chain fatty acid, or a combination thereof. In some instances, the agent that can modulate the host microbiome is a probiotic agent.

[0100] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered.

[0101] In certain embodiments, the cargo is modified to have improved stability compared to the unmodified version of the cargo. The "stability" of the cargo is a unitless ratio of the half-life of the unmodified cargo to the half-life of the modified cargo, where measured under the same environmental conditions. In some embodiments, the environment is experimentally controlled, such as simulated body fluid, RNase-free water, cytoplasm, extracellular space, or "ADAS protoplasm" (i.e., the contents of the internal volume of the ADAS, e.g., after lysis). In some applications, it is an agricultural environment, such as arable soil, river water, or sea water. In other embodiments, the environment is actual or simulated: animal intestine, animal skin, animal reproductive tract, animal respiratory tract, animal bloodstream, or animal extracellular space. In certain embodiments, the ADAS does not significantly degrade the cargo.

[0102] In certain embodiments, the cargo comprises a protein. In certain embodiments, the stability of the protein in the cytoplasm or other environment is greater than about: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 10000000. The protein can be any protein, including growth factors; enzymes; hormones; immunomodulatory proteins; antibiotic proteins, such as antibacterial, antifungal, insecticidal proteins, etc.; targeting agents, such as antibodies or nanobodies, etc. In some embodiments, the protein is a hormone, such as a paracrine, endocrine, autocrine hormone.

[0103] In some embodiments, the cargo comprises a plant hormone, such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.

[0104] In some embodiments, the cargo is an anti-inflammatory agent, such as a cytokine (e.g., a heterologously expressed anti-inflammatory cytokine or a mutant protein thereof (e.g., IL-10, TGF-β, IL-22, IL-2)) or an antibody (e.g., an antibody or antibody fragment targeting tumor necrosis factor (TNF) (e.g., an anti-TNF antibody)); an antibody or antibody fragment targeting IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment targeting IL-23 (e.g., an anti-IL-23 antibody)).

[0105] In certain embodiments, the cargo is an immunomodulator. Immunomodulators include, for example, immunostimulants; checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4); chemotherapeutic agents; immunosuppressive agents; antigens; superantigens; and small molecules (e.g., cyclosporin A, cyclic dinucleotide (CDN), or a STING agonist (e.g., MK-1454)). In some embodiments, the immunomodulator is a moiety that induces tolerance in a subject, such as an allergen, an autoantigen (e.g., a disease-associated autoantigen), or a microbe-specific antigen. In some embodiments, the immunomodulator is a vaccine, such as an antigen from a pathogen (e.g., a virus (e.g., a viral envelope protein) or a bacterium). In some embodiments, the antigen is a cancer neoantigen. In some embodiments, the pathogen is a coronavirus, such as SARS-CoV-2. In some embodiments, the cargo is an adjuvant, such as an immunomodulatory molecule or a molecule that alters the compartmentalization, presentation, or profile of one or more costimulatory molecules associated with a vaccine antigen. In some instances, the adjuvant is an activator of an immune pathway upstream of the desired immune response (e.g., an activator of the innate immune pathway upstream of the adaptive immune response). In other instances, the adjuvant enhances the presentation of an antigen on immune cells or immune moieties (e.g., MHC class I) in a target organism. In some instances, the adjuvant is listeriolysin O (LLO). In some embodiments, the ADAS comprises an antigen and one or more adjuvants.

[0106] In some embodiments, the cargo is an agent for treating or preventing cancer, e.g., an agent that reduces the likelihood of a patient developing cancer or an agent that treats cancer (e.g., an agent that increases the progression-free survival and / or overall survival of an individual with cancer).

[0107] 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, immunotherapy agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as listeriolysin - o), cytotoxins, inflammasome inhibitory agents, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1 or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0108] In some aspects, the ADAS therapeutic composition is administered by oral, intravenous, intradermal, intramuscular, intraperitoneal, peritumoral, intranasal, intraocular, or rectal, and / or subcutaneous administration. In certain embodiments, the ADAS is administered by oral, intravenous, intramuscular, and / or subcutaneous administration. In some embodiments, the ADAS is administered to a subject once, twice, three times, four times, or more. In some embodiments, the ADAS dose is at least 1×10 5 、1×10 6 、1×10 7 、1×10 8 、5×10 8 、6×10 8 、8×10 8 、1×10 9 、2×10 9 、4×10 9 、6×10 9 、8×10 9 、or 1×10 10 ,for example, administration includes administering to a subject at least 1×10 5 、1×10 6 、1×10 7 、1×10 8 、5×10 8 、6×10 8 、8×10 8 、1×10 9 、2×10 9 、4×10 9 、6×10 9 、8×10 9 、or 1×10 10 ADAS.

[0109] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme is an enzyme that exerts catalytic activity in a target cell or organism (e.g., in a human, animal, plant, or 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 for enzyme replacement therapy, such as phenylalanine hydroxylase. In some embodiments, the enzyme is UDP - glucuronosyltransferase. In some embodiments, the enzyme has hepatic enzyme activity (e.g., porphobilinogen deaminase (PBGD), such as human PBGD (hPBGD)). In some embodiments, the enzyme is a protease, an oxidoreductase, or a combination thereof.

[0110] In some embodiments, the enzyme modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered. In some embodiments, the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic di - AMP.

[0111] In some embodiments, the enzyme is chemically conjugated to the ADAS membrane, optionally conjugated to the outer membrane via a linker.

[0112] Alternatively, in some embodiments, the cargo is a nucleic acid encoding any of the enzymes described herein.

[0113] In some embodiments, the cargo is an agent that activates or inhibits the autophagy process (e.g., an activator such as listeriolysin - o, or an inhibitor such as IcsB).

[0114] In some embodiments, the cargo is an anti - infective agent, such as an antimicrobial agent, such as an anti - infective or antimicrobial peptide, protein, and / or natural product.

[0115] In some embodiments, the cargo is a protein that regulates the host transcriptional response, such as a transcription factor; a protein that promotes host cell growth, such as a growth factor; or a protein that inhibits protein function, such as a nanobody. In some embodiments, the transcription factor is a human transcription factor.

[0116] For ADAS containing cargo, in some embodiments, the cargo is RNA, such as circular RNA, mRNA, siRNA, shRNA, ASO, tRNA, dsRNA, or a combination thereof. In certain embodiments, the stability of the RNA in, for example, ADAS protoplasm is greater than approximately: 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, 10000000. In certain embodiments, the RNA cargo can be stabilized, for example, with an additional step-loop structure, such as a tRNA scaffold. For example, non-human tRNALys3 and Escherichia coli tRNAMet (Nat. Methods [Nature Methods], Ponchon 2007). Both have been well characterized and recombinantly expressed. However, various other types can also be used, such as aptamers, lncRNAs, ribozymes, etc. The RNA can also be stabilized when the ADAS is obtained from a parental strain that is ineffective (or hypomorphic) against one or more ribonucleases.

[0117] In some specific embodiments, the RNA is mRNA encoding a protein. In more specific embodiments, the mRNA encoding a protein encodes an enzyme (e.g., an enzyme conferring hepatic enzyme activity, such as human PBGD (hPBGD) mRNA), or an antigen (e.g., an antigen that elicits an immune response (such as eliciting an effective and persistent neutralizing antibody titer), such as mRNA encoding CMV glycoprotein gB and / or pentameric complex (PC)). In certain specific embodiments, the RNA is a small non-coding RNA, such as shRNA, ASO, tRNA, dsRNA, or a combination thereof.

[0118] In certain embodiments, the ADAS provided by the present invention includes a cargo comprising at least one component of a gene editing system. The components of a "gene editing system" include (or encode) a protein (or a nucleic acid encoding said protein) that can, together with a suitable associated nucleic acid and a nucleic acid related to the function of such protein (e.g., guide RNA), modify a target DNA sequence, such as a genomic DNA sequence, whether, for example, by inserting or deleting the target sequence, or by altering the methylation status of the target sequence. Exemplary gene editing systems include gene editing systems based on the Cas system such as Cas9, Cpf1, or other RNA-targeting systems and their associated RNAs (e.g., CRISPR guide RNAs that are sequence complementary), as well as zinc finger nucleases and TAL effectors conjugated to nucleases.

[0119] Other embodiments of the ADAS provided by the present invention include DNA as cargo, including plasmids, optionally, wherein the DNA contains a protein-coding sequence. In certain embodiments, exemplary DNA cargo includes plasmids encoding a target RNA sequence (see examples above), for example, the target RNA sequence can be flanked by tRNA insertion sequences on each side. The present invention encompasses various DNA cargo, including: ADAS production (e.g., driving FTZ overexpression, degrading exonucleases of the genome); long-lived plasmids (ATP synthase expression, rhodopsin expression); cargo expressing stable non-coding RNAs, tRNAs, lncRNAs; expressing secreted system tag proteins, NleE2 effector domains, and localization tags; secreted systems T3 / 4SS, T5SS, T6SS; logic circuits that conditionally express secreted systems; and combinations thereof. In some embodiments, the logic circuit includes inducible expression or repression cassettes, such as the IPTG-inducible Plac promoter and the hrpR portion of the AND gate, and for example, the heat-inducible promoter pL (from bacteriophage λ, which is normally repressed by a heat-labile protein) and the hrpS portion of the AND gate. To engineer an OR gate, the system described by Rosado et al., PLoS Genetics [Public Library of Science · Genetics, USA], 2018 can be used. Briefly, cis-repressive mRNAs encoding RFP can be used under a constitutive promoter. Then repression can be removed in the presence of the RAJ11 sRNA. Then plasmids containing the IPTG-inducible promoter PLac and the heat-inducible promoter pL can be used, both of which induce the expression of the RAJ11 sRNA. Then the output is RFP expression, which is observed in response to either input. These systems can be adjusted for various sensor-type functions.

