WO2024107843A1 - Systèmes actifs dynamiques achromosomiques stabilisés et leurs utilisations - Google Patents

Systèmes actifs dynamiques achromosomiques stabilisés et leurs utilisations Download PDF

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WO2024107843A1
WO2024107843A1 PCT/US2023/079824 US2023079824W WO2024107843A1 WO 2024107843 A1 WO2024107843 A1 WO 2024107843A1 US 2023079824 W US2023079824 W US 2023079824W WO 2024107843 A1 WO2024107843 A1 WO 2024107843A1
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adas
cell
parent
bacterial
loss
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Felipe Oseas BENDEZU
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Flagship Pioneering Innovations Vi, Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/08—Reducing the nucleic acid content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01028—N-Acetylmuramoyl-L-alanine amidase (3.5.1.28)

Definitions

  • achromosomal dynamic active systems and methods of making and using the same.
  • delivery vectors e.g., achromosomal dynamic active systems (ADAS)
  • ADAS achromosomal dynamic active systems
  • the disclosure features an achromosomal dynamic active system (ADAS) derived from a parent bacterial cell, comprising at least one genetic loss-of-function alteration in a lytic enzyme.
  • ADAS achromosomal dynamic active system
  • the genetic loss-of-function in a lytic enzyme results in increased stability of the ADAS, relative to an ADAS derived from a parent bacterial cell not comprising the alteration.
  • the disclosure features an achromosomal dynamic active system (ADAS) derived from a parent bacterial cell, comprising at least one genomic deletion in a lytic enzyme.
  • ADAS achromosomal dynamic active system
  • the genomic deletion in a lytic enzyme results in increased stability of the ADAS, relative to an ADAS derived from a parent bacterial cell not comprising the alteration.
  • the parent bacterial cell has been modified to reduce enzymatic activity.
  • a genetic loss-of-function alteration in the parent bacterial cell reduces enzymatic and/or lytic activity.
  • a genomic deletion in the parent bacterial cell reduces enzymatic and/or lytic activity.
  • the parent bacterial cell has at least one genetic loss-of-function alteration reducing activity of endopeptidases, cell wall lytic enzymes, and/or autolytic enzymes.
  • the parent bacterial cell has at least one genomic deletion reducing activity of one or more endopeptidases, cell wall lytic enzymes, and/or autolytic enzymes.
  • the parent cell comprises a genetic loss-of-function alteration or deletion from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwIK, lytH , CwlS, CwlC, CwlH, MpaA, cwlJ and combinations thereof.
  • the parent cell comprises a genetic loss-of-function alteration or deletion that disrupts sporulation from the group consisting of sigF, sigE, spollAA, spollD, bofA, spoVE, spolVFB, dacB, dapA, dapB, spollGA, spollM, spollR, spoOA and combinations thereof.
  • the parent bacterial cell is a gram-positive bacterial cell.
  • the parent bacterial cell is Bacillus subtilis or of the Bacillus genus.
  • the parent bacterial cell is of the Lactobacillus genus.
  • the parent bacterial cell is a Gram-negative bacterial cell.
  • the parent bacterial cell is from a genus selected from Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas,
  • the parent cell comprises a gene deletion from the group consisting of SPp, skin, PBSX, prophage 1 , pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. 20(12):2076-2090, 2003.)
  • the parent bacterial cell contains a genomic deletion of a cell division topological specificity factor.
  • the genomic deletion is of DivIVA, minC, minD, minE, minCD or the minCDE operon.
  • the ADAS further comprises at least one cargo.
  • the cargo is a protein or a polypeptide.
  • the ADAS or the parent bacterial cell has been modified to increase the level of the cargo in the ADAS.
  • the cargo is an enzyme, a DNA-modifying agent, a chromatinremodeling agent, a gene editing agent, a nuclear targeting agent, a binding agent, an immunogenic agent, or a toxin.
  • the enzyme is a metabolic enzyme.
  • the gene editing agent is a component of a CRISPR system.
  • the nuclear targeting agent is a transcription factor.
  • the binding agent is an antibody or an antibody fragment.
  • the binding agent is a VHH molecule.
  • the immunogenic agent is an immunostimulatory agent.
  • the immunogenic agent is an immunosuppressive agent.
  • Fig. 1 is a set of representative images showing overnight culture of three different parent cell strains derived from Bacillus subtilis. The strains and their distinct genomic deletions are listed in Table 1.
  • Fig. 2 is a bar graph depicting the respective ODeoo measures of each cultivated strain at three different timepoints: tO (after cultivation overnight and achromosomal dynamic active system (ADAS) enrichment), t23 (after cultivation overnight, ADAS enrichment, and 23 hours of incubation at 4°C), and t48 (after cultivation overnight, ADAS enrichment, and 48 hours of incubation at 4°C).
  • tO after cultivation overnight and achromosomal dynamic active system (ADAS) enrichment
  • t23 after cultivation overnight, ADAS enrichment, and 23 hours of incubation at 4°C
  • t48 after cultivation overnight, ADAS enrichment, and 48 hours of incubation at 4°C.
  • Fig. 3A is a pair of representative micrographs showing ADAS and remaining parental cells from two different B. subtilis strains after cultivation overnight.
  • the left panel is strain MACH2347 (described in Table 1 ).
  • the right panel is MACH2403 (described in Table 1 ), which has been modified from MACH2347 with the additional genomic deletion of lytC.
  • the white arrows indicate examples of phase-light ADAS.
  • the arrowheads indicate examples of phase-light parent cells.
  • Fig. 3B is a pair of representative micrographs showing ADAS and remaining parental cells from two different B. subtilis strains after cultivation overnight and ADAS enrichment (tO).
  • Left panel MACH2347.
  • Right panel MACH2403.
  • White arrows exemplary phase-light ADAS.
  • Arrowheads exemplary phase-light parent cells.
  • Fig. 3C is a pair of representative micrographs showing ADAS and remaining parental cells from two different B. subtilis strains after cultivation overnight, ADAS enrichment, and 23 hours of incubation at 4°C (t23).
  • achromosomal dynamic system refers to a genome- free, non-replicating, enclosed membrane system comprising at least one membrane and having an interior volume suitable for containing a cargo (e.g., one or more of a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)).
  • a cargo e.g., one or more of a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)).
  • a cargo e
  • ADAS are minicells or modified minicells derived from a parent bacterial cell (e.g., a gram-negative or a gram-positive bacterial cell).
  • ADAS are derived from parent cells by modifying the parent cell to remove the genome, and are substantially similar in size to the parent cell.
  • ADAS are derived from parent bacteria using any suitable method, e.g., genetic manipulation of the parent cell or exposure to a culture or condition that increases the likelihood of formation of bacterial minicells. Exemplary methods for making ADAS are those that disrupt the cell division machinery of the parent cell.
  • ADAS include one or more endogenous or heterologous features of the parent cell surface, e.g., cell walls, cell wall modifications, flagella, or pilli, and/or one or more endogenous or heterologous features of the interior volume of the parent cell, e.g., nucleic acids, plasmids, proteins, small molecules, transcription machinery, or translation machinery.
  • ADAS may lack one or more features of the parent cell.
  • ADAS are loaded or otherwise modified with a feature not comprised by the parent cell.
  • highly active ADAS refers to an ADAS having high work potential, e.g. an ADAS having the capability to do a significant amount of useful work.
  • work is metabolic work, including chemical synthesis (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), chemical modification (e.g., of proteins, nucleic acids, lipids, carbohydrates, polymers or small molecules), or transport (e.g., import, export, or secretion, e.g., secretion by a bacterial secretion system (e.g., T3SS)) under suitable conditions.
  • 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
  • transport e.g., import, export, or secretion, e.g., secretion by a bacterial secretion system (e
  • highly active ADAS begin with a large pool of energy, e.g., energy in the form of ATP.
  • ADAS have the capacity to take up or generate energy/ ATP from another source.
  • highly active ADAS are identified, e.g., by having increased ATP concentration, increased ability to generate ATP, increased ability to produce a protein, increased rate or amount of production of a protein, and/or increased responsiveness to a biological signal, e.g., induction of a promoter.
  • parent bacterial cell refers to a cell (e.g., a gram-negative or a gram-positive bacterial cell) from which an ADAS is derived.
  • Parent bacterial cells are typically viable bacterial cells.
  • viable bacterial cell refers to a bacterial cell that contains a genome and is capable of cell division.
  • Preferred parent bacterial cells are derived from any of the strains: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psych
  • an ADAS composition or preparation that is “substantially free of” parent bacterial cells and/or viable bacterial cells is defined herein as a composition having no more than 500, e.g., 400, 300, 200, 150, 100 or fewer colony-forming units (CFU) per mL.
  • an ADAS composition that is substantially free of parent bacterial cells or viable bacterial cells includes fewer than 50, fewer than 25, fewer than 10, fewer than 5, fewer than 1 , fewer than 0.1 , or fewer than 0.001 CFU/mL, including no bacterial cells.
  • cell division topological specificity factor refers to a component of the cell division machinery in a bacterial species that is involved in the determination of the site of the septum and functions by restricting the location of other components of the cell division machinery, e.g., restricting the location of one or more Z-ring inhibition proteins.
  • Exemplary cell division topological specificity factors include minE, which was first discovered in E. coli and has since been identified in a broad range of gram-negative bacterial species and gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3: 959-968, 2005). minE functions by restricting the Z-ring inhibition proteins minC and minD to the poles of the cell.
  • a second exemplary cell division topological specificity factor is DivIVA, which was first discovered in Bacillus subtilis (Rothfield et al., Nature Reviews Microbiology, 3: 959-968, 2005).
  • Z-ring inhibition protein refers to a component of the cell division machinery in a bacterial species that is involved in the determination of the site of the septum and functions by inhibiting the formation of a stable FtsZ ring or anchoring such a component to a membrane.
  • the localization of Z-ring inhibition proteins is modulated by cell division topological specificity factors, e.g., minE and DivIVA.
  • Exemplary Z-ring inhibition proteins include minC and minD, which were first discovered in E. coli and have since been identified in a broad range of gramnegative bacterial species and gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3: 959-968, 2005).
  • minC, minD, and minE occur at the same genetic locus, which may be referred to as the “min operon”, the minCDE operon, or the min or minCDE genetic locus.
  • the term “reduction in the level or activity of a cell topological specificity factor,” refers to an overall reduction of any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, in the level or activity of the cell topological specificity factor (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard methods, as compared to the level in a reference sample (for example, an ADAS produced from a wild-type cell or a cell having a wild-type minCDE operon or wild-type divIVA gene), a reference cell (for example, 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.
  • a reference sample for example, an ADAS produced from a wild-type cell or a cell having a wild-type min
  • a reduced level or activity refers to a decrease in the level or activity in the sample which is at least about 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1 x, 0.05x, or 0.01 x the level or activity of the cell topological specificity factor in a reference sample, reference cell, control sample, or control cell.
  • percent identity refers to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in some embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST.
  • the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleotides or amino acids in B.
  • sequence alignment program e.g., BLAST
  • sequence identity for example, in homologues of MinE or DivIVA proteins 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, to a native sequence MinE (or minE) or DivIVA (or divIVA) sequence as disclosed herein.
  • modulating a state of a cell refers to an observable change in the state (e.g., the transcriptome, proteome, epigenome, biological effect, or health or disease state) of the cell (e.g., an animal, plant, or insect cell) as measured using techniques and methods known in the art for such a measurement, e.g., methods to measure the level or expression of a protein, a transcript, an epigenetic mark, or to measure the increase or reduction of activity of a biological pathway.
  • modulating the state of the cell results in a change of at least 1% relative to prior to administration (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% or more relative to prior to administration; e.g., up to 100% relative to prior to administration).
  • modulating the state of the cell involves increasing a parameter (e.g., the level or expression of a protein, a transcript, or activity of a biological pathway) of the cell.
  • increasing the state of the cell results in an increase of the parameter by at least 1% relative to prior to administration (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% or more relative to prior to administration; e.g., up to 100% relative to prior to administration).
  • modulating the state of involves decreasing a parameter (e.g., the level or expression of a protein, a transcript, or activity of a biological pathway) of the cell.
  • decreasing the state of the cell results in a decrease of the parameter by at least 1% relative to prior to administration (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% or more relative to prior to administration; e.g., up to 100% relative to prior to administration).
  • at least 1% relative to prior to administration e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% or more relative to prior to administration; e.g., up to 100% relative to prior to administration.
  • endogenous Type 3 secretion system or “endogenous T3SS” refers to a T3SS that is present on a cell (e.g., a parent cell), or an ADAS derived therefrom, and is naturally encoded by the cell (e.g., is encoded by a wild-type version of the cell).
  • the T3SS may be expressed by the endogenous genes of the cell, and/or may be encoded and expressed by a synthetic construct in the cell.
