ADAS including type 1 villi

Abstract

A non-chromosomal dynamic active system comprising type 1 pili (T1P) and methods for making and using the same are provided herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 354,979, filed on June 23, 2022, which is hereby incorporated by reference in its entirety herein.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety herein. The XML copy created on June 20, 2023, is named 51296 - 056WO2_Sequence_Listing_6_20_23 and is 46,583 bytes in size.

[0003] Provided herein are non - chromosomal dynamic activity systems (ADAS) that include type 1 pili (T1P), as well as methods for creating and using the same.

Background Art

[0004] There is a need for delivery vectors that are capable of targeting mammalian cells and delivering biological agents, compositions containing such delivery vectors, and related methods for delivering said vectors to cells and thereby modulating biological systems including mammalian cells and organisms. In particular, there is a need for delivery vectors that have tropism for target cells.

Summary of the Invention

[0005] In one aspect, the present disclosure features a preparation comprising a plurality of non - chromosomal dynamic activity systems (ADAS) derived from parent bacterial cells genetically engineered to constitutively express type 1 pili (T1P), wherein the plurality of ADAS bind to target cells via T1P.

[0006] In one aspect, the present disclosure features a plurality of non - chromosomal dynamic activity systems (ADAS) that include type 1 pili (T1P), wherein the ADAS are derived from parent bacterial cells that constitutively express components of T1P.

[0007] In some embodiments, the parental cell comprises a modified fimS promoter operably linked to a component of T1P and directing its constitutive expression.

[0008] In some embodiments, the component of T1P is encoded by the fim operon.

[0009] In some embodiments, the parental cell comprises a modified fimS promoter operably linked to the fim operon, the modified fimS promoter comprising a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site.

[0010] In some embodiments, the parental cell expresses the component of T1P at a level that is at least 1.5-fold higher than the level observed in unmodified parental cells.

[0011] In some embodiments, the ADAS comprises T1P at a level that is at least 1.5-fold higher than the level observed in a plurality of ADAS produced from unmodified parental cells.

[0012] In some embodiments, the proportion of the plurality of ADAS comprising T1P is increased compared to the plurality of ADAS produced by parental cells that do not constitutively express the component of T1P.

[0013] In some embodiments, the parental cell comprises an endogenous fim operon.

[0014] In some embodiments, the parental cell is a bacterium that is Escherichia coli (E. coli). In some embodiments, the bacterium that is Escherichia coli (E. coli) is Escherichia coli (E. coli) CFT073.

[0015] In some embodiments, the parental cell comprises one or more heterologous nucleotide sequences encoding components of T1P. In some embodiments, the one or more heterologous nucleotide sequences comprise the fim operon. In some embodiments, the fim operon is the fim operon of Escherichia coli (E. coli) CFT073.

[0016] In some embodiments, the one or more heterologous nucleotide sequences comprise a sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more heterologous nucleotide sequences comprise a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more heterologous nucleotide sequences comprise the nucleotide sequence of SEQ ID NO: 1.

[0017] In some embodiments, the one or more heterologous nucleotide sequences further comprise a constitutive promoter operably linked to the fim operon.

[0018] In some embodiments, the constitutive promoter is a modified fimS promoter that contains a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site.

[0019] In some embodiments, the one or more nucleotide sequences encoding components of T1P are carried on a vector. In some embodiments, the parental bacterial cell is transiently transformed by the vector. In some embodiments, the parental bacterial cell is stably transformed by the vector.

[0020] In some embodiments, the parental cell is a Gram-negative bacterial cell. In some embodiments, the Gram-negative bacterial cell is a bacterial cell of Escherichia coli (E. coli), Salmonella, Yersinia, Vibrio, Pseudomonas, Shigella, or Legionella.

[0021] In some embodiments, the parental cell does not contain the complete endogenous fim operon.

[0022] In some embodiments, the parental cell has not been exposed to culture conditions that promote the expression of the fim operon. In some embodiments, the culture conditions are temperature, pH, osmolarity, agitation, or activation of a stress response or stringent response.