[0120] In some embodiments, the ADAS provided by the present invention includes transporters in the membrane. In certain embodiments, the transporters are specific for glucose, sodium, potassium, metal ions, anionic solutes, cationic solutes, or water.

[0121] In some embodiments, the membrane of the ADAS provided by the present invention contains enzymes. In specific embodiments, the enzymes are proteases, oxidoreductases, or combinations thereof. In some embodiments, the enzymes are chemically conjugated to the ADAS membrane, optionally conjugated to the outer membrane via a linker.

[0122] F. ADAS Containing Secretion Systems

[0123] In certain embodiments, the ADAS provided by the present invention includes a bacterial secretion system (e.g., an endogenous bacterial secretion system or a heterologous secretion system). A "bacterial secretion system" is a protein or protein complex that can export cargo from the cytoplasm of a bacterial cell (or, e.g., an ADAS derived therefrom) to: the extracellular space, the periplasmic space of a Gram-negative bacterium, or the intracellular space of another cell. In some embodiments, the bacterial secretion system functions by an active (e.g., ATP-dependent or PMF-dependent) process, and in certain embodiments, the bacterial secretion system includes a tube or spike that spans the host cell (or ADAS) and the target cell. In other embodiments, the bacterial secretion system is a transmembrane channel. Exemplary bacterial secretion systems include T3SS and T4SS (and T3 / T4SS as defined below), which are tube-containing structures through which cargo passes through the interior of the protein tube, and T6SS, which delivers cargo at the tip of a spike. Other exemplary bacterial secretion systems include T1SS, T2SS, T5SS, T7SS, Sec, and Tat, which are transmembrane.

[0124] In some aspects, the present disclosure features a non-chromosomal dynamic active system (ADAS) derived from a parental bacterial cell, the ADAS including a bacterial type 3 secretion system (T3SS) heterologous to the parental bacterial cell.

[0125] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0126] In some embodiments, the parental bacterial cell does not include an endogenous T3SS.

[0127] In some embodiments, the parental bacterial cell is an Escherichia coli cell. In some embodiments, the Escherichia coli cell is Escherichia coli Nissle.

[0128] In some embodiments, the parental bacterial cell is a probiotic cell.

[0129] 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 the Salmonella enterica T3SS. In some embodiments, the Vibrio T3SS is the Vibrio parahaemolyticus T3SS. In some embodiments, the Escherichia T3SS is the enteropathogenic Escherichia coli (EPEC) T3SS. In some embodiments, the Yersinia T3SS is the Yersinia enterocolitica T3SS. In some embodiments, the Shigella T3SS is the Shigella flexneri T3SS.

[0130] In some embodiments, the parental bacterial cell comprises one or more heterologous nucleotide sequences encoding components of the T3SS. In some embodiments, one or more nucleotide sequences encoding components of the T3SS are carried on a vector. In some embodiments, the parental bacterial cell has been transiently transformed with the vector. In some embodiments, the parental bacterial cell has been stably transformed with the vector. In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS. In some embodiments, the moiety is a transcriptional activator of one or more heterologous nucleotide sequences encoding components of the T3SS.

[0131] In another aspect, the present disclosure features a non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the ADAS comprising a bacterial type III secretion system (T3SS) endogenous to the parental bacterial cell, wherein the parental bacterial cell has been modified to reduce the level of an endogenous protein or polypeptide that can be secreted by the T3SS.

[0132] In some embodiments, the parental bacterial cell has been modified by deletion of a transcriptional activator of an endogenous protein or polypeptide that can be secreted by the T3SS.

[0133] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0134] In some embodiments, the parental bacterial cell is a species of Salmonella, a species of Vibrio, a species of Escherichia, a species of Yersinia, or a species of Shigella. In some embodiments, the species of Salmonella is Salmonella enterica. In some embodiments, the species of Vibrio is Vibrio parahaemolyticus. In some embodiments, the species of Escherichia is enteropathogenic Escherichia coli (EPEC). In some embodiments, the species of Yersinia is Yersinia enterocolitica. In some embodiments, the species of Shigella is Shigella flexneri.

[0135] In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of T3SS in the ADAS. In some embodiments, the moiety is a transcriptional activator of a nucleotide sequence encoding a component of the T3SS.

[0136] In some embodiments, the parental bacterial cell has been modified to reduce the level of a negative regulator of a component of the T3SS. In some embodiments, the chromosomal locus encoding the negative regulator has been deleted from the parental bacterial cell.

[0137] In some embodiments, the parental bacterial cell has been modified to reduce the level of one or more of the following: LPS; metabolically non-essential proteins; toxins not associated with the T3SS; endotoxins; flagella; and pili.

[0138] In some embodiments, the ADAS further comprises at least one cargo, wherein the T3SS is capable of delivering the cargo to a target cell. In some embodiments, the delivery is to the cytoplasm of the target cell.

[0139] In some embodiments, the cargo is a protein or polypeptide.

[0140] In some embodiments, the cargo is endogenously secreted by the T3SS.

[0141] In some embodiments, the ADAS or the parental bacterial cell has been modified to increase the level of the cargo in the ADAS.

[0142] In some embodiments, the cargo is not endogenously secreted by the T3SS.

[0143] In some embodiments, the cargo is endogenously secreted by a T3SS from a species other than the ADAS T3SS species.

[0144] In some embodiments, the cargo is endogenously secreted by a type 4 secretion system (T4SS) or a type 6 secretion system (T6SS).

[0145] In some embodiments, the cargo has been modified for delivery by the T3SS.

[0146] In some embodiments, the cargo is an enzyme, a DNA modifier, a chromatin remodeler, a gene editor, a nuclear targeting agent, a binder, an immunogenic agent, or a toxin. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editor is a component of the CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binder is an antibody or an antibody fragment. In some embodiments, the binder is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulant. In some embodiments, the immunogenic agent is an immunosuppressant.

[0147] In some embodiments, the cargo has been modified by adding a secretion signal.

[0148] In another aspect, the present disclosure features a method for delivering a cargo to the cytoplasm of a target cell, the method comprising contacting the target cell with an ADAS of any of the above aspects.

[0149] In some embodiments, the ADAS comprises a cargo, wherein the cargo comprises a portion that directs the output of the bacterial secretion system, e.g., in some embodiments, the portion is a Pho / D, Tat, or synthetic peptide signal.

[0150] In certain embodiments, the ADAS provided by the present invention is a double-membrane ADAS. In more specific embodiments, the double-membrane ADAS further comprises a bacterial secretion system. In still more specific embodiments, the bacterial secretion system is selected from T3SS, T4SS, T3 / 4SS, or T6SS, optionally, wherein T3SS, T4SS, T3 / 4SS, or T6SS has a weakened or non-functional effector that does not affect the fitness of the target cell.

[0151] In some embodiments, the ADAS provided by the present invention includes a bacterial secretion system.

[0152] In some embodiments, the bacterial secretion system (such as T3SS, T4SS, T3 / T4SS, or T6SS) is capable of exporting the cargo through the outer membrane of the ADAS into the target cell (such as an animal, fungal, bacterial, or plant cell).

[0153] In more specific embodiments, the bacterial secretion system is a T3 / 4SS. "T3 / 4SS" is a secretion system based on T3SS or T4SS, including hybrid systems and unmodified forms, which form a protein tube between the bacterium (or ADAS) and the target cell, thereby connecting the two and delivering one or more effectors. The target cell can be an animal, a plant, a fungus, or a bacterium. In some embodiments, the T3 / 4SS includes effectors, which can be modified effectors. Examples of the T3SS system include the Salmonella SPI-1 system, the Enterohemorrhagic Escherichia coli (EHEC coli) ETT1 system, the Xanthomonas Citri / Campestri T3SS system, and the Pseudomonas syringae T3SS system. Examples of the T4SS system include the Agrobacterium Ti plasmid system, the Helicobacter pylori T4SS. In certain embodiments, the T3 / 4SS has altered effector function, for example, an effector selected from SopD2, SopE, Bop, Map, Tir, EspB, EspF, NleC, NleH2, or NleE2. In more specific embodiments, the altered effector function is for intracellular targeting, such as translocation to the nucleus, Golgi apparatus, mitochondria, actin, microvilli, ZO-1, microtubules, or cytoplasm. In still more specific embodiments, the altered effector function is nuclear targeting based on NleE2 derived from Escherichia coli. In other specific embodiments, the altered effector function is for filopodia formation, tight junction disruption, microvilli elimination, or SGLT-1 deactivation.

[0154] In other embodiments, the ADAS provided by the present invention comprising a bacterial secretion system comprises a T6SS. In some embodiments, the T6SS targets bacteria in its natural host and contains effectors that kill bacteria. In certain specific embodiments, the T6SS is derived from Pseudomonas putida K1-T6SS, and optionally, wherein the effector comprises the amino acid sequence of Tke2 (accession number AUZ59427.1), or a functional fragment thereof. In other embodiments, the T6SS targets fungi in its natural host and contains effectors that kill fungi, for example, the T6SS is derived from Serratia Marcescens, and the effector comprises the amino acid sequence of: Tfe1 (Genbank: SMDB11_RS05530) or Tfe2 (Genbank: SMDB11_RS05390).