  • Expression or abundance of an endogenous T3SS may be increased, e.g., by the addition of a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or reduction or removal of a negative regulator of expression of the T3SS.
  • a moiety that increases the abundance of the T3SS e.g., a transcriptional activator of the T3SS
  • reduction or removal of a negative regulator of expression of the T3SS e.g., a transcriptional activator of the T3SS
  • heterologous Type 3 secretion system or “heterologous T3SS” refers to a T3SS that is present on a cell (e.g., a parent cell), or an ADAS derived therefrom, and is not naturally encoded by the cell (e.g., is not encoded by a wild-type version of the cell).
  • the cell may encode another T3SS, or may not encode any T3SS.
  • the T3SS is expressed by a synthetic construct in the cell.
  • an “endogenous effector” of a secretion system is a moiety (e.g., a protein or polypeptide) that is naturally encoded by a cell from which the secretion system (e.g., T3SS) is derived (e.g., is encoded by a wild-type version of the cell) and is capable of being secreted by the secretion system.
  • a secretion system and one or more of its endogenous effectors may be expressed in the cell in which they naturally occur or may be expressed heterologously, e.g., expressed by a cell that does not naturally encode the endogenous effector or the secretion system.
  • an effector that is heterologous with respect to a secretion system is a moiety (e.g., a protein or polypeptide) that is not naturally encoded by a cell from which the secretion system (e.g., T3SS) is derived (e.g., is not encoded by a wild-type version of the cell) and is capable of being secreted by a secretion system of a cell from which the heterologous effector is derived.
  • the effector is capable of being secreted by the secretion system to which it is heterologous, or is modified to be secreted by the secretion system to which it is heterologous.
  • the heterologous effector is an effector of a T4SS or a T6SS that is secreted by a T3SS.
  • heterologous means not native to a cell or composition in its naturally-occurring state.
  • heterologous refers to a molecule; for example, a cargo or payload (e.g., a polypeptide, a nucleic acid such as a protein-encoding RNA or tRNA, or small molecules) or a structure (e.g., a plasmid or a gene-editing system) that is not found naturally in an ADAS or the parent bacteria from which it is produced (e.g., a gram-negative or gram-positive bacterial cell).
  • a cargo or payload e.g., a polypeptide, a nucleic acid such as a protein-encoding RNA or tRNA, or small molecules
  • a structure e.g., a plasmid or a gene-editing system
  • phase-light ADAS and “phase-light parent cells” refer to the bodies of ADAS or parental cells (e.g., refer to dead ADAS or dead parental cells) that appear lighter or ghosted in images (e.g., photomicrographs taken using a light microscope), indicating disintegration and lysis, as compared to the dark-colored ADAS or parent cells, which will be herein described as “intact ADAS” and “intact parent cells,” respectively.
  • ADAS ADAS e.g., stability of a particular population, strain, or variety of ADAS
  • Stability of an ADAS may be measured as the proportion of “intact” ADAS relative to “phase-light” ADAS (e.g., the percentage of intact ADAS) in one or more representative images of a plurality of the ADAS.
  • increased stability of ADAS derived from a modified strain containing at least one lytic enzyme deletion may be defined as an increased percentage of “intact” ADAS in representative images of ADAS derived from the modified strain as compared to ADAS derived from a control strain (e.g., a strain not comprising a lytic enzyme deletion) , as measured (e.g., scored) with the same conditions and timepoints. For example, if a modified ADAS has greater than 40% stability, then more than 40% of the visible ADAS are intact rather than phase-light.
  • stability of an ADAS is measured as a unitless ratio of half-life of ADAS derived from an unmodified strain (e.g., a strain not comprising a lytic enzyme deletion) and the half-life of ADAS derived from a modified strain (e.g., a strain comprising a lytic enzyme deletion), as measured in the same environmental conditions.
  • an unmodified strain e.g., a strain not comprising a lytic enzyme deletion
  • a modified strain e.g., a strain comprising a lytic enzyme deletion
  • fold change of the optical density at wavelength 600 (OD600) of ADAS derived from a modified strain compared to ADAS derived from a control strain at one or more timepoints is another measure of ADAS stability.
  • the stability of the ADAS is modified (e.g., increased) by at least one genomic deletion of a lytic enzyme in the parent cell.
  • the ADAS with one or more lytic enzyme deletions has stability greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% as measured by percent of intact ADAS compared to ADAS without a lytic enzyme deletion (e.g., derived from a parent cell not comprising a lytic enzyme deletion).
  • a “lytic enzyme” refers to an enzyme (e.g., an endopeptidase, cell wall lytic enzyme, or autolytic enzyme) that regulates the disruption of parent bacterial cell walls and membranes. Genomic deletions of one or more of these enzymes reduce lytic activity in the parental cell and in some embodiments can increase stability and integrity of the ADAS structure, allowing production of a greater number of intact ADAS and/or survival of intact ADAS for increased periods of time.
  • genomic deletions that reduce lytic activity in parental cells include but are not limited to lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwIK, lytH, CwlS, CwlC, CwlH, MpaA, and combinations thereof.
  • sporulation refers to the process in certain parent bacterial strains of forming spores from cells during unfavorable conditions. Genetic deletions that could disrupt sporulation include but are not limited to sigF, sigE, spollAA, spollD, bofA, spoVE, spolVFB, dacB, dapA, dapB, spollGA, spollM, spollR, spoOA and combinations thereof.
  • genetic loss-of-function refers to a substantial reduction or complete ablation of a protein (e.g., a substantial reduction in the function of the protein, a complete loss of function of the protein, a substantial reduction in expression of the protein, or a complete loss of expression of the protein) caused by an alteration (e.g., a genetic mutation) in the gene encoding the protein. Mutations include but are not limited to insertions or deletions of one or more nucleotides, non-silent codon changes, and duplications. Proteins in which genetic loss-of-fu notion may occur include but are not limited to enzymes, such as lytic enzymes, proteases, amylases, lipases, or cellulases.
  • promoter inactivation or chemical inhibition can impact (e.g., decrease or ablate) expression of a protein (e.g., a lytic enzyme, protease, amylase, lipase, or cellulase).
  • a protein e.g., a lytic enzyme, protease, amylase, lipase, or cellulase.
  • ADAS achromosomal dynamic active systems
  • An “ADAS” is a genome-free, non-replicating, enclosed membrane system comprising at least one membrane (in some embodiments, two membranes, where the two membranes are non-intersecting) and having an interior volume suitable for containing a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)).
  • a cargo e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle,
  • ADAS are minicells or modified minicells derived from a parent bacterial cell (e.g., a gram-negative or a gram-positive bacterial cell).
  • ADAS are derived from parent bacteria using any suitable method, e.g., genetic manipulation of the parent cell or exposure to a culture or condition that increases the likelihood of formation of bacterial minicells.
  • an ADAS has a major axis cross section between about 100nm-500 pm (e.g., in certain embodiments, about: 100-600 nm, such as 100-400 nm; or between about 0.5- 10pm, and 10-500 pm).
  • an ADAS has a minor axis cross section between about: 0.001 , 0.01 , 0.1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, up to 100% of the major axis.
  • an ADAS has an interior volume of between about: 0.001 -1 pm 3 , 0.3-5 pm 3 , 5-4000 pm 3 , or 4000-50x10 7 pm 3 .
  • the ADAS is substantially similar in size to the parent cell, e.g., has a size (e.g., interior volume, major axis crosssection, and/or minor axis cross section) that is about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the size of the parent cell, has a size that is identical to that of the parent cell, or has a size that is about 101 %, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 1 10% of the size of the parent cell.
  • the invention provides highly active ADAS.
  • a “highly active” ADAS is an ADAS with high work potential, e.g. an ADAS having the capability to do a significant amount of useful work.
  • work is defined as, e.g., 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.
  • highly active ADAS begin with a large pool of energy, e.g., energy in the form of adenosine triphosphate (ATP).
  • ADAS have the capacity to take up or generate energy (e.g., ATP) from another source.
  • ADAS provided by the invention encompasses all embodiments of ADAS described herein, including, in particular embodiments, highly active ADAS, the set of which can be referenced as “highly active ADAS provided by the invention”, which is a subset of the ADAS provided by the invention.
  • the invention provides a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1 mM and wherein the composition is substantially free of viable bacterial cells.
  • ADAS highly active achromosomal dynamic active systems
  • the invention provides a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 3 mM and wherein the composition is substantially free of viable bacterial cells.
  • ADAS highly active achromosomal dynamic active systems
  • a highly active ADAS has an initial ATP concentration of at least 1 nM, 1.1. nM, 1.2 nM, 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, 2.5 mM, 3 nM, 3.5 nM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 50 mM.
  • ATP concentration can be evaluated by a variety of means including, in certain embodiments, a BacTiter-GloTM assay (Promega) on lysed ADAS.
  • high activity is additionally or alternatively assessed as the rate or amount of increase in ATP concentration in an ADAS over time.
  • the ATP concentration of an ADAS is increased by at least 50%, at least 60%, at least 75%, at least 100%, at least 150%, at least 200%, or more than 200% following incubation under suitable conditions, e.g., incubation at 37°C for 12 hours.
  • a highly active ADAS has a rate of ATP generation greater than about: 0.000001 , 0.00001 , 0.0001 , 0.001 , 0.01 , 0.05, 0.1 , 0.5, 1 .0, 2, 3, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 500, 1000, 10000 ATP/sec/nm 2 for at least about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, or two weeks.
  • high activity is assessed as a rate of decrease in ATP concentration over time.
  • ATP concentration decreases less rapidly in ADAS that are highly active than in ADAS that are not highly active.
  • the drop in ATP concentration in an ADAS or an ADAS composition at 24 hours after preparation is less than about 50% (e.g., less than about: 45, 40, 35, 30, 25, 20, 15, 10, or 5%) compared to the initial ATP concentration (e.g., ATP per cell volume), e.g., as measured using a BacTiter-GloTM assay (Promega).
  • high activity is additionally or alternatively assessed as lifetime index of an ADAS.
  • the lifetime index is calculated as the ratio of the rate of GFP production at 24 hours vs. 30 minutes.
  • a highly active ADAS has a lifetime index of greater than about: 0.13, 0.14, 0.15, 0.16, 0.18, 0.2, 0.25, 0.3,0.35, 0.45, 0.5, 0.60, 0.70, 0.80, 0.90, 1.0 or more.
  • lifetime index is measured in an ADAS containing a functional GFP plasmid with a species-appropriate promoter in which GFP concentration is measured relative to number of ADAS, average number of plasmids per ADAS, and solution volume with a plate reader at 30 minutes and 24 hours.
  • the ADAS produces a protein, e.g., a heterologous protein.
  • high activity is assessed as a rate, amount, or duration of production of a protein or a rate of induction of expression of the protein (e.g., responsiveness of an ADAS to a signal).
  • the ADAS comprises a plasmid, the plasmid comprising 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 results in production of the heterologous protein.
  • the production of the heterologous protein is increased by at least 1 .6-fold in an ADAS, e.g., a highly active ADAS, that has been contacted with the inducer relative to an ADAS that has not been contacted with the inducer.
  • 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 an ADAS, e.g., a highly active ADAS, that has been contacted with the inducer.
  • the rate of production of the heterologous protein by a highly active ADAS reaches a target level within a particular duration following the contacting of the ADAS 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.
  • a protein e.g., a heterologous protein
  • high activity of an ADAS is assessed as a duration for which a protein is produced.
  • a highly active ADAS produces a protein (e.g. a 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 longer than 48 hours.
  • ADAS are derived from bacterial parent cells, as described herein.
  • the invention provides an ADAS and/or a composition comprising a plurality of ADAS derived from a parent bacterium having a reduction in a level, activity, or expression of a cell division topological specificity factor.
  • the invention provides a composition comprising a plurality of ADAS, wherein the ADAS do not comprise a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells.
  • the invention provides a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, and being produced by a process comprising: (a) making, providing, or obtaining a plurality of parent bacteria having a reduction in the level or activity of a cell division topological specificity factor; (b) exposing the parent bacterium to conditions allowing the formation of a minicell, thereby producing the highly active ADAS; and (c) separating the ADAS from the parent bacterium, thereby producing a composition that is substantially free of viable bacterial cells.
  • the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 20% identity to an E. coli minE polypeptide (e.g., as encoded by SEQ ID NO: 1 ), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to a polypeptide encoded by SEQ ID NO: 1 .
  • the cell division topological specificity factor comprises the amino acid sequence encoded by SEQ ID NO: 1 .
  • the cell division topological specificity factor is a minE polypeptide. Exemplary species having minE polypeptides are provided in Rothfield et al., Nature Reviews Microbiology, 3: 959-968, 2005.
  • the parent bacterium is E. coli and the minE polypeptide is E. coli minE. In other embodiments, the parent bacterium is Salmonella typhimurium and the minE polypeptide is S. typhimurium minE.