[0023] In some embodiments, the ADAS contains a cargo.

[0024] In some embodiments, the cargo is a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulatory agent, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP).

[0025] In some embodiments, the cargo is encapsulated within the ADAS. In some embodiments, the cargo is attached to the surface of the ADAS.

[0026] In some embodiments, the ADAS contains a heterologous bacterial secretion apparatus. In some embodiments, the heterologous bacterial secretion apparatus is a type III secretion system (T3SS).

[0027] In some embodiments, the cargo contains a component that directs export by the bacterial secretion apparatus.

[0028] In another aspect, the disclosure features a composition comprising a plurality of ADASs of any one of the above embodiments.

[0029] In some embodiments, the composition is formulated for delivery to a mammal. In some embodiments, the composition is formulated for oral delivery.

[0030] In another aspect, the present disclosure features a method of delivering an ADAS to a cell, the method comprising: (a) providing a composition comprising a plurality of ADASs of any one of the above embodiments; and (b) contacting the cell with the composition of step (a).

[0031] In some embodiments, the delivery of the ADAS to the cell is at least 10% increased compared to the ADAS derived from the unmodified parental cell.

[0032] In some embodiments, an effective amount of the ADAS is delivered to the cell at a dose that is at least 10% lower than the dose required for the ADAS derived from the unmodified parental cell.

[0033] In another aspect, the present disclosure features a method of delivering a cargo to a cell, the method comprising: (a) providing a composition comprising a plurality of ADASs of any one of the above embodiments, wherein the ADAS further comprises the cargo; and (b) contacting the cell with the composition of step (a).

[0034] In some embodiments, the ADAS further comprises a heterologous bacterial secretion apparatus. In some embodiments, the heterologous bacterial secretion apparatus is a T3SS.

[0035] In some embodiments, the delivery is delivery to the cytoplasm of the cell.

[0036] In some embodiments, the delivery of the cargo to the cell is at least 10% increased compared to the ADAS derived from the unmodified parental cell.

[0037] In some embodiments, an effective amount of the cargo is delivered to the cell at a dose that is at least 10% lower than the dose required for the ADAS derived from the unmodified parental cell.

[0038] In another aspect, the present disclosure features a method of modulating a cell, the method comprising (a) providing a composition comprising a plurality of ADASs of any one of the above embodiments; and (b) comprising contacting the cells with the composition of step (a), whereby the cells are modulated.

[0039] In some embodiments, the cells are mammalian cells.

[0040] In some embodiments, the mammalian cells are intestinal cells. In some embodiments, the intestinal cells are gut-associated lymphoid tissue (GALT) cells, Peyer's patch cells, M cells, lamina propria cells, small intestinal cells or large intestinal cells.

[0041] In some embodiments, the mammalian cells are bladder cells.

[0042] In some embodiments, the mammalian cells are immune cells.

[0043] In some embodiments, the mammalian cells are blood-brain barrier cells.

[0044] In some embodiments, the mammalian cells are mannosylated cells.

[0045] In another aspect, the present disclosure features a method for treating a mammal in need thereof, the method comprising: (a) providing a composition comprising a plurality of ADASs of any one of the above embodiments; and (b) contacting the mammal with an effective amount of the composition of step (a), whereby the mammal is treated.

[0046] In some embodiments, the therapeutic effect is achieved at a dose that is at least 10% lower than the dose required for ADASs derived from unmodified parental cells.

[0047] In another aspect, the present disclosure features an ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being produced by a process comprising: (a) providing a parental cell modified to constitutively express a component of the T1P; and (b) producing the ADAS from the parental bacterial cell, wherein the ADAS comprises the T1P.

[0048] In another aspect, the present disclosure features an ADAS that includes a T1P derived from a parental bacterial cell, the ADAS being produced by a process that includes: (a) providing a parental cell modified to express a component of the native T1P at a level that is at least 1.5-fold higher than the level observed in the unmodified parental cell; and (b) producing the ADAS from the parental bacterial cell, the ADAS including a T1P that is native to the parental cell.