[0155] In other embodiments of the ADAS provided by the present invention that contain a bacterial secretion system, the bacterial secretion system is capable of exporting cargo extracellularly. In certain more specific embodiments, the bacterial secretion system is T1SS, T2SS, T5SS, T7SS, Sec, or Tat.

[0156] G. ADAS lacking protease, ribonuclease, and / or LPS

[0157] In another aspect, the present invention provides a composition further comprising a plurality of ADAS (e.g., highly active ADAS), wherein the protease level or activity of the ADAS is reduced relative to the ADAS produced by wild-type parental bacteria. In some aspects, the ADAS is produced by parental bacteria that have been modified to reduce or eliminate the expression of at least one protease.

[0158] In another aspect, the present invention provides a composition comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ribonuclease level or activity of the ADAS is reduced relative to the ADAS produced by wild-type parental bacteria. In some aspects, the ADAS is produced by parental bacteria that have been modified to reduce or eliminate the expression of at least one ribonuclease. In some embodiments, the ribonuclease is an endoribonuclease or an exoribonuclease.

[0159] In another aspect, the present invention provides a composition comprising a plurality of ADAS, wherein the ADAS is modified to have reduced lipopolysaccharide (LPS). In some embodiments, the modification is a lipid A biosynthetic myristoyltransferase (msbB) mutation.

[0160] In certain embodiments, the ADAS provided by the present invention lacks one or more metabolically non-essential proteins. "Metabolically non-essential proteins" non-exhaustively include: pili, flagella, undesired secretion systems, transposases, effectors, phage elements, or regulatory elements thereof, such as flhC or OmpA. In some embodiments, the ADAS provided by the present invention lacks one or more of ribonuclease, protease, or a combination thereof, and in certain embodiments, lacks one or more endoribonucleases (such as ribonuclease A, ribonuclease h, ribonuclease III, ribonuclease L, ribonuclease PhyM) or exoribonucleases (such as ribonuclease R, ribonuclease PH, ribonuclease D); or serine, cysteine, threonine, aspartic acid, glutamic acid, and metalloproteases; or any combination of the foregoing.

[0161] H. ADAS comprising a targeting moiety

[0162] In another embodiment, the present invention provides a composition comprising a plurality of ADASs, wherein the ADASs comprise targeting moieties. In some embodiments, the targeting moiety is a nanobody, a carbohydrate-binding protein, or a tumor-targeting peptide. In some embodiments, the targeting moiety is an endogenous surface ligand of the parental cell (e.g., a surface ligand genetically acquired by the ADAS). In other embodiments, the targeting moiety is an exogenous ligand (e.g., an exogenous tissue-targeting ligand) added to the ADAS using any of the methods described herein for modifying ADASs. In some embodiments, the targeting moiety promotes tissue-related targeting of the ADAS to a tissue type or cell type.

[0163] In certain embodiments, the nanobody is a nanobody against a tumor antigen (such as HER2, PSMA, or VEGF-R). In other embodiments, the carbohydrate-binding protein is a lectin, such as mannose-binding lectin (MBL). In still other embodiments, the tumor-targeting peptide is an RGD motif or a CendR peptide.

[0164] I. ADASs Derived from Symbiotic or Pathogenic Parental Strains

[0165] In another embodiment, the present invention provides a composition comprising a plurality of ADASs (e.g., highly active ADASs), wherein the ADASs are derived from parental bacteria that are mammalian pathogens or mammalian symbiotic bacteria. In some cases, the mammalian symbiotic bacteria are species of Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus, or Actinomyces, or the mammalian pathogenic bacteria are Escherichia coli (EHEC), Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori.

[0166] In another embodiment, the present invention provides a composition comprising a plurality of ADASs (e.g., highly active ADASs), wherein the ADASs are derived from parental bacteria that are plant pathogens or plant symbiotic bacteria. In some cases, the plant symbiotic bacteria are Bacillus subtilis or Pseudomonas putida, or the plant pathogenic bacteria are species of Xanthomonas or Pseudomonas syringae.

[0167] J. ADAS Derived from Auxotrophic Parent Strains

[0168] In another embodiment, the present invention provides a composition comprising multiple ADASs (e.g., highly active ADAS), wherein the ADAS is derived from an auxotrophic parent bacterium, i.e., a parent bacterium that cannot synthesize organic compounds required for growth. Such bacteria can grow only when organic compounds are provided.

[0169] K. ADAS Comprising Additional Moieties

[0170] In certain embodiments, the ADAS includes a functional ATP synthase, and in some embodiments, includes a membrane-embedded proton pump. The ADAS can be derived from different sources, including: parent bacterial strains (the "parent strain") engineered or induced to produce a genome-free closed membrane system, genomically excised bacteria, bacterial cell preparation extracts (e.g., by mechanical or other means), or total synthesis processes, optionally including fractions of bacterial cell preparations. In some embodiments, the ATP synthase concentration of the highly active ADAS is at least: 1 / 10000 nm 2 、1 / 5000 nm 2 、1 / 3500 nm 2 、1 / 1000 nm 2 。

[0171] The ADAS provided by the present invention can include various additional components, including, for example, photovoltaic pumps, one or more retinol-producing cassettes, metabolic enzymes, targeting agents, cargoes, bacterial secretion systems, and transporters, including combinations of the foregoing, including certain specific embodiments described below. In certain embodiments, the ADAS lacks other elements, such as metabolically non-essential genes and / or certain enzymes, nucleases, or proteases.

[0172] In certain embodiments, the ADAS provided by the present invention comprises an ATP synthase, optionally lacking a regulatory domain, such as lacking the ε domain. The deletion can be achieved in various ways. In certain embodiments, the deletion is effected by inducing a natural ε domain deletion. In certain embodiments, the deletion can be achieved by flanking LoxP sites and induced Cre expression or CRISPR knockout, or can be induced (on a plasmid in the presence of the tTa tet transactivator in an ATP synthase knockout strain)

[0173] In some embodiments, the ADAS can include a photovoltaic proton pump. In certain embodiments, the photovoltaic proton pump is proteorhodopsin. In more specific embodiments, the proteorhodopsin contains the amino acid sequence of proteorhodopsin from the uncultured marine bacterial lineage SAR86 (GenBank accession number: AAS73014.1). In other embodiments, the photovoltaic proton pump is bacteriorhodopsin. In certain embodiments, the photovoltaic proton pump is bacteriorhodopsin, δ-rhodopsin, or halorhodopsin from Halobium salinarum, Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica, or Haloarcula marismortui.

[0174] In some embodiments, the ADAS provided by the present invention further contains retinal. In certain embodiments, the ADAS provided by the present invention further contains a retinal synthesis protein (or protein system), or a nucleic acid encoding the same.

[0175] In certain embodiments, the ADAS provided by the present invention further contains one or more glycolytic pathway proteins. In some embodiments, the glycolytic pathway protein is phosphofructokinase (PfK-A), for example, phosphofructokinase containing the amino acid sequence of UniProt accession number P0A796 or a functional fragment thereof. In other embodiments, the glycolytic pathway protein is triosephosphate isomerase (tpi), for example, triosephosphate isomerase containing the amino acid sequence of UniProt accession number P0A858 or a functional fragment thereof.

[0176] L. ADAS Compositions and Formulations

[0177] The present invention provides a composition or formulation containing the ADAS provided by the present invention, which particularly includes the highly active ADAS formulation provided by the present invention, or an ADAS formulation as follows, in which various individual ADAS lack a cell division topological specificity factor (for example, lack the minE gene product), and optionally wherein the ADAS formulation is substantially free of live cells. These are collectively referred to as "various compositions provided by the present invention" or "a composition provided by the present invention", etc., and can contain any ADAS provided by the present invention and any combination of the ADAS provided by the present invention.

[0178] For example, in some embodiments, the compositions provided by the present invention 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 ADAS containing a bacterial secretion system. In certain embodiments, the bacterial secretion system is one of T3SS, T4SS, T3 / 4SS or T6SS.

[0179] In some embodiments, the compositions provided by the present invention contain ADAS containing T3SS, wherein the ADAS has an average T3SS membrane density greater than 1 at about 40000, 35000, 30000, 25000, 19600, 15000, 10000 or 5000 nm 2 In certain specific embodiments, the ADAS is derived from a Salmonella typhimurium or Escherichia coli parental strain.

[0180] Certain embodiments of the compositions provided by the present invention contain ADAS containing T3SS, wherein the ADAS has an average T3SS membrane density greater than 1 at about 300000, 250000, 200000, 150000, 100000, 50000, 20000, 10000, 5000 nm 2 In certain specific embodiments, the ADAS is derived from an Agrobacterium tumefacien parental strain.

[0181] In another aspect, the present invention provides a composition of ADAS, wherein at least about: 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the ADAS contain a bacterial secretion system, the bacterial secretion system includes T3, T4, T3 / 4SS, T6SS, and optionally includes one or more of the following: exogenous carbohydrates, phosphate-producing synthase, photoreactive proteins, importins, enzymes, functional cargo, organism-specific effectors, fusion proteins.

[0182] It will be apparent that the compositions and formulations provided by the present invention may contain any ADAS provided by the present invention, such as highly active ADAS or ADAS lacking the minE gene product.