  • the parent bacterium is an Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter,
  • the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 20% identity to a Bacillus subtilis DivIVA polypeptide (e.g., as encoded by SEQ ID NO: 5), e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to a polypeptide encoded by SEQ ID NO: 5.
  • the cell division topological specificity factor comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 5.
  • the cell division topological specificity factor is a DivIVA polypeptide.
  • Exemplary species having DivIVA polypeptides are provided in Rothfield et al., Nature Reviews Microbiology, 3: 959-968, 2005.
  • the parent bacterium is Bacillus subtilis and the cell division topological specificity factor is B. subtilis DivIVA.
  • the ADAS or parent bacterium having the reduction in a level or activity of the cell division topological specificity factor also has a reduction in a level of one or more Z- ring inhibition proteins.
  • the Z ring inhibition protein is a polypeptide having an amino acid sequence with at least 20% identity to an E. coli minC polypeptide (e.g., as encoded by SEQ ID NO:
  • the Z ring inhibition protein comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 2.
  • the Z ring inhibition protein is a minC polypeptide.
  • the Z ring inhibition protein is a polypeptide having an amino acid sequence with at least 20% identity to an E. coli minD polypeptide (e.g., as encoded by SEQ ID NO:
  • the Z ring inhibition protein comprises the amino acid sequence of the polypeptide encoded by SEQ ID NO: 3. In some embodiments, the Z ring inhibition protein is a minD polypeptide.
  • the ADAS or parent bacterium has a reduction in the level, activity, or expression of at least two Z-ring inhibition proteins. In some embodiments, the ADAS or parent bacterium has a reduction in expression of a minC polypeptide and a minD polypeptide. In some embodiments, the ADAS or parent bacterium has a reduction in expression of a minC polypeptide, a minD polypeptide, and a minE polypeptide, e.g., a deletion of the minCDE operon (AminCDE).
  • a reduction in the level, activity, or expression of a cell division topological specificity factor or a Z-ring inhibition protein is achieved using any suitable method.
  • the reduction in the level or activity is caused by a loss-of-function mutation, e.g., a gene deletion.
  • the loss-of-function mutation is an inducible loss-of-function mutation and loss of function is induced by exposing the parent cell to an inducing condition, e.g., the inducible loss-of- function mutation is a temperature-sensitive mutation and wherein the inducing condition is a temperature condition.
  • the parent cell has a deletion of the minCDE operon (AminCDE) or homologous operon.
  • the parent bacterial cell has one or more genetic loss-of-function alterations that stabilize ADAS derived from the cell (e.g., result in increased stability in ADAS derived from the cell).
  • the parent bacterial cell has been modified to reduce enzymatic activity.
  • ADAS stability is improved through genetic deletion of one or more enzymes.
  • the parent cell comprises one or more genetic loss-of-function alterations that reduce or abrogate enzymatic and/or lytic activity (e.g., reduce or abrogate an enzymatic or lytic activity carried out by a protein encoded by a gene comprising the genetic loss-of- function alteration).
  • the parent cell comprises a genomic deletion that reduces or abrogates enzymatic and/or lytic activity (e.g., reduces or abrogates an enzymatic or lytic activity carried out by a protein encoded by the deleted genomic region).
  • the parent bacterial cell comprises one or more genomic deletions reducing activity of one or more endopeptidases, cell wall lytic enzymes, and/or autolytic enzymes.
  • the parent cell comprises a genetic loss-of-function (e.g., a genomic deletion) from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, CwIK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ and combinations thereof.
  • a genetic loss-of-function e.g., a genomic deletion
  • the parent cell comprises a genetic loss-of-function in lytC.
  • the parent cell of the invention may comprise a deletion as shown by comparison of SEQ ID NOs: 8 and 9.
  • the parent 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 to lytC may comprise deletion of all or a portion of the coding region of the gene.
  • the parent cell comprises a genetic loss-of-function in sigF.
  • a wild-type parent cell comprises a sigF sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4
  • the parent cell of the invention may comprise a deletion as shown by comparison of SEQ ID NOs: 4 and 5.
  • the parent cell may comprise a genomic sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.
  • the loss of function alteration to sigF may comprise deletion of all or a portion of the coding region of the gene.
  • a protein e.g., a protein with enzymatic and/or lytic activity, e.g., a lytic enzyme
  • a protein with enzymatic and/or lytic activity e.g., a lytic enzyme
  • the parent cell contains the genetic loss-of-function in lytC.
  • the parent cell contains the genomic deletion of lytC.
  • the parent bacterial cell comprises at least one genetic loss-of- function (e.g. genomic deletion) of gene a encoding a lytic enzyme, a gene impacting a sporulation mechanism, or a cell division topological factor and combinations thereof (e.g., comprises two or more genetic loss-of-function alterations impacting one or more lytic enzymes, one or more genes impacting a sporulation mechanism, and/or one or more cell division topological factors).
  • a genetic loss-of- function e.g. genomic deletion
  • a genetic loss-of-function e.g. genomic deletion
  • a cell division topological factor e.g., comprises two or more genetic loss-of-function alterations impacting one or more lytic enzymes, one or more genes impacting a sporulation mechanism, and/or one or more cell division topological factors.
  • the parent bacterial cell comprises the genetic loss-of-function (e.g. genomic deletion) of lytC, sigF, and divIVa. In some embodiments, the parent bacterial cell comprises genomic deletions of lytC, sigF, and divIVa.
  • the parent bacterial cell is a gram-positive bacterial cell.
  • the parent bacterial cell is Bacillus subtilis or is of the Bacillus genus (e.g., is a Bacillus species).
  • the parent bacterial cell is of the Lactobacillus genus (e.g., is a Lactobacillus species). In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell.
  • the parent bacterial cell is a bacterium in the genus Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomona
  • the parent cell comprises a gene deletion from the group consisting of SPp, skin, PBSX, prophage 1 , pks::cat, prophage 3, and combinations thereof (Westers et al., Mol. Biol. Evol. 20(12):2076-2090, 2003). Additional deletions of genomic sections in parental bacterial cells can lead to the reduction of many lytic elements through the removal of prophage and prophagelike segments. These deletions are not imperative, as certain lytic enzyme deletions or loss-of- function mutations are (e.g., lytC), to establish intact and stable ADAS.
  • lytic enzyme deletions or loss-of- function mutations are (e.g., lytC), to establish intact and stable ADAS.
  • the parent cell comprises a gene deletion or a loss-of-function alteration (e.g., loss-of-function mutation) that interrupts sporulation (e.g., decreases or abrogates sporulation).
  • the loss-of-function alteration is selected from the group consisting of sigF, sigE, spollAA, spollD, bofA, spoVE, spolVFB, dacB, dapA, dapB, spollGA, spollM, spollR, spoOA and combinations thereof. While genomic deletions used to interrupt sporulation and genomic deletions used to reduce enzymatic activity in ADAS are not necessary or dependent on each other, each provides unique benefits to ADAS formation and use.
  • parent cells provided herein comprise both one or more alterations (e.g., loss-of-function mutations) that reduce enzymatic activity and one or more alterations (e.g., loss-of-function mutations) that interrupt sporulation.
  • an ADAS provided by the invention includes a cargo contained in the interior of the ADAS.
  • a cargo is any moiety disposed in the interior of an ADAS (e.g., encapsulated by the ADAS) or conjugated to the surface of the ADAS.
  • the cargo comprises a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP) or a combination of the foregoing.
  • the cargo is delivered by a secretion system (e.g., T3SS). In other aspects, the cargo is not delivered by a T3SS.
  • the nucleic acid is a DNA, an RNA, or a plasmid.
  • the nucleic acid e.g., DNA, RNA (e.g., mRNA, ASO, circular RNA, siRNA, shRNA, tRNA, dsRNA, or a combination thereof), or plasmid
  • the protein is transcribed and/or translated in the ADAS.
  • the nucleic acid inhibits translation of a protein or polypeptide, e.g., is an siRNA or an antisense oligonucleotide (ASO).
  • the cargo is an agent that can modulate the microbiome of the target organism (e.g., a human, animal, plant, or fungi microbiome), e.g., a polysaccharide,, an amino acid, an anti-microbial agent (e.g., e.g., an anti-infective or antimicrobial peptide, protein, and/or natural product), a short chain fatty acid, or a combination thereof.
  • the agent that can modulate the host microbiome is a probiotic agent.
  • the cargo is an enzyme.
  • the enzyme alters a substrate to produce a target product.
  • the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in a target cell or environment to which the ADAS is delivered.
  • the cargo is modified for improved stability compared to an unmodified version of the cargo.
  • “Stability” of a cargo is a unitless ratio of half-life of unmodified cargo and modified cargo half-life, as measured in the same environmental conditions.
  • the environment is experimentally controlled, e.g., a simulated body fluid, RNAse free water, cell cytoplasm, extracellular space, or “ADAS plasm” (i.e., the content of the interior volume of an ADAS, e.g., after lysis).
  • ADAS plasm i.e., the content of the interior volume of an ADAS, e.g., after lysis.
  • it is an agricultural environment, e.g., variable field soil, river water, or ocean water.
  • the environment is an actual or simulated: animal gut, animal skin, animal reproductive tract, animal respiratory tract, animal blood stream, or animal extracellular space.
  • the ADAS does not substantially degrade the cargo.
  • the cargo comprises a protein.
  • the protein has stability greater than about: 1.01 , 1.1 , 10, 100, 1000, 10000, 100000, 100000, 10000000 in cell cytoplasm or other environments.
  • the protein can be any protein, including growth factors; enzymes; hormones; immune-modulatory proteins; antibiotic proteins, such as antibacterial, antifungal, insecticidal proteins, etc.; targeting agents, such as antibodies or nanobodies, etc.
  • the protein is a hormone, e.g., paracrine, endocrine, autocrine.
  • the cargo comprises a plant hormone, such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.
  • a plant hormone such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.
  • the cargo is an anti-inflammatory agent, e.g., a cytokine (e.g., a heterologously expressed anti-inflammatory cytokine or mutein thereof (e.g., IL-10, TGF-Beta, IL-22, IL-2) or 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).
  • a cytokine e.g., a heterologously expressed anti-inflammatory cytokine or mutein thereof (e.g., IL-10, TGF-Beta, IL-22, IL-2) or 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
  • the cargo is an immune modulator.
  • Immune modulators include, e.g., immune stimulators; checkpoint inhibitors (e.g., inhibitors of PD-1 , PD-L1 , or CTLA-4); chemotherapeutic agents; immune suppressors; antigens; super antigens; and small molecules (e.g., cyclosporine A, cyclic dinucleotides (CDNs), or STING agonists (e.g., MK-1454)).
  • the immune modulator is a moiety that induces tolerance in a subject, e.g., an allergen, a self-antigen (e.g., a disease-associated self-antigen), or a microbe-specific antigen.
  • the immune modulator is a vaccine, e.g., an antigen from a pathogen (e.g., a virus (e.g., a viral envelope protein) or a bacteria).
  • the antigen is a cancer neoantigen.
  • the pathogen is a coronavirus, e.g., SARS-CoV-2.
  • the cargo an adjuvant, e.g., an immunomodulatory molecule or a molecule that alters the compartmentalization, presentation, or profile of one or more co-stimulatory molecules associated with a vaccine antigen.
  • the adjuvant is an activator of an immune pathway upstream of a desired immune response (e.g., an activator of an innate immune pathway upstream of an adaptive immune response).
  • the adjuvant enhances the presentation of an antigen on an immune cell or immune moiety (e.g., MHO class 1 ) in the target organism.
  • the adjuvant is listeriolysin O (LLO).
  • an ADAS comprises an antigen and one or more adjuvants.
  • the cargo is an agent for treatment or prevention of a cancer, e.g., an agent that decreases the likelihood that a patient will develop a cancer or an agent that treats a cancer (e.g., an agent that increases progression-free survival and/or overall survival in an individual having a cancer).
  • a cancer e.g., an agent that decreases the likelihood that a patient will develop a cancer or an agent that treats a cancer (e.g., an agent that increases progression-free survival and/or overall survival in an individual having a cancer).
  • Agents for the prevention of cancer include, but are not limited to anti-inflammatory agents and growth inhibitors.
  • Agents for the treatment of cancer include, but are not limited to anti-inflammatory agents, growth inhibitors, chemotherapy agents, immunotherapy agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neo-antigens)), cancer vaccines (e.g., vaccines comprising a cancer neo-antigen), agents that induce autophagy (e.g., activators such as listeria-lysin-o), cytotoxins, inflammasome inhibiting agents, immune checkpoint inhibitors (e.g., inhibitors of PD-1 , PD-L1 , or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.
  • cancer antigens e.g., cancer neo-antigens
  • cancer vaccines e.g., vaccines comprising a cancer neo-antigen
  • the ADAS therapeutic composition is administered by oral, intravenous, intradermal, intramuscular, intraperitoneal, peritumoral, intranasal, intraocular, or rectal, and/or subcutaneous administration.