[0049] In another aspect, the present disclosure features an ADAS that includes a T1P derived from a parental bacterial cell, the ADAS being produced by a process that includes: (a) providing a parental cell modified to express a component of a heterologous T1P; and (b) producing the ADAS from the parental bacterial cell, the ADAS including a T1P that is heterologous to the parental cell.

[0050] In another aspect, the present disclosure features an engineered bacterium that constitutively expresses a component of a T1P, the engineered bacterium including the T1P at a level that is at least 1.5-fold higher compared to the level of the T1P included by an unengineered bacterium.

[0051] In some embodiments, the T1P is a native T1P. In other aspects, the T1P is a heterologous T1P.

[0052] In another aspect, the present disclosure features an engineered bacterium that constitutively expresses a component of a T1P, the bacterium being modified to produce an ADAS.

[0053] In another aspect, the present disclosure features a method for producing an ADAS, the method including: (a) providing an engineered bacterium that constitutively expresses a component of a T1P; and (b) producing the ADAS from the bacterium.

[0054] In another aspect, the present disclosure features an ADAS produced according to any one of the methods described above.

[0055] Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief Description of the Drawings

[0056]

Figure 1

Figure 2

Figure 3

Figure 4

DETAILED DESCRIPTION OF THE INVENTION

[0057] I. Definitions As used herein, the term "achromosomal dynamic system" or "ADAS" refers to a genome-free, non-replicating, closed membrane system that contains at least one membrane and has an internal volume suitable for accommodating a cargo (e.g., one or more of nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulatory agents, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs)). In some embodiments, the ADAS is a minicell or modified minicell derived from a parental bacterial cell (e.g., a gram-negative or gram-positive bacterial cell). In other aspects, the ADAS is obtained from a parental cell by modifying the parental cell such that its genome is removed, and the size is substantially the same as the parental cell. The ADAS may be obtained from a parental bacterium using any suitable method, e.g., genetic manipulation of the parental cell or exposure to a culture medium or conditions that increase the formation potential of bacterial minicells. Exemplary methods of producing the ADAS disrupt the cell division mechanism of the parental cell. In some embodiments, the ADAS may contain one or more endogenous or heterologous features on the surface of the parental cell, e.g., cell wall, cell wall modification, flagella, or pili (e.g., type 1 pili (T1P)) and / or one or more endogenous or heterologous features of the internal volume of the parental cell, e.g., nucleic acids, plasmids, proteins, small molecules, transcriptional machinery, or translational machinery. In other embodiments, the ADAS may lack one or more features of the parental cell. In still other embodiments, the ADAS may be loaded with features not contained in the parental cell or otherwise modified thereby.

[0058] As used herein, the term "T1P-ADAS" refers to an ADAS that includes type 1 pili (T1P) above a detectable threshold. T1P may be detected, for example, using a red blood cell (RBC) agglutination assay, as described herein.

[0059] As used herein, the terms "constitutive expression" and "constitutively expressed", when used with respect to T1P, refer to a state in which a nucleic acid sequence encoding a component of T1P is expressed (e.g., transcribed and translated, e.g., expressed by a bacterial cell, ADAS or other system containing the nucleic acid sequence) regardless of environmental conditions, i.e., a state in which the expression of the nucleic acid sequence encoding a component of T1P cannot be turned off.

[0060] As used herein, the term "endogenous type 1 pili" or "endogenous T1P" refers to T1P that is present in a cell (e.g., a parent cell) or an ADAS derived therefrom and is naturally encoded by the cell (e.g., encoded by the wild-type version of the cell). T1P may be expressed by an endogenous gene of the cell (e.g., the endogenous fim operon) and / or encoded and expressed by a synthetic construct in the cell. The expression or abundance of endogenous T1P may be increased, for example, by the addition of a moiety that increases the abundance of T1P (e.g., a transcriptional activator of T1P) or by the decrease or removal of a negative regulator of T1P expression. In some embodiments, the expression of T1P is increased by modification or replacement of the natural promoter of the endogenous gene encoding T1P, e.g., modification of the fimS promoter (e.g., by a modification that locks the fimS promoter in the "ON" configuration).