[0183] The compositions provided by the present invention can be prepared in any suitable formulation. For example, the formulation can be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosolized or nebulized administration. In some embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.

[0184] In some embodiments, the ADAS compositions described herein contain less than 100 colony forming units (CFU / mL) of viable bacterial cells, e.g., less than 50 CFU / mL, less than 20 CFL / mL, less than 10 CFU / mL, less than 1 CFU / mL or less than 0.1 CFU / mL of viable bacterial cells.

[0185] In some embodiments, the present invention provides an ADAS composition, wherein the ADAS is lyophilized and reconstituted, and wherein the ATP concentration of the reconstituted ADAS is at least 90% of the ATP concentration of the non-lyophilized ADAS, e.g., at least 95%, 98% or at least equal to the ATP concentration of the non-lyophilized ADAS.

[0186] In some embodiments, the present invention provides an ADAS composition, wherein the ADAS is stored, e.g., stored at 4°C, and wherein the ATP concentration of the ADAS after storage is at least 90% of the ATP concentration of the non-stored ADAS, e.g., at least 95%, 98% or at least equal to the ATP concentration of the non-stored ADAS. In some embodiments, the storage lasts for at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least six months or at least one year.

[0187] In some embodiments, the ADAS is stored in a "quiescent" state or otherwise treated and then rapidly activated.

[0188] In some embodiments, the ADAS composition is formulated for delivery to an animal, e.g., formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosolized or nebulized administration.

[0189] In some embodiments, the ADAS composition is formulated for delivery to a plant. In some aspects, the composition includes adjuvants such as surfactants (e.g., nonionic surfactants, surfactant plus nitrogen source, silicone surfactants, or high surfactant oil concentrates), crop oil concentrates, vegetable oil concentrates, modified vegetable oils, nitrogen sources, deposition (drift control) and / or retention aids (with or without ammonium sulfate and / or defoamers), compatibilizers, buffers and / or acidifying agents, water conditioners, alkaline mixtures, sticker-spreaders and / or extenders, adjuvant plus foliar fertilizer, defoamers, foam markers, odorants, or tank cleaners and / or neutralizers. In some embodiments, the adjuvant is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (13th Edition), Purdue University).

[0190] In some embodiments, the ADAS composition is formulated for delivery to an invertebrate (e.g., an arthropod (e.g., an insect or arachnid), nematode, protozoan, or annelid). In some embodiments, the ADAS composition is formulated for delivery to an insect.

[0191] In some embodiments, the composition is formulated as a liquid, solid, aerosol, paste, gel, or gas composition.

[0192] M. ADAS Comprising Enzymes

[0193] In one aspect, the invention features a composition comprising a plurality of ADAS, wherein the ADAS comprises an enzyme, and wherein the enzyme modifies a substrate to produce a target product. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or the environment to which the ADAS is delivered. In some embodiments, the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic di-AMP.

[0194] III. Methods of Making ADAS

[0195] A. Preparation of ADAS and Highly Active ADAS

[0196] In some aspects, the production of ADAS is characterized by a method for manufacturing a composition comprising multiple ADAS, the composition being substantially free of live bacterial cells, the method comprising (a) preparing, providing, or obtaining a plurality of parental bacteria with a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of minicells, thereby producing the highly active ADAS; and (c) separating the highly active ADAS from the parental bacteria, thereby producing a composition substantially free of live bacterial cells.

[0197] The parental bacteria include any suitable bacterial species that can generate ADAS (e.g., species that can be modified using the methods described herein to produce ADAS). The following provides a non-limiting list of suitable genera from which ADAS can be derived: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacter, Desulfitobacterium, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wegenerella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacteria.

[0198] In some aspects, use the method for manufacturing any of the ADAS compositions described in Part I herein (e.g., the highly active ADAS composition). For example, provided herein are methods for preparing highly active ADAS; methods for preparing ADAS lacking a cell division topology-specific factor and optionally lacking a Z-ring inhibitory protein (e.g., a method for preparing ADAS from ΔminCDE parental bacteria), and methods for preparing any of the ADAS mentioned herein, wherein the ADAS comprises cargo.

[0199] In some embodiments, the ADAS (high-activity ADAS) is prepared from a parental strain that is a plant bacterium, such as a plant symbiotic bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogenic bacterium (e.g., Xanthomonas species or Pseudomonas syringae), or a bacterium capable of colonizing the plant rhizosphere and / or forming root nodules, such as Rhizobium.

[0200] In some embodiments, the ADAS (high-activity ADAS) is prepared from a parental strain that is an invertebrate symbiont, such as a symbiont of an arthropod (e.g., an insect or an arachnid), a nematode, a protozoan, or an annelid. In an embodiment, the invertebrate is a pest or pathogen of a plant or an animal.

[0201] In some embodiments, the ADAS (e.g., high-activity ADAS) is prepared from a parental strain capable of genetic transformation (e.g., Agrobacterium).

[0202] In some embodiments, the ADAS (e.g., high-activity ADAS) is prepared from a parental strain that is a human bacterium, such as a symbiotic human bacterium (e.g., Escherichia coli, Staphylococcus species, Bifidobacterium species, Micrococcus species, Lactobacillus species, or Actinomyces species) or a human pathogenic bacterium (e.g., Escherichia coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica, or Helicobacter pylori) or an extremophile.

[0203] In some embodiments, the ADAS and / or the parental strain is a functionalized derivative of any of the foregoing, e.g., including a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial agent, digest plastic, secrete an insecticide, survive in an extreme environment, manufacture nanoparticles, integrate in other organisms, respond to the environment, and produce a reporter signal.

[0204] In some embodiments, the parental bacterium includes a functionalized derivative of any of the foregoing, e.g., including a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial agent, digest plastic, secrete an insecticide, survive in an extreme environment, manufacture nanoparticles, integrate in other organisms, respond to the environment, and produce a reporter signal.

[0205] In some embodiments, the ADAS is derived from a parental strain engineered or induced to overexpress ATP synthase. In some more specific embodiments, the ATP synthase is heterologous to the parental strain. In certain specific embodiments, the parental strain is modified to express functional F o F1 ATP synthase.

[0206] In certain embodiments, the ADAS provided by the present invention is obtained from a parental strain cultured under conditions selected from the following: applied voltage (e.g., 37 mV), non-atmospheric oxygen concentration (e.g., 1%-5% O2, 5%-10% O2, 10%-15% O2, 25%-30% O2), low pH (about: 4.5, 5.0, 5.5, 6.0, 6.5), or a combination thereof.

[0207] The highly active ADAS according to any one of the preceding claims, which is prepared from an extremophilic microorganism, includes a functionalized derivative of any one of the foregoing, for example, includes a functional cassette, such as a functional cassette that induces the bacterium to perform one or more of the following: secrete an antimicrobial agent, digest plastics, secrete an insecticide, survive in an extreme environment, manufacture nanoparticles, integrate into other organisms, respond to the environment, and generate a reporting signal.

[0208] Due to the diversity of bacteria, ADAS with a modified membrane can be prepared, for example, to improve the biodistribution of ADAS after administration to target cells. In certain embodiments, the membrane is modified to have less immunogenicity or immunostimulatory properties in plants or animals. For example, in certain embodiments, the ADAS is obtained from a parental strain, wherein the immunostimulatory ability of the parental strain is reduced or eliminated by post-production treatment with a detergent, an enzyme, or PEG functionalization. In certain embodiments, the ADAS is prepared from a parental strain, and the membrane is modified by knocking out the LPS synthesis pathway in the parental strain, for example, by knocking out msbB. In other specific embodiments, the ADAS is prepared from a parental strain that produces cell wall-deficient particles by exposure to hypertonic conditions.

[0209] In some embodiments, the method includes transforming the parental strain with an inducible DNA enzyme system, such as the exoI (NCBI GeneID: 946529) & sbcD (NCBI GeneID: 945049) nucleases, or the I-CeuI (e.g., Swissprot: P32761.1) nuclease. In more specific embodiments, the method includes using one, two, three, or four auxotrophic strains and having complementary genes on a plasmid encoding the inducible nuclease.

[0210] In some embodiments, the parental strain is cultured under conditions selected from the following: applied voltage (e.g., 37 mV), non-atmospheric oxygen concentration (e.g., 1%-5% O2, 5%-10% O2, 10%-15% O2, 25%-30% O2), low pH (4.5 - 6.5), or a combination thereof.

[0211] In certain embodiments, the parental strain lacks flagella and undesired secretion systems, optionally wherein the flagella and undesired secretion systems are removed using λred recombination engineering.

[0212] In some embodiments, the flagellar control components are excised from the parental strain genome by, for example, inserting a plasmid containing a CRISPR domain targeting flagellar control genes such as flhD and flhC.

[0213] In certain embodiments, the methods provided are for preparing highly active ADASs, where the ADASs containing a plasmid encoding a rhodopsin gene are cultured in the presence of light. In more specific embodiments, the rhodopsin is proteorhodopsin or a functional fragment thereof having the amino acid sequence with GenBank accession number: AAS73014.1 from uncultured bacterium SAR86. In still more specific embodiments, the culture is supplemented with retinal. In other more specific embodiments, the rhodopsin is proteorhodopsin and the plasmid additionally contains a gene for synthesizing retinal (such a plasmid is the pACYC-RDS plasmid from Kim et al., Microb Cell Fact [Microbial Cell Factories], 2012).