  • the ADAS is administered by oral, intravenous, intramuscular, and/or subcutaneous administration.
  • the ADAS is administered once, twice, three times, four times or more to the subject.
  • the ADAS dosage is at least 1 x10 5 , 1 x10 6 , 1 x10 7 , 1 x10 8 , 5x10 8 , 6x10 8 , 8x10 8 , 1 x10 9 , 2x10 9 , 4x10 9 , 6x10 9 , 8x10 9 , or 1 x10 10 , e.g., dosing comprises administering at least 1 x10 5 , 1 x10 6 , 1 x10 7 , 1 x10 s , 5x10 8 , 6x10 8 , 8x10 8 , 1 x10 9 , 2x10 9 , 4x10 9 , 6x10 9 , 8x10 9 , or 1 x10 10 ADAS to the subject.
  • the cargo is an enzyme.
  • the enzyme is an enzyme that performs a catalytic activity in a target cell or organism (e.g., in a human, animal, plant, or fungi, or insect).
  • the catalytic activity is extracellular matrix (ECM) digestion (e.g., the enzyme is hyaluronidase and the catalytic activity is ECM digestion) or removal of toxins.
  • ECM extracellular matrix
  • the enzyme is an enzyme replacement therapy, e.g., is phenylalanine hydroxylase.
  • the enzyme is a UDP-glucuronosyltransferase.
  • the enzyme has hepatic enzymatic activity (e.g., porphobilinogen deaminase (PBGD), e.g., human PBGD (hPBGD)).
  • PBGD porphobilinogen deaminase
  • hPBGD human PBGD
  • the enzyme is a protease, oxidoreductase, or a combination thereof.
  • the enzyme alters a substrate to produce a target product.
  • the substrate is present in the ADAS and the target product is produced in the ADAS.
  • the substrate is present in a target cell or environment to which the ADAS is delivered.
  • the enzyme is diadenylate cyclase A
  • the substrate is ATP
  • the target product is cyclic-di-AMP.
  • the enzyme is chemically conjugated to the ADAS membrane, optionally via a linker to the exterior membrane.
  • the cargo is a nucleic acid that encodes any of the enzymes described herein.
  • the cargo is an agent that activates or inhibits autophagic processes (e.g., an activator such as listeria-lysin-o or an inhibitor such as IcsB).
  • an activator such as listeria-lysin-o or an inhibitor such as IcsB.
  • the cargo is an anti-infective agent, e.g., an anti-microbial agent, e.g., an anti-infective or antimicrobial peptide, protein, and/or natural product.
  • an anti-infective agent e.g., an anti-microbial agent, e.g., an anti-infective or antimicrobial peptide, protein, and/or natural product.
  • the cargo is a protein that modulates host transcriptional response e.g., a transcription factor; a protein that promotes host cell growth, e.g., a growth factor; or a protein that inhibits protein function, e.g., a nanobody.
  • the transcription factor is a human transcription factor.
  • the cargo is an RNA, such as circular RNA, mRNA, siRNA, shRNA, ASO, tRNA, dsRNA, or a combination thereof.
  • the RNA has stability greater than about: 1 .01 , 1 .1 ,10, 100, 1000, 10000, 100000, 100000, 10000000, e.g., in ADAS plasm.
  • the RNA cargo can be stabilized, in certain embodiments, e.g., with an appended step-loop structure, such as a tRNA scaffold.
  • a tRNA scaffold for example, non-human tRNALys3 and E. coli tRNAMet (Nat. Methods, Ponchon 2007).
  • RNA can also be stabilized where the ADAS is obtained from a parental strain null (or hypomorphic) for one or more ribonucleases.
  • the RNA is a protein-coding mRNA.
  • the protein-coding mRNA encodes an enzyme (e.g., and enzyme that imparts hepatic enzymatic activity, such as human PBGD (hPBGD) mRNA) or an antigen, e.g., that elicits an immune response (such as eliciting a potent and durable neutralizing antibody titer), such as mRNA encoding CMV glycoproteins gB and/or pentameric complex (PC)).
  • the RNA is a small non-coding RNA, such as shRNA, ASO, tRNA, dsRNA, or a combination thereof.
  • the ADAS provided by the invention includes cargo comprising at least one component of a gene editing system.
  • Components of a “gene editing system” include (or encode) proteins (or nucleic acids encoding said proteins) that can, with suitable associated nucleic acids, modify a DNA sequence of interest, such as a genomic DNA sequence, whether e.g., by insertion or deletion of a sequence of interest, or by altering the methylation state of a sequence of interest, as well as nucleic acids associated with the function of such proteins, e.g., guide RNAs.
  • Exemplary gene editing systems include those based on a Cas system, such as Cas9, Cpf 1 or other RNA-targeted systems with their companion RNA (e.g., sequence-complementary CRISPR guide RNA), as well as Zinc finger nucleases and TAL-effectors conjugated to nucleases.
  • a Cas system such as Cas9, Cpf 1 or other RNA-targeted systems with their companion RNA (e.g., sequence-complementary CRISPR guide RNA), as well as Zinc finger nucleases and TAL-effectors conjugated to nucleases.
  • ADAS include DNA as the cargo, including as a plasmid, optionally wherein the DNA comprises a protein-coding sequence.
  • Exemplary DNA cargo includes, in certain embodiments, a plasmid encoding an RNA sequence of interest (see examples above), e.g., which may be flanked on each side by an tRNA insert.
  • ADAS producing e.g., driving FTZ overexpression, genome degrading exonucleases
  • longevity plasmids ATP synthase expressing, rhodopsin- expressing
  • those expressing stabilized non-coding RNA, tRNA, IncRNA expressing secretion system tag proteins, NleE2 effector domain and localization tag
  • secretion systems T3/4SS, T5SS, T6SS logic circuits, conditionally expressed secretion systems; and combinations thereof.
  • a logic circuit includes inducible expression or suppression cassettes, such as IPTG- inducible Plac promoter and the hrpR portion of the AND gate, and, for example, the heat-induced promoter pL (from phage lambda, which is usually suppressed by a thermolabile protein) and the hrpS portion of the AND gate.
  • inducible expression or suppression cassettes such as IPTG- inducible Plac promoter and the hrpR portion of the AND gate, and, for example, the heat-induced promoter pL (from phage lambda, which is usually suppressed by a thermolabile protein) and the hrpS portion of the AND gate.
  • Plasmids containing the IPTG-inducible promoter PLac and heat-induced promoter pL, both of which induce the expression of RAJ11 sRNA, can then be used.
  • the output would then be RFP expression, which is seen in response to either input.
  • ADAS provided by the invention include a transporter in the membrane.
  • the transporter is specific for glucose, sodium, potassium, a metal ion, an anionic solute, a cationic solute, or water.
  • the membrane of an ADAS provided by the invention comprises an enzyme.
  • the enzyme is a protease, oxidoreductase, or a combination thereof.
  • the enzyme is chemically conjugated to the ADAS membrane, optionally via a linker to the exterior membrane.
  • F. ADAS comprising a secretion system
  • an ADAS provided by the invention comprises 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 a cargo from the cytoplasm of a bacterial cell (or, for example, an ADAS derived therefrom) into: the extracellular space, the periplasmic space of a gram-negative bacterium, or the intracellular space of another cell.
  • the bacterial secretion system works by an active (e.g., ATP-dependent or PMF-dependent) process, and in certain embodiments the bacterial secretion system comprises a tube or a spike spanning the host cell (or ADAS) to a target cell. In other embodiments the bacterial secretion system is a transmembrane channel.
  • Exemplary bacterial secretion systems include T3SS and T4SS (and T3/T4SS, as defined, below), which are tube-containing structures where the cargo traverses through the inside of a protein tube and T6SS, which carries the cargo at the end of a spike.
  • Other exemplary bacterial secretion systems include T1 SS, T2SS, T5SS, T7SS, Sec, and Tat, which are transmembrane.
  • the disclosure features an achromosomal dynamic active system (ADAS) derived from a parent bacterial cell, the ADAS comprising a bacterial Type 3 secretion system (T3SS) that is heterologous to the parent bacterial cell.
  • ADAS achromosomal dynamic active system
  • T3SS Type 3 secretion system
  • the parent bacterial cell is a Gram-negative bacterial cell.
  • the parent bacterial cell does not comprise an endogenous T3SS.
  • the parent bacterial cell is an E. co// cell. In some embodiments, the E. co// cell is a Nissle E. co// cell.
  • the parent bacterial cell is a probiotic cell.
  • 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.
  • the Salmonella T3SS is a Salmonella enterica T3SS.
  • the Vibrio T3SS is a Vibrio parahaemolyticus T3SS.
  • the Escherichia T3SS is an enteropathogenic E. co// (EPEC) T3SS.
  • the Yersinia T3SS is a Yersinia enterocolitica T3SS.
  • the Shigella T3SS is a Shigella flexneri T3SS.
  • the parent bacterial cell comprises one or more heterologous nucleotide sequences encoding the components of the T3SS.
  • the one or more nucleotide sequences encoding the components of the T3SS are carried on a vector.
  • the parent bacterial cell has been transiently transformed with the vector.
  • the parent bacterial cell has been stably transformed with the vector.
  • the parent bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS.
  • the moiety is a transcriptional activator of the one or more heterologous nucleotide sequences encoding a component of theT3SS.
  • the disclosure features an achromosomal dynamic active system (ADAS) derived from a parent bacterial cell, the ADAS comprising a bacterial Type 3 secretion system (T3SS) that is endogenous to the parent bacterial cell, wherein the parent bacterial cell has been modified to reduce the level of an endogenous protein or polypeptide capable of being secreted by the T3SS.
  • ADAS achromosomal dynamic active system
  • T3SS Type 3 secretion system
  • the parent cell bacterial has been modified by deleting a transcriptional activator of the endogenous protein or polypeptide capable of being secreted by the T3SS.
  • the parent bacterial cell is a Gram-negative bacterial cell.
  • the parent bacterial cell is a Salmonella species, a Vibrio species, an Escherichia species, a Yersinia species, or a Shigella species.
  • the Salmonella species is Salmonella enterica.
  • the Vibrio species is a Vibrio parahaemolyticus.
  • the Escherichia species is an enteropathogenic E. coli (EPEC).
  • the Yersinia species is Yersinia enterocolitica.
  • the Shigella species is Shigella flexneri.
  • the parent bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS.
  • the moiety is a transcriptional activator of a nucleotide sequence encoding a component of the T3SS.
  • the parent bacterial cell has been modified to reduce the level of a negative regulator of a component of the T3SS.
  • a chromosomal locus encoding the negative regulator has been deleted from the parent bacterial cell.
  • the parent bacterial cell has been modified to reduce the level of one or more of LPS; a metabolically non-essential protein; a toxin not associated with a T3SS; an endotoxin; a flagella; and a pillus.
  • the ADAS further comprises at least one cargo, wherein the T3SS is capable of delivering the cargo to a target cell.
  • the delivery is to the cytoplasm of the target cell.
  • the cargo is a protein or a polypeptide.
  • the cargo is endogenously secreted by the T3SS.
  • the ADAS or the parent bacterial cell has been modified to increase the level of the cargo in the ADAS.
  • the cargo is not endogenously secreted by the T3SS.
  • the cargo is endogenously secreted by a T3SS from a species other than the ADAS T3SS species.
  • the cargo is endogenously secreted by a Type 4 secretion system (T4SS) or a Type 6 secretion system (T6SS).
  • T4SS Type 4 secretion system
  • T6SS Type 6 secretion system
  • the cargo has been modified for delivery by the T3SS.
  • the cargo is an enzyme, a DNA-modifying agent, a chromatinremodeling agent, a gene editing agent, a nuclear targeting agent, a binding agent, an immunogenic agent, or a toxin.
  • the enzyme is a metabolic enzyme.
  • the gene editing agent is a component of a CRISPR system.
  • the nuclear targeting agent is a transcription factor.
  • the binding agent is an antibody or an antibody fragment.
  • the binding agent is a VHH molecule.
  • the immunogenic agent is an immunostimulatory agent.
  • the immunogenic agent is an immunosuppressive agent.
  • the cargo has been modified by addition of a secretion signal.
  • the 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 one of the above aspects.
  • the ADAS comprises a cargo, wherein the cargo comprises a moiety that directs export by the bacterial secretion system, e.g., in some embodiments the moiety is Pho/D, Tat, or a synthetic peptide signal.
  • the ADAS provided by the invention are two-membrane ADAS.
  • the two-membrane ADAS further comprises a bacterial secretion system.
  • the bacterial secretion system is selected from T3SS, T4SS, T3/4SS, or T6SS, optionally wherein the T3SS, T4SS, T3/4SS, or T6SS have an attenuated or non-functional effector that does not affect fitness of a target cell.
  • ADAS provided by the invention include a bacterial secretion system.