[0061] As used herein, the term "heterologous type 1 pilus" or "heterologous T1P" refers to a T1P that is present in a cell (e.g., a parental cell) or an ADAS derived therefrom and is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell). The cell may or may not encode another T1P. In some embodiments, the T1P is expressed by one or more synthetic constructs in the cell.

[0062] As used herein, the term "highly active ADAS" refers to an ADAS (e.g., a T1P-ADAS) with high potential for function, e.g., an ADAS having the ability to perform a large amount of useful functions. The functions may be metabolic functions 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., intracellular translocation, extracellular translocation or secretion, e.g., secretion by a bacterial secretion apparatus (e.g., T3SS)) under suitable conditions. In certain embodiments, the highly active ADAS starts from a large pool of energy in the form of energy, e.g., ATP. In other embodiments, the ADAS has the ability to take up or generate energy / ATP from another source. The highly active ADAS may be identified, for example, by an increase in ATP concentration, an increase in the ability to generate ATP, an increase in the ability to produce proteins, an increase in protein production rate or production amount and / or an increase in responsiveness to biological signals, e.g., induction of a promoter.

[0063] As used herein, the term "parental bacterial cell" refers to a cell (e.g., a gram-negative or gram-positive bacterial cell) from which an ADAS (e.g., a T1P-ADAS) is derived. The parental bacterial cell is typically a viable bacterial cell. The term "viable bacterial cell" refers to a bacterial cell that contains a genome and has the ability to divide. Preferred parental bacterial cells are derived from any of the strains in Table 1.

[0064] An ADAS composition or preparation that is "substantially free" of parental bacterial cells and / or viable bacterial cells is defined herein as a composition having 500 or fewer colony-forming units (CFU) per mL, for example, 400, 300, 200, 150, 100 or fewer. An ADAS composition substantially free of parental bacterial cells or viable bacterial cells may contain fewer than 50 CFU / mL of bacterial cells, fewer than 25 CFU / mL, fewer than 10 CFU / mL, fewer than 5 CFU / mL, fewer than 1 CFU / mL, fewer than 0.1 CFU / mL or fewer than 0.001 CFU / mL, or may contain none at all.

[0065] As used herein, the term "endogenous type III secretion apparatus" or "endogenous T3SS" refers to a T3SS that is present in a cell (e.g., a parental cell), or an ADAS derived therefrom, and that is naturally encoded by that cell (e.g., encoded by the wild-type version of that cell). The T3SS may be expressed by an endogenous gene of the cell and / or may be encoded and expressed by a synthetic construct in the cell. The expression or abundance of the endogenous T3SS can be increased, for example, by addition of a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by reduction or removal of a negative regulator of the expression of the T3SS.

[0066] As used herein, the term "heterologous type III secretion apparatus" or "heterologous T3SS" refers to a T3SS that is present in a cell (e.g., a parental cell), or an ADAS derived therefrom, and that is not naturally encoded by that cell (e.g., not encoded by the wild-type version of that cell). This cell may or may not encode another T3SS. In some embodiments, the T3SS is expressed by a synthetic construct in the cell.

[0067] As used herein, an "endogenous effector" of a secretion apparatus (e.g., T3SS, T4SS, or T6SS) is a moiety (e.g., a protein or polypeptide) that is naturally encoded (e.g., encoded by a wild-type version of the cell) by the cell from which the secretion apparatus (e.g., T3SS) is derived and that has the ability to be secreted by that secretion apparatus. One or more of the secretion apparatus and its endogenous effector may be expressed in the cell in which they naturally occur or may be heterologously expressed, e.g., by a cell that does not naturally encode the endogenous effector or its secretion apparatus.