[0214] In certain specific embodiments, the parental strain contains a nucleic acid sequence encoding a nanobody, and then the nanobody is expressed on the membrane of the ADAS.

[0215] In some embodiments of the methods provided by the present invention, the parental strain contains a nucleic acid sequence encoding one or more bacterial secretion system operons. Exemplary plasmids include Salmonella SPI-1 T3SS, Shigella flexneri T3SS, Agrobacterium Ti plasmid, and Pseudomonas putida K1-T6SS system.

[0216] In certain embodiments, the parental strain contains cargo. In some embodiments, the parental strain contains a nucleic acid sequence encoding a set of synthetic small molecule cargo.

[0217] IV. Purification of ADAS and ADAS Compositions

[0218] In some embodiments of the methods and compositions provided herein, ADAS is purified from a composition (e.g., a culture) containing live bacteria (e.g., parental bacteria). For example, the present invention features a method for making a composition containing multiple ADASs that is substantially free of live bacterial cells, the method comprising (a) preparing, providing, or obtaining a plurality of parental bacteria with a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of microcells, thereby producing the ADAS; and (c) separating the ADAS from the parental bacteria, thereby producing a composition that is substantially free of live bacterial cells.

[0219] Purification separates the ADAS from the live parental bacterial cells, which are larger and contain a genome. Separation of highly active ADAS from the parental bacteria can be carried out using a variety of methods as described herein. Exemplary methods for the purification described herein include centrifugation, selective growth, and buffer exchange / concentration methods.

[0220] In some aspects, the present disclosure provides ADAS compositions and methods for comparing such compositions, wherein the compositions are substantially free of parental bacterial cells and / or live bacterial cells, e.g., having no more than 500, e.g., 400, 300, 200, 150, or 100 or fewer than 50, fewer than 25, fewer than 10, fewer than 5, fewer than 1, fewer than 0.1 colony forming units (CFU) / mL. In some embodiments, an ADAS composition that is substantially free of parental bacterial cells does not include bacterial cells.

[0221] Auxotrophic parental strains can be used to prepare the ADAS provided by the present invention. As described in more detail below, such manufacturing methods can be used to purify ADAS. For example, in some embodiments, after ADAS generation, the parental bacterial cells are removed by growing in a medium lacking nutrients (e.g., amino acids) necessary for the survival of the parental bacteria. In some embodiments, the ADAS provided by the present invention is derived from at least 1, 2, 3, 4 or more auxotrophic parental strains of the following: arginine (e.g., argA knockout, such as strains JW2786-1 and NK5992), cysteine (cysE knockout, such as strains JW3582-2 and JM15), glutamine (e.g., glnA knockout, such as strains JW3841-1 and M5004), glycine (e.g., glyA knockout, such as strains JW2535-1 and AT2457), histidine (e.g., hisB knockout, such as strains JW2004-1 and SB3930), isoleucine (e.g., ilvA knockout, such as strains JW3745-2 and AB1255), leucine (e.g., leuB knockout, such as strains JW5807-2 and CV514), lysine (e.g., lysA knockout, such as strains JW2806-1 and KL334), methionine (e.g., metA knockout, such as strains JW3973-1 and DL41), phenylalanine (e.g., pheA knockout, such as strains JW2580-1 and KA197), proline (e.g., proA knockout, such as strains JW0233-2 and NK5525), serine (e.g., serA knockout, such as strains JW2880-1 and JC158), threonine (e.g., thrC knockout, such as strains JW0003-2 and Gif 41), tryptophan (e.g., trpC knockout, such as strains JW1254-2 and CAG18455), tyrosine (e.g., tyrA knockout, such as strains JW2581-1 and N3087), valine / isoleucine / leucine (e.g., ilvd knockout, such as strains JW5605-1 and CAG18431).

[0222] In certain embodiments, the method comprises using one, two, three or four auxotrophic parental strains, optionally wherein the parental strains further comprise a plasmid expressing ftsZ.

[0223] V. Methods of Using ADAS

[0224] A. Methods of Delivering ADAS

[0225] In one aspect, the present invention features a method for delivering an ADAS (e.g., a highly active ADAS) to a target cell, the method comprising (a) providing a composition comprising a plurality of ADASs, wherein the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a), wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0226] In another aspect, the present invention features a method for delivering an ADAS to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADASs; and (b) contacting the target cell with the composition of step (a), wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS.

[0227] In some embodiments, the target cell is, for example, an animal cell, a plant cell, or a fungal cell.

[0228] B. Methods of Delivering Cargo

[0229] In another aspect, the present invention features a method for delivering cargo (e.g., nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADASs, wherein the ADAS comprises the cargo and the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a), wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS. In some embodiments, the plurality of ADASs are a plurality of highly active ADASs, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0230] In another aspect, the present invention features a method for delivering a cargo (e.g., nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADAS; and (b) contacting the target cell with the composition of step (a), wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS.

[0231] In another aspect, the present invention features a method for delivering a cargo (e.g., nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS, the ADAS comprises the cargo, and the composition is substantially free of live bacterial cells; and (b) contacting the target cell with the composition of step (a). In some aspects, the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topological specificity factor.

[0232] In some embodiments, the target cell to which the cargo is to be delivered is, for example, an animal cell, a plant cell, or a fungal cell.

[0233] C. Method for modulating cell state

[0234] In one aspect, the present invention features a method for modulating the state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal cell with the composition of step (a) to modulate the state of the animal cell, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS. In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0235] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant cell with the composition of step (a) to thereby modulate the state of the plant cell, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0236] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the insect cell with the composition of step (a) to thereby modulate the state of the insect cell, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0237] In another aspect, the present invention features a method of modulating the state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal cell with the composition of step (a) to thereby modulate the state of the animal cell, wherein the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topoispecificity factor.

[0238] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant cell with the composition of step (a) to thereby modulate the state of the plant cell, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topoispecificity factor.

[0239] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the insect cell with the composition of step (a) to thereby modulate the state of the insect cell, wherein the ADAS is derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topoispecificity factor.

[0240] In one aspect, the present invention features a method of modulating the state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of acentric dynamic activity systems (ADAS); and (b) contacting the animal cell with the composition of step (a) to thereby modulate the state of the animal cell. In some embodiments, the ADAS is derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase).

[0241] In another aspect, the present invention features a method of modulating the state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of acentric dynamic activity systems (ADAS); and (b) contacting the plant cell with the composition of step (a) to thereby modulate the state of the plant cell. In some embodiments, the ADAS is derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase).

[0242] In another aspect, the present invention features a method of modulating the state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of acentric dynamic activity systems (ADAS); and (b) contacting the insect cell with the composition of step (a) to thereby modulate the state of the insect cell. In some embodiments, the ADAS is derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase).

[0243] In some embodiments, modulation is any observable change in the state of a cell (e.g., an animal, plant, or insect cell) (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state) as measured using techniques and methods known in the art for such measurements (e.g., methods for measuring the level or expression of a protein, transcript, epigenetic marker, or for measuring an increase or decrease in biological pathway activity). In some embodiments, modulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway).

[0244] D. Method for treating an animal, plant, insect, or fungus

[0245] In some aspects, the invention features a method for treating an animal in need thereof, the method comprising (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a) to thereby treat the animal, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the plurality of ADAS are a plurality of highly active ADAS, wherein the initial ATP concentration of the ADAS is at least 1.25 mM.

[0246] In some embodiments, the invention features the use of ADAS in the manufacture of a medicament for treating an animal, plant, insect, or fungus.

[0247] In some embodiments, the invention features the use of ADAS in the manufacture of a medicament for treating an animal, wherein the initial ATP concentration of the ADAS is at least 1.25 mM and wherein the composition is substantially free of live bacterial cells.

[0248] In some embodiments, the invention features the use of ADAS in the manufacture of a medicament for treating an animal, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase) and wherein the composition is substantially free of live bacterial cells.

[0249] In other aspects, the invention features a method of treating an animal in need thereof, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a) to treat the animal, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topoispecificity factor.

[0250] In some aspects, the invention features a method of treating an animal in need thereof, the method comprising (a) providing a composition comprising a plurality of acentric dynamic activity systems (ADAS); and (b) contacting the animal with an effective amount of the composition of step (a) to treat the animal. In some embodiments, the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase).

[0251] In some embodiments, the animal in need of treatment has a disease, such as cancer. In some embodiments, the ADAS carries a chemotherapeutic cargo or an immunotherapeutic cargo.

[0252] In some aspects, the invention features a method of treating a plant in need thereof, the method comprising (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) to treat the plant, wherein the ADAS is derived from a parental cell comprising one or more genetic loss-of-function alterations that stabilize the ADAS (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the ADAS is derived from a parental bacterium with a reduced level or activity of a cell division topoispecificity factor.

[0253] In other aspects, the present invention features a method of treating a plant in need thereof, the method comprising: (a) providing a composition comprising a plurality of ADASs, wherein the ADASs are derived from a parental bacterium having a reduced level or activity of a cell division topoispecificity factor, and wherein the composition is substantially free of live bacterial cells; and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) so as to treat the plant, wherein the ADASs are derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADASs (e.g., one or more loss-of-function alterations in a lyase). In some embodiments, the ADASs are derived from a parental bacterium having a reduced level or activity of a cell division topoispecificity factor.