  • the bacterial secretion system is capable of exporting a cargo across the ADAS outer membrane into a target cell, such as an animal, fungal, bacterial, or plant cell, such as T3SS, T4SS, T3/T4SS, or T6SS.
  • a target cell such as an animal, fungal, bacterial, or plant cell, such as T3SS, T4SS, T3/T4SS, or T6SS.
  • the bacterial secretion system is a T3/4SS.
  • a “T3/4SS” is a secretion system based on T3SS or T4SS, including hybrid systems as well as unmodified versions, which forms a protein tube between a bacterium (or ADAS) and a target cell, connecting the two and delivering one or more effectors.
  • the target cell can be an animal, plant, fungi, or bacteria.
  • a T3/4SS includes an effector, which may be a modified effector.
  • T3SS systems include the Salmonella SPI-1 system, the EHEC coli ETT1 system, the Xanthamonas Citri/Campestri 3SS system, and the Pseudomonas syringae T3SS system.
  • T4SS systems include the Agrobacterium Ti plasmid system, Helicobacter pylori ASS.
  • the T3/4SS has modified effector function, e.g., an effector selected from SopD2, SopE, Bop, Map, Tir, EspB, EspF, NleC, NleH2, or NleE2.
  • the modified effector function is for intracellular targeting such as translocation into the nucleus, golgi, mitochondria, actin, microvilli, ZO-1 , microtubules, or cytoplasm.
  • the modified effector function is nuclear targeting based on NleE2 derived from E. Coli.
  • the modified effector function is for filopodia formation, tight junction disruption, microvilli effacement, or SGLT-1 inactivation.
  • an ADAS provided by the invention comprising a bacterial secretion system comprises a T6SS.
  • the T6SS in its natural host, targets a bacterium and contains an effector that kills the bacteria.
  • the T6SS is derived from P. putida K1 -T6SS and, optionally, wherein the effector comprises the amino acid sequence of Tke2 (Accession AUZ59427.1 ), or a functional fragment thereof.
  • the T6SS in its natural host, targets a fungi and contains an effector that kills fungi, e.g., the T6SS is derived from Serratia Marcescens and the effectors comprise the amino acid sequences of: Tfe1 (Genbank: SMDB11_RS05530) or Tfe2 (Genbank: SMDB11_RS05390).
  • the bacterial secretion system is capable of exporting a cargo extracellularly.
  • the bacterial secretion system is T1 SS, T2SS, T5SS, T7SS, Sec, or Tat.
  • ADAS lacking proteases, RNases, and/or LPS
  • the invention provides a composition further comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS have a reduced protease level or activity relative to an ADAS produced from a wild-type parent bacterium.
  • ADAS e.g., highly active ADAS
  • the ADAS is produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one protease.
  • the invention provides a composition comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS have a reduced RNAse level or activity relative to an ADAS produced from a wild-type parent bacterium.
  • ADAS e.g., highly active ADAS
  • the ADAS is produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one RNAse.
  • the RNase is an endoribonuclease or an exoribonuclease.
  • the invention provides a composition comprising a plurality of ADAS, wherein the ADAS has been modified to have reduced lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the modification is a mutation in Lipid A biosynthesis myristoyltransferase (msbB).
  • an ADAS provided by the invention lacks one or more metabolically non-essential proteins.
  • a “metabolically non-essential protein” non-exhaustively includes: fimbriae, flagella, undesired secretion systems, transposases, effectors, phage elements, or their regulatory elements, such as flhC or OmpA.
  • an ADAS provided by the invention lacks one or more of an RNAse, a protease, or a combination thereof, and, in particular embodiments, lacks one or more endoribonucleases (such as RNAse A, RNAse h, RNAse III, RNAse L, RNAse PhyM) or exoribonucleases (such as RNAse R, RNAse PH, RNAse D); or serine, cysteine, threonine, aspartic, glutamic and metallo-proteases; or a combination of any of the foregoing.
  • endoribonucleases such as RNAse A, RNAse h, RNAse III, RNAse L, RNAse PhyM
  • exoribonucleases such as RNAse R, RNAse PH, RNAse D
  • H. ADAS comprising a targeting moiety
  • the invention provides a composition comprising a plurality of ADAS, wherein the ADAS comprises a targeting moiety.
  • the targeting moiety is a nanobody, a carbohydrate binding protein, or a tumor-targeting peptide.
  • the targeting moiety is an endogenous surface ligand of the parent cell (e.g., a surface ligand that is inherited by the ADAS).
  • the targeting moiety is an exogenous ligand (e.g., an exogenous tissue-targeting ligand) that is added to the ADAS using any of the methods for modifying ADAS described herein.
  • the targeting moiety promotes tissue-associated targeting of the ADAS to a tissue type or cell type.
  • the nanobody is a nanobody directed to a tumor antigen, such as HER2, PSMA, or VEGF-R.
  • a tumor antigen such as HER2, PSMA, or VEGF-R.
  • the carbohydrate binding protein is a lectin, e.g. Mannose Binding Lectin (MBL).
  • the tumor-targeting peptide is an RGD motif or CendR peptide.
  • the invention provides a composition comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS is derived from a parent bacterium that is a mammalian pathogen or a mammalian commensal bacterium.
  • ADAS e.g., highly active ADAS
  • the mammalian commensal bacterium is a Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus, or Actinomyces species or the mammalian pathogenic bacterium is enterohemorrhagic Escherichia coli (EH EC), Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori.
  • EH EC enterohemorrhagic Escherichia coli
  • Salmonella typhimurium Shigella flexneri
  • Yersinia enterolitica or Helicobacter pylori.
  • the invention provides a composition comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS is derived from a parent bacterium that is a plant pathogen or a plant commensal bacterium.
  • ADAS e.g., highly active ADAS
  • the plant commensal bacterium is Bacillus subtilis or Psuedomonas putida or the plant pathogenic bacterium is a Xanthomonas species or Pseudomonas syringae.
  • the invention provides a composition comprising a plurality of ADAS (e.g., highly active ADAS), wherein the ADAS is derived from an auxotrophic parent bacterium, i.e., a parent bacterium that is unable to synthesize an organic compound required for growth. Such bacteria are able to grow only when the organic compound is provided.
  • ADAS e.g., highly active ADAS
  • An ADAS in certain embodiments, includes a functional ATP synthase and, in some embodiments, a membrane embedded proton pump.
  • ADAS can be derived from different sources including: a parental bacterial strain (“parental strain”) engineered or induced to produce genome-free enclosed membrane systems, a genome-excised bacterium, a bacterial cell preparation extract (e.g., by mechanical or other means), or a total synthesis, optionally including fractions of a bacterial cell preparation.
  • a highly active ADAS has an ATP synthase concentration of at least: 1 per 10000 nm 2 , 1 per 5000 nm 2 , 1 per 3500 nm 2 , 1 per 1000 nm 2 .
  • ADAS provided by the invention can include a variety of additional components, including, for example, photovoltaic pumps, retinals and retinal-producing cassettes, metabolic enzymes, targeting agents, cargo, bacterial secretion systems, and transporters, including combinations of the foregoing, including certain particular embodiments described, below.
  • the ADAS lack other elements, such as metabolically non-essential genes and/or certain enzymes, nucleases or proteases.
  • an ADAS provided by the invention comprises an ATP synthase, optionally lacking a regulatory domain, such as lacking an epsilon domain.
  • Deletion can be accomplished by a variety of means.
  • the deletion in by inducible deletion of the native epsilon domain.
  • deletion can be accomplished by flanking with LoxP sites and inducible Cre expression or CRISPR knockout, or be inducible (place on plasmid under a tTa tet transactivator in an ATP synthase knockout strain)
  • An ADAS in some embodiments, can include a photovoltaic proton pump.
  • the photovoltaic proton pump is a proteorhodopsin.
  • the proteorhodopsin comprises the amino acid sequence of proteorhodopsin from the uncultured marine bacterial clade SAR86, GenBank Accession: AAS73014.1 .
  • the photovoltaic proton pump is a gloeobacter rhodopsin.
  • the photovoltaic proton pump is a bacteriorhodopsin, deltarhodopsin, or halorhodopsin from Halobium salinarum Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica, or Haloarcula marismortui.
  • an ADAS provided by the invention further comprising retinal.
  • an ADAS provided by the invention further comprises a retinal synthesizing protein (or protein system), or a nucleic acid encoding the same.
  • an ADAS provided by the invention further comprises one or more glycolysis pathway proteins.
  • the glycolysis pathway protein is a phosphofructokinase (Pfk-A), e.g., comprising the amino acid sequence of UniProt accession P0A796 or a functional fragment thereof.
  • the glycolysis pathway protein is triosephosphate isomerase (tpi), e.g., comprising the amino acid sequence of UniProt accession P0A858, or a functional fragment thereof.
  • compositions or preparations that contain an ADAS provided by the invention, including, inter alia, a highly active ADAS preparation provided by the invention or an ADAS preparation wherein a plurality of individual ADAS lack a cell division topological specificity factor, e.g., lack a minE gene product, and optionally wherein the ADAS preparation is substantially free of viable cells.
  • compositions provided by the invention or “a composition provided by the invention”, or the like and can contain any ADAS provided by the invention and any combination of ADAS provided by the invention.
  • a composition provided by the invention contains at least about: 80, 81 , 82, 83, 84, 85, 90, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 %, or more ADAS that contain a bacterial secretion system.
  • the bacterial secretion system is one of T3SS, T4SS, T3/4SS, or T6SS.
  • a composition provided by the invention contains ADAS that contain a T3SS, where the ADAS comprise a mean T3SS membrane density greater than 1 in about: 40000, 35000,30000, 25000, 19600, 15000, 10000, or 5000 nm 2 .
  • the ADAS is derived from a S. typhimurium or E. coli parental strain.
  • compositions provided by the invention contain ADAS that contain a T3SS, where the ADAS comprise a mean T3SS membrane density greater than 1 in about: 300000, 250000, 200000, 150000, 100000, 50000, 20000, 10000, 5000 nm 2 .
  • the ADAS is derived from an Agrobacterium tumefaciens parental strain.
  • the invention provide 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, including T3, T4, T3/4SS, T6SS, and optionally including one or more of: exogenous carbohydrates, phosphate producing synthases, light responsive proteins, import proteins, enzymes, functional cargo, organism-specific effectors, fusion proteins.
  • compositions and preparations provided by the invention can contain any ADAS provided by the invention, such as highly active ADAS or ADAS that lack a minE gene product.
  • compositions provided by the invention can be prepared in any suitable formulation.
  • the formulation can be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosolized, or nebulized administration.
  • a formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.
  • an ADAS composition described herein comprises 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.
  • CFU/mL colony-forming units
  • the invention provides an ADAS composition wherein the ADAS are lyophilized and reconstituted, and wherein the reconstituted ADAS have an ATP concentration that is at least 90% of the ATP concentration of an ADAS that has not been lyophilized, e,g, at least 95%, 98%, or at least equal to the ATP concentration of an ADAS that has not been lyophilized.
  • the invention provides an ADAS composition wherein the ADAS are stored, e.g., stored at 4°C, wherein after storage, the ADAS have an ATP concentration that is at least 90% of the ATP concentration of an ADAS that has not been stored, e.g., at least 95%, 98%, or at least equal to the ATP concentration of an ADAS that has not been stored.
  • the storage is 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 a year.
  • ADAS is preserved or otherwise in a “quiescent” state and then rapidly become activated.
  • the ADAS composition is formulated for delivery to an animal, e.g., formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosolized, or nebulized administration. In some embodiments, the ADAS composition is formulated for delivery to a plant.
  • the composition includes an adjuvant, e.g., a surfactant (e.g., a nonionic surfactant, a surfactant plus nitrogen source, an organo-silicone surfactant, or a high surfactant oil concentrate), a crop oil concentrate, a vegetable oil concentrate, a modified vegetable oil, a nitrogen source, a deposition (drift control) and/or retention agent (with or without ammonium sulfate and/or defoamer), a compatibility agent, a buffering agent and/or acidifier, a water conditioning agent, a basic blend, a spreader-sticker and/or extender, an adjuvant plus foliar fertilizer, an antifoam agent, a foam marker, a scent, or a tank cleaner and/or neutralizer.
  • the adjuvant is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (
  • the ADAS composition is formulated for delivery to an invertebrate, (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid). In some embodiments, the ADAS composition is formulated for delivery to an insect.
  • an invertebrate e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid).
  • the ADAS composition is formulated for delivery to an insect.
  • the composition is formulated as a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
  • the invention features a composition comprising a plurality of ADAS, wherein the ADAS comprise an enzyme and wherein the enzyme alters a substrate to produce a target product.
  • the substrate is present in the ADAS and the target product is produced in the ADAS.
  • the substrate is present in a target cell or environment to which the ADAS is delivered.
  • the enzyme is diadenylate cyclase A
  • the substrate is ATP
  • the target product is cyclic-di-AMP.