[0068] As used herein, an effector that is heterologous to a secretion apparatus ("heterologous effector") is a moiety (e.g., a protein or polypeptide) that is not naturally encoded (e.g., not encoded by a wild-type version of the cell) by the cell from which the secretion apparatus (e.g., T3SS) is derived and that has the ability to be secreted by the secretion apparatus of the cell from which the heterologous effector is derived. The effector may have the ability to be secreted by a secretion apparatus that is heterologous to it or may be modified to be secreted by a secretion apparatus that is heterologous to it. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0069] As used herein, the "cargo" of a secretion apparatus (e.g., T3SS) is a moiety that has the ability to be secreted by that secretion apparatus (e.g., delivered into the cytoplasm of a host cell or into the extracellular space). The cargo may be an endogenous effector, a heterologous effector, or a moiety that is not secreted by any secretion apparatus naturally. The cargo may be, for example, a protein, or a polypeptide, such as an enzyme (e.g., a metabolic enzyme), a DNA modifying agent (e.g., a component of a CRISPR system), a chromatin remodeling agent, a gene editing agent, a nuclear targeting agent (e.g., a transcription factor), a binding agent (e.g., an antibody or antibody fragment, such as a VHH molecule), an immunogenic agent (e.g., an immunostimulatory or immunosuppressive agent), or a toxin. The cargo may be modified, for example, by the addition of a secretion signal (e.g., an N-terminal secretion signal), such as those provided in Table 3, to be secreted by that secretion apparatus.

[0070] As used herein, the term "percent identity" refers to the percent (%) sequence identity to a reference polynucleotide or polypeptide sequence after alignment by standard techniques. Alignments for the determination of percent nucleic acid or amino acid sequence identity are within the capabilities of those of skill in the art and can be accomplished by various methods, e.g., using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. Those of skill in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or as compared to a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to, with, or as compared to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(ratio X / Y) (Wherein, X is the number of nucleotides or amino acids that are evaluated as a perfect match in the alignment of A and B by an array alignment program (e.g., BLAST), and wherein Y is the total number of nucleotides or amino acids of B). In some embodiments, the sequence identity, e.g., the sequence identity of a homolog of the MinE or DivIVA protein, is at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, or even 95% or more, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acid or nucleic acid sequence identity with the native sequence MinE (or minE) or DivIVA (or divIVA) sequence as disclosed herein, or amino acid sequence or nucleic acid identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0071] As used herein, the expression "regulating the state of a cell" refers to an observable change in the state of a cell (e.g., a mammalian cell) (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state) when measured using techniques and methods known in the art for such measurements, such as methods for measuring the level or expression of proteins, transcripts, epigenetic markers, or methods for measuring an increase or decrease in the activity of a biological pathway. Regulating the state of a cell can result in a change of at least 1% (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; e.g., up to 100% compared to before administration) compared to before administration. In some embodiments, regulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). Increasing the state of a cell can result in an increase in the parameter of at least 1% (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; e.g., up to 100% compared to before administration) compared to before administration. In other embodiments, regulating the state of a cell involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). Decreasing the state of a cell can result in a decrease in the parameter of at least 1% (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; e.g., up to 100% compared to before administration) compared to before administration.

[0072] As used herein, the term "heterologous" means not natural to a cell or composition in its naturally occurring state. In some embodiments, "heterologous" refers to a molecule not naturally found in an ADAS or the parental bacterium (e.g., a gram-negative or gram-positive bacterial cell) from which it is produced; e.g., a T1P, a component thereof, or a nucleic acid encoding the same, or a cargo or payload (e.g., a nucleic acid such as an RNA encoding a polypeptide, protein or tRNA, or a small molecule), or a structure (e.g., a plasmid or a gene editing system).