[0254] In some aspects, the present invention features a method of treating a plant in need thereof, the method comprising (a) providing a composition comprising a plurality of acentric dynamic activity systems (ADASs); and (b) contacting the plant or its pest (e.g., a pest) with an effective amount of the composition of step (a) so as to treat the plant. In some embodiments, the ADASs are derived from a parental cell comprising one or more loss-of-function genetic alterations that stabilize the ADASs (e.g., one or more loss-of-function alterations in a lyase).

[0255] In another aspect, the present invention provides methods of modulating a target cell. The target cell can be any cell, including animal cells (e.g., including human and non-human animals, including farm animals or livestock, pests), plant cells (including plant cells from crops or pests), fungal cells or bacterial cells. In some embodiments, the cells are isolated, e.g., in vitro, or in other embodiments, in vivo, within an organism. These methods require providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention to approach the target cell. In some embodiments, approaching the target cell is direct, e.g., wherein the target cell is directly modulated by the ADAS, such as by secreting a certain agent adjacent to the target cell or injecting the agent into the target cell, or is indirect. Indirect modulation of the target cell can be effected by targeting different cells, e.g., by modulating a cell adjacent to the target cell, which adjacent cell can be symbiotic with or pathogenic to the target cell. The adjacent cell can be in vitro or in vivo, just like the target cell - i.e., within an organism, it can be symbiotic or pathogenic. These methods are collectively referred to as "usage methods provided by the present invention" and the like. In related aspects, the present invention provides target uses of the ADAS and composition provided by the present invention, which target uses are consistent with the usage methods provided by the present invention.

[0256] For example, in some embodiments, the present invention provides a method of modulating the state 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 in proximity to the animal cell. In certain embodiments, the ADAS or composition is provided to be in proximity to the animal cell in an animal (such as a mammal, such as a human). In some embodiments, animal cells in a healthy animal are exposed to bacteria. In more specific embodiments, the animal cell is a lung epithelial cell, an immune cell, a skin cell, an oral epithelial cell, an intestinal epithelial cell, a reproductive tract epithelial cell, or a urinary tract cell. In still more specific embodiments, the animal cell is an intestinal epithelial cell, such as an intestinal epithelial cell from a human subject suffering from an inflammatory bowel disease (such as Crohn's disease or colitis). In yet more specific embodiments, the animal cell is an intestinal epithelial cell from a subject suffering from an inflammatory bowel disease, and the ADAS comprises a bacterial secretion system and a cargo comprising an anti-inflammatory agent, such as an antibody or antibody fragment that targets tumor necrosis factor (TNF) (e.g., an anti-TNF antibody); an antibody or antibody fragment that targets IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment that targets IL-23 (e.g., an anti-IL-23 antibody)).

[0257] In other embodiments, animal cells in a diseased state are exposed to bacteria. In certain embodiments, the animal cell is pathogenic, such as a tumor. In other embodiments, animal cells in a diseased state, such as a wound, ulcer, tumor, or inflammatory disease, are exposed to bacteria.

[0258] In certain embodiments, the ADAS is derived from an animal symbiotic parental strain. In other embodiments, the ADAS is derived from an animal pathogenic parental strain.

[0259] In certain specific 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 into the animal cell. In some specific embodiments, the animal cell is provided to be in proximity to an effective amount of an ADAS comprising a cargo and a secretion system, wherein the cargo is secreted extracellularly and contacts the animal cell.

[0260] In some embodiments, the state of the animal cell is regulated by: providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention to approach a bacterial or fungal cell near the animal cell. That is, these methods require indirectly regulating the state of the animal cell. In certain embodiments, the bacterial or fungal cell is pathogenic. In more specific embodiments, the adaptability of the pathogenic bacterial or fungal cell is reduced. In certain other embodiments, the bacterial or fungal cell is symbiotic. In more specific embodiments, the adaptability of the symbiotic bacterial or fungal cell is increased. In still more specific embodiments, the adaptability of the symbiotic bacterial or fungal strain is increased by reducing the adaptability of a large number of competing bacteria or fungi that may be neutral, symbiotic, or pathogenic.

[0261] In certain specific embodiments, the bacterial or fungal cell near 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 into the bacterial or fungal cell. In other specific embodiments, the bacterial or fungal cell near the animal cell is provided to approach an effective amount of an ADAS that extracellularly secretes a cargo, and the cargo contacts the bacterial or fungal cell.

[0262] In some embodiments, the ADAS is derived from a parental strain that is a competitor of the bacterial or fungal cell. In other embodiments, the ADAS is derived from a parental strain that is a mutualistic bacterium of the bacterial or fungal cell.

[0263] As will be appreciated, the various methods of use provided by the present invention for regulating the state of an animal cell can be readily adapted to corresponding methods for regulating the state of a plant, fungal, or bacterial cell. For illustrative purposes, methods for regulating a plant cell or a fungal cell will be described more particularly.

[0264] Accordingly, in a related aspect, the present invention provides a method for regulating the state of a plant or fungal cell by providing an effective amount of the ADAS provided by the present invention or the composition provided by the present invention to approach: a) a plant or fungal cell, b) an adjacent bacterial or adjacent fungal cell near the plant or fungal cell, or c) an invertebrate (e.g., an arthropod (e.g., an insect or an arachnid), a nematode, a protozoan, or an annelid) cell near the plant or fungal cell.

[0265] In certain embodiments, the ADAS is provided in situ, such as within a plant proximate to plant cells, e.g., crop plants such as row crops including corn, wheat, soybeans, and rice, and vegetable crops including Solanaceae such as tomatoes and peppers; Cucurbitaceae such as melons and cucumbers; Brassicaceae such as cabbage and broccoli; leafy greens such as kale and lettuce; roots and tubers such as potatoes and carrots; large-seeded vegetables such as beans and corn; and mushrooms. In some embodiments, plant or fungal cells in healthy plants or fungi are exposed to bacteria. In other embodiments, plant or fungal cells in a diseased state are exposed to bacteria.

[0266] In certain embodiments, the plant or fungal cells divide, such as meristematic cells, or are pathogenic, such as tumors. In some embodiments, plant or fungal cells in a diseased state such as a wound are exposed to bacteria, or where the plant or fungal cells are not part of human food.

[0267] In certain embodiments, the ADAS is derived from a symbiotic parental strain. In other embodiments, the ADAS is derived from a plant- or fungal-pathogenic parental strain.

[0268] In some embodiments, the ADAS comprises a T3 / 4SS or T6SS and cargo, and the cargo is delivered into the plant or fungal cell. In other embodiments, the plant or fungal cell is provided in proximity to an effective amount of an ADAS comprising a bacterial secretion system and cargo, wherein the bacterial secretion system extracellularly secretes the cargo such that the plant or fungal cell is contacted with the cargo.

[0269] In some embodiments, the methods require providing an effective amount of an ADAS or composition in proximity to adjacent bacterial or adjacent fungal cells near the plant or fungal cell. In more specific embodiments, the adjacent bacterial or adjacent fungal cells are pathogenic, optionally, wherein the fitness of the pathogenic adjacent bacterial or adjacent fungal cell is reduced. In other more specific embodiments, the adjacent bacterial or adjacent fungal cells are symbiotic, optionally, wherein the fitness of the symbiotic adjacent bacterial or adjacent fungal cell is increased. In still more specific embodiments, the fitness is increased by reducing competing bacteria or competing fungi that may be neutral, symbiotic, or pathogenic.

[0270] In some embodiments, the adjacent bacterial or adjacent fungal cell is contacted with an effective amount of an ADAS comprising a T3 / 4SS or T6SS and cargo, wherein the cargo is delivered into the adjacent bacterial or adjacent fungal cell.

[0271] In other embodiments, an effective amount of an ADAS comprising a bacterial secretion system and a cargo is provided in proximity to the adjacent bacterial or adjacent fungal cells, wherein the bacterial secretion system extracellularly secretes the cargo such that the adjacent bacterial or adjacent fungal cells are contacted with the cargo.

[0272] In some embodiments, the ADAS is derived from a parental strain that is a competitor to the adjacent bacterial or adjacent fungal cells. In other embodiments, the ADAS is derived from a parental strain that is a mutualistic bacterium to the adjacent bacterial or adjacent fungal cells.

[0273] In certain embodiments, the method includes providing an effective amount of an ADAS or composition in proximity to invertebrate (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cells near a plant or a fungus. In more specific embodiments, the invertebrate is pathogenic. In still more specific embodiments, the fitness of the pathogenic invertebrate cells is reduced. In yet more specific embodiments, the fitness of the pathogenic invertebrate cells is reduced by modulating symbionts in the invertebrate cells. In other specific embodiments, the invertebrate is symbiotic. In more specific embodiments, the fitness of the symbiotic invertebrate cells is increased. In still more specific embodiments, the fitness is increased by reducing competing bacteria or fungi that may be neutral, symbiotic, or pathogenic.

[0274] In yet another aspect, the present invention provides a method for removing one or more undesired materials from an environment, the method comprising contacting the environment with an effective amount of an ADAS provided by the present invention or a composition provided by the present invention, wherein the ADAS comprises one or more molecules (such as proteins, polymers, nanoparticles, binders, or combinations thereof) that solubilize, chelate, or degrade one or more undesired materials. "Environment" is defined as a non-cellular target such as an ocean, soil, Superfund contaminated site, skin, pond, gut lumen, and food in a container.