  • production of ADAS features a method for manufacturing a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, the method comprising (a) making, providing, or obtaining a plurality of parent bacteria having a reduction in the level or activity of a cell division topological specificity factor; (b) exposing the parent bacteria to conditions allowing the formation of a minicell, thereby producing the highly active ADAS; and (c) separating the highly active ADAS from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.
  • Parent bacteria include any suitable bacterial species from which an ADAS may be generated (e.g., species that may be modified using methods described herein to produce ADAS).
  • ADAS e.g., species that may be modified using 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, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizob
  • any of the ADAS compositions e.g., highly active ADAS compositions, described in Section I herein.
  • methods for making highly active ADAS are used.
  • methods for making ADAS lacking a cell division topological specificity factor and, optionally, lacking a Z-ring inhibition protein e.g., methods of making ADAS from AminCDE parent bacteria
  • methods for making any of the ADAS mentioned herein, wherein the ADAS comprises a cargo are used.
  • the ADAS (e.g., highly active ADAS) is made from a parental strain that is a plant bacterium, such as a plant commensal bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogen bacterium (e.g., Xanthomonas sp. or Pseudomonas syringae), or a bacterium that is capable of plant rhizosphere colonization and/or root nodulation, e.g., a Rhizobia bacterium.
  • a plant bacterium such as a plant commensal bacterium (e.g., Bacillus subtilis or Pseudomonas putida), a plant pathogen bacterium (e.g., Xanthomonas sp. or Pseudomonas syringae), or a bacterium that is capable of plant rhizosphere colon
  • the ADAS (e.g., highly active ADAS) is made from a parental strain that is a symbiont of an invertebrate, e.g., a symbiont of an arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid.
  • the invertebrate is a pest or a pathogen of a plant or of an animal.
  • the ADAS e.g., highly active ADAS
  • the ADAS is made from a parental strain that is capable of genetic transformation, e.g., Agrobacterium.
  • the ADAS (e.g., highly active ADAS) is made from a parent strain that is a human bacterium, such as a commensal human bacterium (e.g., E. coli, Staphylococcus sp., Bifidobacterium sp., Micrococcus sp., Lactobacillus sp., or Actinomyces sp.) or a pathogenic human bacterium (e.g., E scherichia coli EH EC, Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori), or an extremophile.
  • a human bacterium such as a commensal human bacterium (e.g., E. coli, Staphylococcus sp., Bifidobacterium sp., Micrococcus sp., Lactobacillus sp., or Acti
  • the ADAS and/or parent strain is a functionalized derivative of any of the foregoing, for example including a functional cassette, such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • a functional cassette such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • parent bacteria includes functionalized derivatives of any of the foregoing, for example including a functional cassette, such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • a functional cassette such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • an ADAS is derived from a parental strain engineered or induced to overexpress ATP synthase.
  • the ATP synthase is heterologous to the parental strain.
  • the parental strain is modified to express a functional F0F1 ATP synthase.
  • an ADAS provided by the invention is obtained from a parental strain cultured under a condition selected from: applied voltage (e.g., 37 mV), non-atmospheric oxygen concentration (e.g., 1 -5% O2, 5-10% O2, 10-15% O2, 25-30% O2), low pH (about: 4.5, 5.0, 5.5, 6.0, 6.5), or a combination thereof.
  • 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
  • ADAS of any one of the preceding claims which is made from an extremophile, including functionalized derivatives of any of the foregoing, for example including a functional cassette, such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • a functional cassette such as a functional cassette that induces the bacterium to do one or more of: secrete antimicrobials, digest plastic, secrete insecticides, survives extreme environments, make nanoparticles, integrate within other organisms, respond to the environment, and create reporter signals.
  • ADAS can be made with modified membranes, e.g., to improve the biodistribution of the ADAS upon administration to a target cell.
  • the membrane is modified to be less immunogenic or immunostimulatory in plants or animals.
  • the ADAS is obtained from a parental strain, wherein the immunostimulatory capabilities of the parental strain are reduced or eliminated through postproduction treatment with detergents, enzymes, or functionalized with PEG.
  • the ADAS is made from a parental strain and the membrane is modified through knockout of LPS synthesis pathways in the parental strain, e.g., by knocking out msbB.
  • the ADAS is made from a parental strain that produces cell wall-deficient particles through exposure to hyperosmotic conditions.
  • the methods include transforming a parental strain with an inducible DNAse system, such as the exol (NCBI GenelD: 946529) & sbcD (NCBI GenelD: 945049) nucleases, or the l-Ceul (e.g., Swissprot: P32761 .1 ) nuclease.
  • the methods include using a single, double, triple, or quadruple auxotrophic strain and having the complementary genes on the plasmid encoding the inducible nucleases.
  • the parental strain is cultured under a condition selected from: applied voltage (e.g., 37 mV), non-atmospheric oxygen concentration (e.g., 1 -5% O2, 5-10% O2, 10-15% O2, 25-30% O2), low pH (4.5-6.5), or a combination thereof.
  • 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
  • the parental strain lacks flagella and undesired secretion systems, optionally where the flagella and undesired secretion systems are removed using lambda red recombineering.
  • flagella control components are excised from the parental strain genome via, for example, insertion of a plasmid containing a CRISPR domain that is targeted towards flagella control genes, such as flh D and flhC.
  • the methods provided are for making a highly active ADAS, where an ADAS comprising a plasmid containing a rhodopsin-encoding gene is cultured in the presence of light.
  • the rhodopsin is proteorhodopsin from SAR86 uncultured bacteria, having the amino acid sequence of GenBank Accession: AAS73014.1 , or a functional fragment thereof.
  • the culture is supplemented with retinal.
  • the rhodopsin is proteorhodopsin and the plasmid additionally contains genes synthesizing retinal (such a plasmid is the pACYC-RDS plasmid from Kim et al., Microb Cell Fact, 2012).
  • the parental strain contains a nucleic acid sequence encoding a nanobody that is then expressed on the membrane of the ADAS.
  • the parental strain contains a nucleic acid sequence encoding one or more bacterial secretion system operons.
  • Exemplary plasmids include the Salmonella SPI-1 T3SS, the Shigella flexneri T3SS, the Agro Ti plasmid, and the P. putida K1 -T6SS system.
  • the parental strain comprises a cargo.
  • the parent strain contains a nucleic acid sequence encoding a set of genes that synthesize a small molecule cargo.
  • ADAS are purified from compositions (e.g., cultures) comprising viable bacteria, e.g., parental bacteria.
  • the invention features a method for manufacturing a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, the method comprising (a) making, providing, or obtaining a plurality of parent bacteria having a reduction in the level or activity of a cell division topological specificity factor; (b) exposing the parent bacteria to conditions allowing the formation of a minicell, thereby producing the ADAS; and (c) separating the ADAS from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.
  • Purification separates ADAS from viable parent bacterial cells, which are larger and contain a genome. Separating the highly active ADAS from the parent bacteria can be performed using a number of methods, as described herein. Exemplary methods for purification described herein include centrifugation, selective outgrowth, and buffer exchange/concentration processes.
  • ADAS compositions and methods of comparing such compositions, wherein the compositions are substantially free of parent bacterial cells and/or viable bacterial cells, e.g., have 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) per mL.
  • an ADAS composition that is substantially free of parent bacterial cells includes no bacterial cells.
  • Auxotrophic parental strains can be used to make ADAS provided by the invention. As described in more detail below, such manufacturing methods are useful for purification of the ADAS. For example, in some embodiments, following ADAS generation, parent bacterial cells are removed by growth in media lacking the nutrient (for example, amino acid) necessary for viability of the parent bacterium.
  • nutrient for example, amino acid
  • an ADAS provided by the invention is derived from a parental strain auxotrophic for at least 1 , 2, 3, 4, or more of: arginine (e.g., knockout in argA, such as strains JW2786-1 and NK5992), cysteine knockout in cysE (such as strains JW3582-2 and JM15), glutamine e.g., knockout in glnA (such as strains JW3841 -1 and M5004), glycine e.g., knockout in glyA (such as strains JW2535-1 and AT2457), Histidine e.g., knockout in hisB (such as strains JW2004-1 and SB3930), isoleucine e.g., knockout in ilvA (such as strains JW3745-2 and AB1255), leucine e.g., knockout in leuB (such as strains JW5807-2 and CV514), lys
  • the methods include using a single, double, triple, or quadruple auxotrophic parental strain, optionally wherein said parental strain further includes a plasmid expressing a ftsZ.
  • the 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 ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a), wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM.
  • the invention features a method for delivering an ADAS 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 are derived from a parent 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 lytic enzyme).
  • the target cell is, e.g., an animal cell, a plant cell, or a fungal cell.
  • the invention features a method for delivering a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the ADAS comprise a cargo, and the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a), wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly repetitive polypeptide,
  • the invention features a method for delivering a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADAS; and (b) contacting the target cell with the composition of step (a), wherein the ADAS are derived from a parent 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 lytic enzyme).
  • a cargo e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small
  • the invention features a method for delivering a cargo (e.g., a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)) to a target cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from a parent 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 lytic enzyme), the ADAS comprise a cargo, and the composition is substantially free of viable bacterial cells; and (b) contacting the target cell with the composition of step (a).
  • the ADAS are derived from a parent bacterium having a
  • the target cell to which the cargo is delivered is, e.g., an animal cell, a plant cell, or a fungal cell.
  • the invention features a method of modulating a state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the animal cell with the composition of step (a), whereby a state of the animal cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM.
  • the invention features a method of modulating a state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant cell with the composition of step (a), whereby a state of the plant cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM.
  • the invention features a method of modulating a state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), whereby a state of the insect cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM.
  • the invention features a method of modulating a state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the animal cell with the composition of step (a), whereby a state of the animal cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of modulating a state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant cell with the composition of step (a), whereby a state of the plant cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of modulating a state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the composition is substantially free of viable bacterial cells; and (b) contacting the insect cell with the composition of step (a), whereby a state of the insect cell is modulated, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of modulating a state of an animal cell, the method comprising: (a) providing a composition comprising a plurality of achromosomal dynamic active systems (ADAS); and (b) contacting the animal cell with the composition of step (a), whereby a state of the animal cell is modulated.
  • ADAS achromosomal dynamic active systems
  • the ADAS are derived from a parent 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 lytic enzyme).
  • the invention features a method of modulating a state of a plant cell, the method comprising: (a) providing a composition comprising a plurality of achromosomal dynamic active systems (ADAS); and (b) contacting the plant cell with the composition of step (a), whereby a state of the plant cell is modulated.
  • ADAS achromosomal dynamic active systems
  • the ADAS are derived from a parent 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 lytic enzyme).
  • the invention features a method of modulating a state of an insect cell, the method comprising: (a) providing a composition comprising a plurality of achromosomal dynamic active systems (ADAS); and (b) contacting the insect cell with the composition of step (a), whereby a state of the insect cell is modulated.
  • ADAS achromosomal dynamic active systems
  • the ADAS are derived from a parent 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 lytic enzyme).
  • the modulating is any observable change in the state (e.g., the transcriptome, proteome, epigenome, biological effect, or health or disease state) of the cell (e.g., an animal, plant, or insect cell) as measured using techniques and methods known in the art for such a measurement, e.g., methods to measure the level or expression of a protein, a transcript, an epigenetic mark, or to measure the increase or reduction of activity of a biological pathway.
  • modulating the state of the cell involves increasing a parameter (e.g., the level or expression of a protein, a transcript, or activity of a biological pathway) of the cell.
  • modulating the state of involves decreasing a parameter (e.g., the level or expression of a protein, a transcript, or activity of a biological pathway) of the cell.
  • 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 viable bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the plurality of ADAS is a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM.
  • the invention features a use of ADAS in the manufacture of a medicament for the treatment of an animal, a plant, insect or a fungi.
  • the invention features a use of ADAS in the manufacture of a medicament for the treatment of an animal, wherein the ADAS have an initial ATP concentration of at least 1 .25 mM and wherein the composition is substantially free of viable bacterial cells.
  • the invention features a use of ADAS in the manufacture of a medicament for the treatment of an animal, wherein the ADAS are derived from a parent 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 lytic enzyme) and wherein the composition is substantially free of viable bacterial cells.
  • ADAS derived from a parent 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 lytic enzyme) and wherein the composition is substantially free of viable bacterial cells.
  • 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 viable bacterial cells; and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of treating an animal in need thereof, the method comprising (a) providing a composition comprising a plurality of achromosomal dynamic active systems (ADAS); and (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.
  • ADAS achromosomal dynamic active systems
  • the ADAS are derived from a parent 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 lytic enzyme).
  • the animal in need of treatment has a disease, e.g., a cancer.
  • the ADAS carries a chemotherapy cargo or an immunotherapy cargo.
  • 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 viable bacterial cells; and (b) contacting the plant or a pest (e.g., an insect pest) thereof with an effective amount of the composition of step (a), thereby treating the plant, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of treating an plant in need thereof, the method comprising: (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and (b) contacting the plant or a pest (e.g., an insect pest) thereof with an effective amount of the composition of step (a), thereby treating the plant, wherein the ADAS are derived from a parent 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 lytic enzyme).
  • the ADAS are derived from a parent bacterium having a reduction in the level or activity of a cell division topological specificity factor.
  • the invention features a method of treating a plant in need thereof, the method comprising (a) providing a composition comprising a plurality of achromosomal dynamic active systems (ADAS); and (b) contacting the plant or a pest (e.g., an insect pest) thereof with an effective amount of the composition of step (a), thereby treating the plant.
  • ADAS achromosomal dynamic active systems
  • the ADAS are derived from a parent 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 lytic enzyme).
  • the invention provides methods of modulating a target cell.
  • the target cell can be any cell, including an animal cell (e.g., including humans and non-human animals, including farm or domestic animals, pests), a plant cell (including from a crop or a pest), a fungal cell, or a bacterial cell.
  • the cell is isolated, e.g., in vitro or, in other embodiments, within an organism, in vivo. These methods entail providing an ADAS provided by the invention or a composition provided by the invention with access to the target cell, in an effective amount.
  • the access to the target cell is either direct, e.g., where the target cell is modulated directly by the ADAS, such as by proximate secretion of some agent proximate to the target cell or injection of the agent into the target cell, or indirect.
  • the indirect modulation of the target cell can be by targeting a different cell, for example, by modulating a cell adjacent to the target cell, which adjacent cell may be commensal or pathogenic to the target cell.
  • the adjacent cell like the target cell may be either in vitro or in vivo — i.e., in an organism, which may be commensal or pathogenic.
  • the invention provides method of modulating a state of an animal cell, by providing an effective amount of an ADAS provided by the invention or composition provided by the invention access to the animal cell.
  • the ADAS or composition is provided access to the animal cell in vivo, in an animal, such as a mammal, such as a human.
  • the animal cell is exposed to bacteria in a healthy animal.
  • the animal cell is lung epithelium, an immune cell, skin cell, oral epithelial cell, gut epithelial cell, reproductive tract epithelial cell, or urinary tract cell.
  • the animal cell is a gut epithelial cell, such as a gut epithelial cell from a human subject with an inflammatory bowel disease, such as Crohn’s disease or colitis.
  • the animal cell is a gut epithelial cell from a subject with an inflammatory bowel disease
  • the ADAS comprises a bacterial secretion system and a cargo comprising an antiinflammatory agent, 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).
  • TNF tumor necrosis factor
  • IL-12 e.g., an anti-IL-12 antibody
  • IL-23 e.g., an anti-IL-23 antibody
  • the animal cell is exposed to bacteria in a diseased state.
  • the animal cell is pathogenic, such as a tumor.
  • the animal cell is exposed to bacteria in a diseased state, such as a wound, an ulcer, a tumor, or an inflammatory disorder
  • the ADAS is derived from an animal commensal parental strain. In other embodiments, the ADAS is derived from animal pathogenic parental strain.
  • the animal cell is contacted to an effective amount of an ADAS comprising a T3/4SS or T6SS and a cargo, wherein the cargo is delivered into the animal cell.
  • the animal cell is provided access 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.
  • the state of the animal cell is modulated by providing an effective amount of an ADAS provided by the invention or a composition provided by the invention with access to a bacterial or fungal cell in the vicinity of the animal cell. That is, these methods entail indirectly modulating the state of the animal cell.
  • the bacterial or fungal cell is pathogenic.
  • the fitness of the pathogenic bacterial or fungal cell is reduced.
  • the bacterial or fungal cell is commensal.
  • the fitness of the commensal bacterial or fungal cell is increased.
  • the fitness of the commensal bacterial or fungal strain is increased via reduction in fitness of number of a competing bacteria or fungi, which may be neutral, commensal, or pathogenic.
  • the bacterial or fungal cell in the vicinity of the animal cell is contacted to an effective amount of ADAS comprising a T3/4SS or T6SS and a cargo, wherein the cargo is delivered into the bacterial or fungal cell.
  • the bacterial or fungal cell in the vicinity of the animal cell is provided access to an effective amount of ADAS secreting cargo extracellularly that contacts the bacterial or fungal cell.
  • 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 from a parental strain that is mutualistic bacteria of the bacterial or fungal cell.
  • the various method of use provided by the invention that modulate the state of an animal cell can readily be adapted to corresponding methods for modulating the state of a plant, fungal, or bacterial cell.
  • methods for modulating the cell of a plant or fungal cell will be recited more particularly.
  • the invention provide methods of modulating a state of a plant or fungal cell by providing an effective amount of an ADAS provided by the invention or composition provided by the invention access to: a) the plant or fungal cell, b) an adjacent bacterial or adjacent fungal cell in the vicinity of the plant or fungal cell, or c) an invertebrate, (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cell in the vicinity of the plant or fungal cell.
  • an invertebrate e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cell in the vicinity of the plant or fungal cell.
  • the ADAS is provided access to the plant cell in planta, e.g., in a crop plant such as row crops, including corn, wheat, soybean, and rice, and vegetable crops including Solanaceae, such as tomatoes and peppers; cucurbits, such as melons and cucumbers; Brassicas, such as cabbages and broccoli; leafy greens, such as kale and lettuce; roots and tubers, such as potatoes and carrots; large seeded vegetables, such as beans and corn; and mushrooms.
  • the plant or fungal cell is exposed to bacteria in a healthy plant or fungus. In other embodiments, the plant or fungal cell is exposed to bacteria in a diseased state.
  • the plant or fungal cell is dividing, such as a meristem cell, or is pathogenic, such as a tumor. In some embodiments, the plant or fungal cell is exposed to bacteria in a diseased state, such as a wound, or wherein the plant or fungal cell is not part of a human foodstuff.
  • the ADAS is derived from a commensal parental strain. In other embodiments, the ADAS is derived from a plant or fungal pathogenic parental strain.
  • the ADAS comprises an T3/4SS or T6SS and a cargo, and the cargo is delivered into the plant or fungal cell.
  • the plant or fungal cell is provided access to an effective amount of an ADAS comprising a bacterial secretion system and a cargo, wherein the bacterial secretion system secretes the cargo extracellularly, thereby contacting the plant or fungal cell with the cargo.
  • the methods entail providing an effective amount of an ADAS or composition access to the adjacent bacterial or adjacent fungal cell in the vicinity of the plant or fungal cell.
  • the adjacent bacterial or adjacent fungal cell is pathogenic, optionally wherein the fitness of the pathogenic adjacent bacterial or adjacent fungal cell is reduced.
  • the adjacent bacterial or adjacent fungal cell is commensal, optionally wherein the fitness of the commensal adjacent bacterial or adjacent fungal cell is increased.
  • the fitness is increased via reduction of a competing bacteria or competing fungi, which may be neutral, commensal, or pathogenic.
  • the adjacent bacterial or adjacent fungal cell is contacted with an effective amount of ADAS comprising a T3/4SS or T6SS and a cargo, wherein the cargo is delivered into the adjacent bacterial or adjacent fungal cell.
  • the adjacent bacterial or adjacent fungal cell is provided access to an effective amount of ADAS comprising a bacterial secretion system and a cargo, wherein the bacterial secretion system secretes the cargo extracellularly, thereby contacting the adjacent bacterial or adjacent fungal cell with the cargo.
  • the ADAS is derived from a parental strain that is a competitor of the adjacent bacterial or adjacent fungal cells. In other embodiments, the ADAS is derived from a parental strain that is a mutualistic bacterium of the adjacent bacterial or adjacent fungal cell.
  • the methods include providing an effective amount of the ADAS or composition access to an invertebrate, (e.g., arthropod (e.g., insect or arachnid), nematode, protozoan, or annelid) cell in the vicinity of the plant or fungus.
  • the invertebrate is pathogenic.
  • the fitness of the pathogenic invertebrate cell is reduced.
  • the fitness of the pathogenic invertebrate cell is reduced via modulation of symbiotes in the invertebrate cell.
  • the invertebrate is commensal.
  • the fitness of the commensal invertebrate cell is increased.
  • the fitness is increased via reduction of a competing bacteria or fungi, which may be neutral, commensal, or pathogenic.
  • the invention provide methods of removing one or more undesirable materials from an environment comprising contacting the environment with an effective amount of an ADAS provided by the invention or composition provided by the invention, wherein the ADAS comprises one or more molecules (such as proteins, polymers, nanoparticles, binding agents, or a combination thereof) that take up, chelate, or degrade the one or more undesirable materials.
  • ADAS comprises one or more molecules (such as proteins, polymers, nanoparticles, binding agents, or a combination thereof) that take up, chelate, or degrade the one or more undesirable materials.
  • “Environments” are defined as targets that are not cells, such as the ocean, soil, superfund sites, skin, ponds, the gut lumen, and food in a container.
  • the undesirable material includes a heavy metal, such as mercury
  • the ADAS includes one or more molecules (such as proteins, polymers, nanoparticles, binding agents, or a combination thereof) that bind heavy metals, such as MerR for mercury.
  • the undesirable material includes a plastic, such as PET, and the ADAS includes one or more plastic degrading enzymes, such as PETase.
  • the undesirable material comprises one or more small organic molecules and the ADAS comprise one or more enzymes capable of metabolizing said one or more small organic molecules.
  • the invention provides a composition containing a bacterium or ADAS provided by the invention, wherein the bacterium or ADAS includes a T4SS, an RNA binding protein cargo, and an RNA cargo that is bound by the RNA binding protein and is suitable for delivery into a target cell through the T4SS.
  • the RNA binding protein is a Cas9 fused to VirE2 and VirF
  • the RNA cargo is a guide RNA
  • the T4SS is the Ti system from Agrobacterium.
  • the RNA binding protein is p19 from Carnation Italian Ringspot Virus fused to VirE2 or VirF
  • the RNA cargo is an siRNA
  • the T4SS is the Ti system from Agrobacterium.
  • the invention provides methods of making these particular compositions, such methods entailing transfecting a plasmid containing the Cas9 fused to VirE2 and VirF and RNA cargo into an Agrobacterium cell.
  • the invention provides methods for delivering RNA to a plant cell or animal cell comprising contacting said plant cell or animal cell with a bacterium or ADAS, wherein the bacteria or ADAS comprises a T4SS, an RNA binding protein cargo, and an RNA cargo, wherein the RNA is delivered to the plant cell or animal cell.
  • the RNA-binding protein cargo is also delivered to the plant cell or animal cell.
  • the ADAS are derived from a parent 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 lytic enzyme).
  • Embodiment 1 An achromosomal dynamic active system (ADAS) derived from a parent bacterial cell comprising at least one genetic loss-of-function alteration in a lytic enzyme.
  • ADAS achromosomal dynamic active system
  • Embodiment 2 The ADAS of embodiment 1 , wherein the genetic loss-of-function alteration results in increased stability of the ADAS, relative to an ADAS derived from a parent bacterial cell not comprising the alteration.
  • Embodiment 3 The ADAS of embodiment 1 , wherein the genetic loss-of-function alteration is a non- silent codon change, deletion, insertion, mutation or combination of any of these.
  • Embodiment 4 The ADAS of embodiment 1 , wherein the genetic loss-of-function alteration is a deletion.
  • Embodiment 5 The ADAS of embodiment 1 , wherein the lytic enzyme is an endopeptidase, a cell wall lytic enzyme, and/or an autolytic enzyme.
  • Embodiment 6 The ADAS of embodiment 2, wherein the lytic enzyme is selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwIK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ and combinations thereof.
  • the lytic enzyme is selected from the group consisting of lytC (cwlB), lytF (cwlE), lytE (cwlF), lytM, lytD, CwIK, lytH, CwlS, CwlC, CwlH, MpaA, cwlJ and combinations thereof.
  • Embodiment 7 The ADAS of embodiment 5, wherein the genetic loss-of-function alteration is in lytC.
  • Embodiment 8 The ADAS of embodiment 1 , wherein the parent bacterial cell further comprises a loss-of-function alteration in a cell division topological specificity factor.
  • Embodiment 9 The ADAS of embodiment 8, wherein the cell division topological specificity factor is DivIVA, minC, minD, minE, minCD, or the minCDE operon.
  • Embodiment 10 The ADAS of embodiment 1 , wherein the parent bacterial cell is gram-positive.
  • Embodiment 1 1 The ADAS of embodiment 1 , wherein the parent bacterial cell is gram-negative.
  • Embodiment 12 The ADAS of embodiment 1 , wherein the parent bacterial cell further comprises a genetic loss-of-function alteration that disrupts sporulation.
  • Embodiment 13 The ADAS of embodiment 12, wherein the genetic 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, spollAA, spollD, bofA, spoVE, spolVFB, dacB, dapA, dapB, spollGA, spollM, spollR, spoOA or combinations thereof.