[0275] In certain embodiments, the undesired material includes heavy metals such as mercury, and the ADAS comprises one or more molecules (such as proteins, polymers, nanoparticles, binders, or combinations thereof) that bind heavy metals, such as MerR for mercury. In some embodiments, the undesired material includes plastics such as PET, and the ADAS comprises one or more plastic-degrading enzymes such as PETase. In certain embodiments, the undesired material includes one or more small organic molecules, and the ADAS comprises one or more enzymes capable of metabolizing the one or more small organic molecules.

[0276] E. RNA Delivery Method

[0277] In another aspect, the present invention provides a composition comprising the bacterium or ADAS provided by the present invention, wherein the bacterium or ADAS comprises a T4SS, an RNA-binding protein cargo, and an RNA cargo that is bound by the RNA-binding protein and is suitable for delivery to a target cell via the T4SS. In certain embodiments, the RNA-binding protein is Cas9 fused to VirE2 and VirF, the RNA cargo is a guide RNA, and optionally, the T4SS is the Ti system from Agrobacterium. In other embodiments, the RNA-binding protein is p19 from Carnation Italian Ringspot Virus fused to VirE2 or VirF, the RNA cargo is siRNA, and optionally, the T4SS is the Ti system from Agrobacterium.

[0278] In a related aspect, the present invention provides a method for preparing these specific compositions, such methods requiring transfection of a plasmid containing Cas9 fused to VirE2 and VirF and an RNA cargo into Agrobacterium cells.

[0279] In another related aspect, the present invention provides a method for delivering RNA to a plant cell or an animal cell, the method comprising contacting the plant cell or the animal cell with a bacterium or ADAS, wherein the bacterium or ADAS comprises a T4SS, an RNA-binding protein cargo, and an RNA cargo, wherein the RNA is delivered to the plant cell or the animal cell. In more specific embodiments, the RNA-binding protein cargo is also delivered to the plant cell or the animal cell. In some embodiments, the ADAS is derived from a parental cell comprising one or more loss-of-function genetic alterations (e.g., one or more loss-of-function alterations in a lyase) that stabilize the ADAS.

[0280] VI. Other Embodiments

[0281] Some embodiments of the techniques described herein can be defined according to any of the following numbered embodiments:

[0282] Embodiment 1. A non-chromosomal dynamic activity system (ADAS) derived from a parental bacterial cell, the parental bacterial cell comprising at least one loss-of-function genetic alteration in a lyase.

[0283] Embodiment 2. The ADAS according to Embodiment 1, wherein the loss-of-function genetic alteration results in an increase in the stability of the ADAS relative to an ADAS derived from a parental bacterial cell that does not comprise the alteration.

[0284] Example 3. The ADAS as described in Example 1, wherein the loss-of-function genetic alteration is a non-silent codon change, deletion, insertion, mutation, or any combination thereof.

[0285] Example 4. The ADAS as described in Example 1, wherein the loss-of-function genetic alteration is a deletion.

[0286] Example 5. The ADAS as described in Example 1, wherein the lyase is an endopeptidase, a cell wall lyase, and / or an autolysin.

[0287] Example 6. The ADAS as described in Example 2, wherein the lyase 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.

[0288] Example 7. The ADAS as described in Example 5, wherein the loss-of-function genetic alteration is in lytC.

[0289] Example 8. The ADAS as described in Example 1, wherein the parental bacterial cell further comprises a loss-of-function alteration in a cell division topology-specific factor.

[0290] Example 9. The ADAS as described in Example 8, wherein the cell division topology-specific factor is DivIVA, minC, minD, minE, minCD, or the minCDE operon.

[0291] Example 10. The ADAS as described in Example 1, wherein the parental bacterial cell is Gram-positive.

[0292] Example 11. The ADAS as described in Example 1, wherein the parental bacterial cell is Gram-negative.

[0293] Example 12. The ADAS as described in Example 1, wherein the parental bacterial cell further comprises a loss-of-function genetic alteration that disrupts sporulation.

[0294] Example 13. The ADAS as described in Example 12, wherein the loss-of-function genetic alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, or combinations thereof.

[0295] Example 14. The ADAS as described in Example 13, wherein the loss-of-function alteration in the sporulation gene is in SigF.

[0296] Example 15. The ADAS as described in Example 1, wherein the parental bacterial cell is from the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azospirillum, Azorhizobium, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Cyanothece, Dechloromonas, Desulfobacterium, Desulfitobacterium, Dickeya, Erwinia, Francisella, Fusobacterium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Phoxinus, Phyllobacterium, Psychrobacter, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Roseobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Weeksella, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Syntrophobacter, or Thermoanaerobacter bacteria.

[0297] Example 16. The ADAS as described in Example 15, wherein the parental bacterial cell is Bacillus subtilis.

[0298] Example 17. The ADAS as described in Example 2, wherein the stability of the ADAS is greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%, or 100% as measured by the percentage of intact ADAS.

[0299] Example 18. The ADAS as described in any one of Examples 1-17, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or both of SEQ ID NO:5 and SEQ ID NO:9.

[0300] Example

[0301] A general description of the present invention will now be given, and the present invention will be more easily understood by reference to the following examples, which are only used to illustrate certain aspects and embodiments of the present invention and are not intended to limit the present invention. It should be understood that various other embodiments can be practiced in view of the general description provided above.

[0302] Example 1. Generation of ADAS

[0303] The production of ADAS from parental bacterial cells can be achieved using the methods disclosed in International Patent Application Publication No. WO 2020 / 123569 (which is incorporated herein by reference in its entirety). Briefly, these methods and their variants are described below.

[0304] In this example, ADAS is produced by disrupting one or more genes involved in regulating the partitioning function of the parental cell (i.e., ΔminC, ΔminD, ΔminCDE, ΔminCdivIVA, or ΔdivIVA). This example details the genetic means of producing ADAS strains via disruption of the min operon or overexpression of the septum machinery component FtsZ.

[0305] A. Generation of ADAS via min mutation

[0306] To disrupt the min operon, the λ-RED recombineering method was used according to the protocol designed by Datsenko and Wanner, PNAS [Proceedings of the National Academy of Sciences of the United States of America], 97(12):6640-6645, 2000. Strains harboring plasmids with the λ-RED system for engineering were obtained from the Coli Genetic Stock Center (CGSC) at Yale University. Briefly, primers were designed to non-polar delete the coding sequences of Escherichia coli minC, minD, or the complete minCDE operon by encoding approximately 40 genomic homology base pairs into the 5'-end of the primers. The 3'-ends of these primers were homologous to the plasmids pKD3 and pKD4 of the λ-RED system, and these ends provided antibiotic markers for selecting the parental bacterial strains with genetic target mutations. The primer sequences for the deletions are provided in Table 2. According to the method of Datsenko and Wanner, PNAS [Proceedings of the National Academy of Sciences of the United States of America], 97(12):6640-6645, 2000, after standard PCR using the primers with pKD3 as the DNA template, the purified amplicons were transformed into bacteria prepared with pKD46 (a plasmid containing the λ-RED homologous recombination system derived from a phage) via electroporation. Transformants were selected on LB agar with 35 μg / mL chloramphenicol. These resulting colonies were confirmed to have a genetic disruption (i.e., ΔminC, ΔminD, or ΔminCDE) using standard allele-specific PCR.

[0307] B. Generation of ADAS via divIVA mutation

[0308] Briefly, primers were designed to delete the coding sequences of Bacillus subtilis divIVA or divIVA and minC. The primer sequences for the deletions are listed in Table 2, including both the WT and divIVA deletions. Combining the sigF deletion (sequences in Table 2) to interfere with sporulation, the divIVA deletion generated the parental strain MACH2347. The strain genotypes are provided in Table 1. Introduction of the erm (erythromycin) cassette together with the selected deletion primers into growing bacteria allowed selection of transformed colonies by growth on LB-erm5 plates. Once the transformed bacteria were cultured, colonies with the erm cassette were treated with a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. Transformed bacteria were selected by plating on LB-Spec plates and incubating at 30 °C. Then, the selected bacteria were streaked onto LB plates without antibiotics and incubated at the high temperature of 42 °C, resulting in the loss of the temperature-sensitive plasmid. The isolated colonies were confirmed to contain the expected deletions.

[0309] Table 1. Strains

[0310]

[0311] C. Generating ADAS by overexpressing ftsZ

[0312] To create ADAS from overexpressing the septum machinery, a plasmid was constructed that drives the expression of the FtsZ Z-ring protein from wild-type Escherichia coli. Briefly, computational tools were used to de novo optimize the strong ribosome binding site and the coding sequence of the Escherichia coli FtsZ protein from scratch. This translation unit was customized for de novo DNA synthesis from Integrated DNA Technologies (IDT TM ) and cloned into the backbone using standard cloning techniques. The resulting plasmid pFtsZ (Table 3) has a TetR repressor, a TetA promoter repressed by the TetR protein, a kanamycin resistance marker, and a pMB1 origin of replication. When transformed into compatible bacteria, overproduction of the FtsZ protein by pFtsz can be induced by adding anhydrous tetracycline to the culture. Then, this protein is able to form spontaneous filaments that cause asymmetric division of the parental bacterial cells, thus generating ADAS.

[0313] Example 2. Purifying ADAS from Bacillus subtilis

[0314] Purification of ADAS from the parental bacterial cells can be achieved using the methods disclosed in International Patent Application Publication No. WO 2020 / 123569, which is incorporated herein by reference in its entirety. Briefly, these methods and their variants are described below.