  • a sporulation gene selected from the group consisting of sigF, sigE, spollAA, spollD, bofA, spoVE, spolVFB, dacB, dapA, dapB, spollGA, spollM, spollR, spoOA or combinations thereof.
  • Embodiment 14 The ADAS of embodiment 13, wherein the loss-of-function alteration in a sporulation gene is in SigF.
  • Embodiment 15 The ADAS of embodiment 1 , wherein the parent bacterial cells are Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudom
  • Embodiment 16 The ADAS of embodiment 15, wherein the parent bacterial cell is Bacillus subtilis.
  • Embodiment 17 The ADAS of embodiment 2, wherein the stability of the ADAS, as measured by percent of intact ADAS, is greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or is 100%.
  • Embodiment 18 The ADAS of any one of embodiments 1 -17, wherein parent bacterial cell comprises a genomic region having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to one or both of SEQ ID NO: 5 and SEQ ID NO: 9.
  • ADAS ADAS from parent bacterial cells
  • ADAS are produced by disruption of one or more genes involved in regulating parent cell partitioning functions, i.e., AminC, AminD, AminCDE, AminCdivIVA, or AdivIVA.
  • This example details genetic means of creating ADAS-producing strains via disruption of the min operon or over-expression of the septum machinery component FtsZ.
  • Lambda-RED recombineering methodology was adopted according to protocols laid out in Datsenko and Wanner, PNAS, 97(12): 6640-6645, 2000.
  • Strains for engineering and containing the plasmids for the Lambda-RED system were acquired from the Coli Genetic Stock Center (CGSC) at Yale University. Briefly, primers were designed to nonpolarly delete the coding sequences of E. coli minC, minD, or the entire minCDE operon by encoding approximately 40 base pairs of genomic homology into the 5' ends of primers.
  • primers pKD3 and pKD4 of the Lambda-RED system which provide antibiotic markers that were used to select for parent bacterial strains inheriting the target mutations.
  • Primer sequences used for deletion are provided in Table 2.
  • the purified amplicon was transformed via electroporation into bacteria prepared with pKD46, the plasmid containing the phage-derived Lambda-RED homologous recombination system, according to the methods of Datsenko and Wanner, PNAS, 97(12): 6640- 6645, 2000.
  • Transformants were selected on LB agar with 35 pg/mL chloramphenicol. These resulting colonies were confirmed to have the genetic disruption (i.e., AminC, AminD, or AminCDE) using standard allele-specific PCR.
  • primers were designed to delete the coding sequence of B. subtilis divIVA or divIVA and minC. Primer sequences used for deletion are listed in Table 2, with both WT and deletion of divIVA included. In conjunction with the deletion of sigF (sequences in Table 2) to interfere with sporulation, the deletion of divIVA generated the parental strain MACH2347. Strain genotypes are provided in Table 1 .
  • the introduction of an erm (erythromycin) cassette along with the selected deletion primers into the growing bacteria allowed for the selection of transformed colonies through growth on LB-erm5 plates. Once transformed bacteria were cultured, colonies were cured of the erm cassette using a temperature sensitive plasmid conferring resistance to spectinomycin (Spec).
  • Transformed bacteria were selected by plating on LB-Spec plates and incubated at 30°C. Then selected bacteria were streaked onto LB plates containing no antibiotics and incubated at an elevated temperature of 42°C, leading to loss of the temperature sensitive plasmid. Isolated colonies were confirmed to contain the intended deletion.
  • a plasmid that drives expression of the FtsZ Z-ring protein from wild-type E. coli.
  • a strong ribosome binding site and the coding sequence for the E. coli FtsZ protein were de novo optimized using computational tools from De Novo DNA.
  • This translational unit was ordered for de novo DNA synthesis from Integrated DNA Technologies (IDTTM) and cloned into a backbone using standard cloning techniques.
  • IDTTM Integrated DNA Technologies
  • the resulting plasmid, pFtsZ (Table 3), features a TetR repressor, a TetA promoter that is repressed by the TetR protein, a kanamycin resistance marker, and a pMB1 origin of replication.
  • pFtsz When transformed into a compatible bacterium, pFtsz can be induced to overproduce the FtsZ protein via addition of anhydrotetracycline to the culture. This protein is then capable of forming spontaneous protofilaments, which cause asymmetric division of parent bacterial cells and, thereby, ADAS production.
  • ADAS-producing strains are described. This method may be employed to purify any of the ADAS-producing strains described herein, including the strains of Example 1 and Table 1 . Purification separates ADAS from viable parent bacterial cells, which are larger and contain a genome. ADAS were purified from high cell density cultures of ADAS-producing strains via combinations of 1 ) high or low-speed centrifugation, 2) selective outgrowth, and 3) buffer exchange / concentration. Centrifugation procedures were used to selectively deplete viable parent bacterial cells and large cellular debris, while enriching ADAS in a mixed suspension.
  • Selective outgrowth procedures were used to reduce the number of viable parent bacterial cells present in the sample via the addition of compounds that are directly anti-microbial (i.e., toxic to cells having a microbial genome) and/or compounds that enhance viable cell sedimentation via centrifugation.
  • Buffer exchange I concentration procedures were used to transition ADAS from larger volumes of bacterial culture media into smaller volumes of 1 x PBS while removing culture additives and cellular debris.
  • ADAS-producing strains were generated using the molecular cloning procedures described in Example 1 , then cultured to high cell density in culture medium. Cultures may be scaled up, e.g., from 1 mL to 1000 mL or more culture medium.
  • Cultures were transferred to centrifuge tubes and subjected to a high or low-speed centrifugation procedure, aimed at pelleting intact cells and large cell debris while maintaining ADAS in the supernatant. Centrifugation procedures were performed either at 4°C or at room temperature. In some instances, the low-speed centrifugation procedure was used, involving a sequence of sequential 10-minute spins at 1 ,000xg, 2,000xg, 3,000xg, and 4,000xg performed on an Allegra® X14R benchtop centrifuge (Beckman Coulter) or an EppendorfTM 5424 R benchtop centrifuge (Fisher Scientific).
  • the low-speed centrifugation procedure consisted of sequential 2,000xg spins for 20 minutes at 4°C in which the supernatant of the first spin was decanted into a sterile centrifuge bottle prior to the second spin.
  • a low-speed centrifugation procedure was a single 40-minute spin at 4,000xg in a SorvallTM Lynx 6000 Superspeed Centrifuge (Thermo ScientificTM) in which the rate of rotor acceleration was set to the lowest possible setting.
  • a high-speed centrifugation procedure was used, involving sequential pulses at 20,000xg where the spin was halted as soon as the required speed was reached and the supernatant was transferred to a new high-speed bottle prior to the following spin.
  • the high-speed centrifugation procedure consisted of a 30-minute spin at 4°C and 17,000xg after which the pellet was resuspended in outgrowth media.
  • culture supernatants were decanted into sterile culture tubes and subjected to a selective outgrowth process.
  • culture supernatants were decanted and pellets were resuspended and subjected to a selective outgrowth process.
  • concentrated antibiotic solutions e.g., spectinomycin, clindamycin, tetracycline, ceftriaxone, kanamycin, carbenicillin, gentamicin, and/or ciprofloxacin
  • other concentrated chemical solutions e.g., sodium chloride, sodium hydroxide, M hydrochloric acid, glucose, cas-amino acids, and/or D-amino acids
  • the culture supernatants were pelleted via high-speed centrifugation for 5 to 60 minutes at 10,000xg to 20,000xg and the pellets were resuspended in fresh culture media containing concentrations of antibiotics or other chemical solutions that were inhibitory to viable cells.
  • Selective outgrowth was performed by incubating ADAS at 4°C to 42°C for 1 to 3 hours with agitation at 250rpm. ADAS were then transferred to sterile centrifuge tubes and subjected to an additional round of centrifugation.
  • aPES asymmetrical polyethersulfone
  • Thermo Fisher asymmetrical polyethersulfone
  • ADAS were pelleted via centrifugation at 10,000xg to 20,000xg for 5 to 60 minutes, washed in 1 to 9 volumes of 1 x PBS, pelleted again, and resuspended in 1x PBS at 1 to 100,000x concentration from the starting culture volume.
  • ADAS were pelleted via sequential high-speed pulses of 1 -minute intervals at 16,000xg, followed by a long high-speed spin of 20,000xg for 20 min at 4°C, after which the pellet was resuspended and then washed several times. In some cases, washes consisted of 5 minute spins at 4°C, 15,000xg.
  • ADAS-producing parental strains that are auxotrophic, i.e., are unable to synthesize an organic compound required for growth, are useful for the manufacturing of ADAS. Such strains are able to grow only when the organic compound is provided. Auxotrophic parental strains may thus be selected against by storing or incubating an ADAS preparation in media lacking the organic compound, thus providing an additional method for reducing parental burden in the ADAS preparation.
  • primers were designed to delete the coding sequences of interest (e.g., deletion of genomic sequences encoding lytic enzymes). Sequences targeted for deletion are listed in Table 2, with both wild-type (WT) sequences and sequences showing the deletion of each gene of interest included.
  • WT wild-type
  • SEQ ID NO: 4 shows a wildtype genomic region comprising SigF
  • SEQ ID NO: 5 shows the genomic region following a loss- of-function deletion
  • SEQ ID NO: 8 shows a wild-type genomic region comprising lytC
  • SEQ ID NO: 9 shows the genomic region following a loss-of-fu notion deletion.
  • Strain genotypes are provided in Table 1 .
  • the introduction of an erm (erythromycin) cassette along with the selected deletion primers into the growing bacteria allowed for the selection of transformed colonies through growth on LB-erm5 plates. Once transformed bacteria were cultured, colonies were cured of the erm cassette using a temperature sensitive plasmid conferring resistance to spectinomycin (Spec). Transformed bacteria were selected by plating on LB-Spec plates and incubated at 30°C. Then selected bacteria were streaked onto LB plates containing no antibiotics and incubated at an elevated temperature of 42°C, leading to loss of the temperature sensitive plasmid. Isolated colonies were confirmed to contain the intended deletion (e.g., lytC), and if further genomic deletions were required (such as sigF), a similar process was used.
  • Spec temperature sensitive plasmid conferring resistance to spectinomycin
  • ADAS-producing strains were generated and purified using the procedures described in Examples 1 -3 and are listed in Table 1 .
  • Bacillus subtilis strains were precultured in LB broth and incubated at 37°C, 250 RPM for 6 hours. Cultures were then diluted and incubated overnight at 30°C, 250 RPM.
  • Representative images from overnight cultures highlight differences in parent cell growth prior to ADAS enrichment, with fewer breaks in rod-length in parent bacterial cells comprising the AlytC mutation (MACH2403; Fig. 1 ).
  • Representative images of two different cultures at three different timepoints were also taken to determine ADAS stability visually.
  • ADAS and remaining parental cells are shown from two different B. subtilis strains after cultivation overnight (Fig. 1 and Fig. 3A), cultivation overnight and ADAS enrichment (tO, Fig. 3B), or cultivation overnight, or 23h after cultivation overnight, ADAS enrichment, and incubation at 4°C (t23, Fig. 3C).
  • the left panels are strain MACH2347 (described in Table 1 ).
  • the right panels are MACH2403 (described in Table 1 ), which has been modified from MACH2347 with the additional genomic deletion of lytC.
  • the white arrows indicate examples of phase-light ADAS (ADAS that appear lighter or ghosted, indicating disintegration and lysis).
  • the arrowheads indicate examples of phase-light parent cells.
  • the ADAS populations derived from the parent cell line comprising deletion of lytC show visual evidence of increased stability of ADAS via the increased number of intact (non phase-light) ADAS.
  • the listed strains and shown images should not be considered limiting.

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Abstract

L'invention concerne un système actif dynamique chromosomique (ADAS) dérivé d'une cellule bactérienne parente, comprenant au moins une modification de perte de fonction génétique dans une enzyme lytique pour augmenter la stabilité de l'ADAS. L'invention concerne également des procédés d'interruption de la sporulation dans des cellules bactériennes parentes en combinaison avec une délétion d'enzyme lytique ou une autre mutation de perte de fonction.
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JP2012200168A (ja) * 2011-03-24 2012-10-22 Kao Corp 遺伝子欠損株及びそれを用いたタンパク質の製造方法
WO2020123569A1 (fr) 2018-12-10 2020-06-18 Flagship Pioneering Innovations Vi, Llc Systèmes actifs dynamiques achromosomiques
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JP2012200168A (ja) * 2011-03-24 2012-10-22 Kao Corp 遺伝子欠損株及びそれを用いたタンパク質の製造方法
WO2020123569A1 (fr) 2018-12-10 2020-06-18 Flagship Pioneering Innovations Vi, Llc Systèmes actifs dynamiques achromosomiques
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