[0315] In this example, a method for purifying a population of ADAS from a culture of an ADAS-producing bacterial parental strain is described. This method can be used to purify any ADAS-producing strain described herein, including the strains of Example 1 and Table 1. Purification separates ADAS from the live parental bacterial cells, which are larger and contain a genome. ADAS is purified from a high cell density culture of an ADAS-producing strain via a combination of 1) high-speed or low-speed centrifugation, 2) selective growth, and 3) buffer exchange / concentration. Centrifugation procedures are used to selectively remove the live parental bacterial cells and large cell debris while enriching ADAS in the mixed suspension. Using the selective growth procedure, the number of live parental bacterial cells present in the sample is reduced by adding compounds that are directly antimicrobial (i.e., toxic to cells with a microbial genome) and / or compounds that enhance the sedimentation of live cells via centrifugation. The buffer exchange / concentration procedure transfers ADAS from a larger volume of bacterial medium to a smaller volume of 1x PBS while removing culture additives and cell debris.

[0316] A. ADAS Purification

[0317] Generate the ADAS-producing strain using the molecular cloning procedure described in Use Example 1, and then culture it in a medium to a high cell density. The culture can be scaled up, for example, from 1 mL to 1000 mL or more of the medium.

[0318] Transfer the culture to a centrifuge tube and perform a high-speed or low-speed centrifugation procedure to precipitate intact cells and large cell debris, while maintaining ADAS in the supernatant. The centrifugation procedure is carried out at 4 °C or at room temperature. In some cases, a low-speed centrifugation procedure is used, which involves a process of sequentially spinning at 1,000 × g, 2,000 × g, 3,000 × g, and 4,000 × g for 10 minutes each on an X14R benchtop centrifuge (Beckman Coulter) or an Eppendorf TM 5424R benchtop centrifuge (Fisher Scientific). In some cases, the low-speed centrifugation procedure consists of sequentially spinning at 2,000 × g for 20 minutes at 4 °C, where the supernatant from the first spin is decanted into a sterile centrifuge bottle before the second spin. In some cases, the low-speed centrifugation procedure is a single spin at 4,000 × g for 40 minutes in a Sorvall TM Lynx 6000 ultracentrifuge (Thermo Scientific TM ), where the rate of rotor acceleration is set to the lowest possible setting value. In some cases, a high-speed centrifugation procedure involving sequentially pulsing at 20,000 × g is used, where the rotation is stopped once the desired speed is reached and the supernatant is transferred to a new high-speed bottle before the next spin. In some cases, the high-speed centrifugation procedure includes spinning at 4 °C and 17,000 × g for 30 minutes, after which the pellet is resuspended in the growth medium.

[0319] After low-speed centrifugation, the culture supernatant is decanted into a sterile culture tube and a selective growth process is performed. After high-speed centrifugation, the culture supernatant is decanted and the pellet is resuspended, and a selective growth process is performed. In some cases, a concentrated antibiotic solution (e.g., spectinomycin, clindamycin, tetracycline, ceftriaxone, kanamycin, carbenicillin, gentamicin, and / or ciprofloxacin) or other concentrated chemical solution (e.g., sodium chloride, sodium hydroxide, M hydrochloric acid, glucose, casein amino acids, and / or D-amino acids) is added directly to the culture supernatant. In other cases, the culture supernatant is pelleted by high-speed centrifugation at 10,000×g to 20,000×g for 5 to 60 minutes, and the pellet is resuspended in fresh medium containing a concentrate of an antibiotic or other chemical solution that inhibits live cells. Selective growth is performed by incubating the ADAS at 250 rpm with stirring at 4°C to 42°C for 1 to 3 hours. The ADAS is then transferred to a sterile centrifuge tube and another round of centrifugation is performed.

[0320] After selective growth and centrifugation, a buffer exchange / concentration procedure is performed on the supernatant. In some cases, this is done by passing the supernatant through a 0.2 pm asymmetric polyethersulfone (aPES) membrane filter (Thermo Fisher), and then through 1 to 9 volumes of 1×PBS. In some cases, the ADAS is pelleted by centrifugation at 10,000×g to 20,000×g for 5 to 60 minutes, washed in 1 to 9 volumes of 1×PBS, pelleted again, and resuspended in 1×PBS at a concentration 1 to 100,000 times the starting culture volume. In other cases, the ADAS is pelleted by sequential high-speed pulses at 16,000×g at 1-minute intervals, then spun at high speed for 20 minutes at 20,000×g at 4°C, after which the pellet is resuspended and then washed several times. In some cases, the wash includes spinning at 15,000×g at 4°C for 5 minutes.

[0321] B. Purification of ADAS from an auxotrophic ADAS-producing parental strain

[0322] Auxotrophic (i.e., unable to synthesize organic compounds required for growth) ADAS-producing parental strains can be used to produce ADAS. Such strains can only grow when organic compounds are provided. Thus, auxotrophic parental strains can be selected by storing or incubating the ADAS preparation in a medium lacking organic compounds, thereby providing an additional method for reducing the parental load in the ADAS preparation.

[0323] Example 3: Lyase deletion in ADAS

[0324] Using a method similar to Example 1B, primers were designed to delete the target coding sequence (e.g., the genomic sequence encoding the lyase). The sequences targeted for deletion are listed in Table 2, and both the wild-type (WT) sequences and the sequences showing the deletion of each target gene are included. For example, SEQ ID NO:4 shows the wild-type genomic region containing SigF, and SEQ ID NO:5 shows the genomic region after a loss-of-function deletion. SEQ ID NO:8 shows the wild-type genomic region containing lytC, and SEQ ID NO:9 shows the genomic region after a loss-of-function deletion.

[0325] The strain genotypes are provided in Table 1. Introducing the erm (erythromycin) cassette together with the selected deletion primers into growing bacteria allows the selection of transformed colonies by growth on LB-erm5 plates. Once the transformed bacteria are cultured, colonies of the erm cassette are treated with a temperature-sensitive plasmid conferring spectinomycin (Spec) resistance. Transformed bacteria are selected by plating on LB-Spec plates and incubating at 30 °C. Then, the selected bacteria are streaked onto LB plates without antibiotics and incubated at a high temperature of 42 °C, resulting in the loss of the temperature-sensitive plasmid. The isolated colonies were confirmed to contain the expected deletion (e.g., lytC), and if further genomic deletions (such as sigF) were needed, a similar method was used.

[0326] Table 2. Sequences

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] Example 4: Stability of ADAS

[0336] The strain producing ADAS was generated and purified using the procedures described in Examples 1-3 and is listed in Table 1. The Bacillus subtilis strain was pre-cultured in LB broth and incubated at 37 °C, 250 RPM for 6 hours. Then the culture was diluted and incubated overnight at 30 °C, 250 RPM.

[0337] A. Imaging and Absorbance

[0338] The absorbance of various cultures at an optical density of 600 (OD600) was measured after the following three different time points: (1) after overnight culture and ADAS enrichment (t0); (2) after overnight culture, ADAS enrichment, and incubation at 4 °C for 23 hours (t23); and (3) after overnight culture, ADAS enrichment, and incubation at 4 °C for 48 hours (t48), as Figure 2 shown. Generally, a higher OD600 at a later time point compared to the control strain indicates a more stable ADAS chassis.

[0339] Representative images from overnight cultures highlight the differences in parental cell growth before ADAS enrichment, with fewer breaks in rod length in parental bacterial cells (MACH2403; Figure 1 ) containing the ΔlytC mutation. Representative images of two different cultures at three different time points were also taken to visually determine ADAS stability. Shown are ADAS and remaining parental cells from two different Bacillus subtilis strains after overnight culture ( Figure 1 and Figure 3A ), after overnight culture and ADAS enrichment (t0, Figure 3B ), or after overnight culture, ADAS enrichment, and incubation at 4 °C for 23 hours (t23, Figure 3C ). The left subpanel is strain MACH2347 (described in Table 1). The right subpanel is MACH2403 (described in Table 1), which was modified from MACH2347 to have an additional lytC genomic deletion. White arrows indicate instances of light-phase ADAS (ADAS that appears brighter or has a ghosting, indicating rupture and lysis). Wedge symbols indicate instances of light-phase parental cells. The ADAS population derived from the parental cell line containing the lytC deletion shows visual evidence of increased ADAS stability via an increase in the number of intact (non-light-phase) ADAS. The strains listed and the images shown should not be considered limiting.

[0340] Although, for purposes of clarity of understanding, the foregoing invention has been described in detail by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety. Other embodiments are within the claims.

Claims

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

2. The ADAS of claim 1, wherein the loss-of-function alteration results in an increase in the stability of the ADAS relative to an ADAS derived from a parental bacterial cell that does not comprise the alteration.

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

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

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

6. 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 loss-of-function alteration is in lytC.

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

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

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

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

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

13. The ADAS of claim 12, wherein the loss-of-function alteration that disrupts sporulation is a loss-of-function alteration in a sporulation gene selected from the group consisting of: sigF, sigE, spoIIAA, spoIID, bofA, spoVE, spoIVFB, dacB, dapA, dapB, spoIIGA, spoIIM, spoIIR, spo0A, or combinations thereof.

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

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

16. The ADAS according to claim 15, wherein the parental bacterial cell is Bacillus subtilis.

17. The ADAS according to claim 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.

18. The ADAS according to any one of claims 1-17, wherein the parental bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or both of SEQ ID NO:5 and SEQ ID NO:9.

Images