[0073] II. Compositions A. ADAS Containing Type 1 Pili "ADAS" is a genome-free, non-replicating, closed membrane system that contains at least one membrane (in some embodiments, two membranes that do not intersect) and has an internal volume suitable for accommodating a cargo (e.g., a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulatory agent, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP)). In some embodiments, the ADAS is a mini-cell or a modified mini-cell derived from a parental bacterial cell (e.g., a gram-negative or gram-positive bacterial cell). The ADAS may be obtained from the parental bacterium using any suitable method, e.g., genetic manipulation of the parental cell or exposure to a culture medium or conditions that increase the ability to form bacterial mini-cells. Exemplary methods for the production of ADAS are provided in WO 2020 / 123569 pamphlet.

[0074] The present invention is based, at least in part, on the Applicant's development of an ADAS that includes type 1 pili (T1P-ADAS). T1P is a hair-like structure found on the surface of bacteria. T1P has been shown to promote tropism to the intestine (e.g., Peyer's patches) and areas within other body sites and cell types. The ADAS containing T1P on its surface may be derived from the parent bacteria containing T1P on its surface. However, the expression of the gene (fim operon) encoding the components of T1P is controlled by a phase-variable promoter, and the production of T1P can be switched off in response to growth conditions (Zhang et al., Proc Natl Acad Sci USA, 113(15):4182-4187, 2016), thus making it difficult to sustain the production of T1P-ADAS derived from the parent cells. Furthermore, not all desired parent bacteria have a functional copy of the fim operon. The present invention provides an ADAS derived from a parent bacterial cell that constitutively expresses the components of T1P and / or includes one or more heterologous nucleotide sequences encoding the components of T1P; a method for producing the same; and a method for delivering such an ADAS and / or its cargo to a cell.

[0075] In some embodiments, the ADAS has a major axis cross-section between about 100 nm and 500 μm (e.g., in certain embodiments, about 100 - 600 nm, such as 100 - 400 nm; or between about 0.5 - 10 μm and 10 - 500 μm). In certain embodiments, the ADAS has a minor axis cross-section of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, up to 100% of the major axis. In certain embodiments, the ADAS is about 0.001 - 1 μm 3 , 0.3 - 5 μm 3 , 5 - 4000 μm 3 or 4000 - 50×10 7 μm 3It has an internal volume therebetween. In some embodiments, the ADAS is substantially the same size as the parental cell, e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the size of the parental cell (e.g., internal volume, major axis cross-section and / or minor axis cross-section), has the same size as the parental cell or has a size that is about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or 110% of the size of the parental cell.

[0076] The ADAS can be derived from various sources, including a parental bacterial strain (the "parent strain") engineered or induced to produce a genome-free closed membrane system; a bacterium from which the genome has been excised; a bacterial cell preparation extract (e.g., by mechanical or other means); or a fully synthetic composition optionally including a fraction of a bacterial cell preparation.

[0077] ADAS derived from a parental cell that constitutively expresses T1P components In some aspects, the present disclosure features a plurality of extrachromosomal motility active systems (T1P-ADAS) that include type 1 pili, and the ADAS is derived from a parental bacterial cell that constitutively expresses components of the T1P. In some embodiments, the components of the T1P include the FimA, FimI, FimC, FimD, FimF, FimG, and FimH genes.

[0078] In some embodiments, the parental cell includes a modified fimS promoter operably linked to the components of the T1P and directing its constitutive expression. In some embodiments, the components of the T1P are encoded by the fim operon. In some aspects, the fim operon includes genes encoding FimA, FimI, FimC, FimD, FimF, FimG, and FimH.

[0079] In the fimS promoter, two recombination sites are adjacent, and by cleavage and recombination of these sites, the promoter can switch between an "ON" configuration and an "OFF" configuration. In some embodiments, the parental cell comprises a modified fimS promoter operably linked to the fim operon, the modified fimS promoter comprising a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site. In some embodiments, the recombination site containing the mutation is upstream of the fimS promoter. In other embodiments, the recombination site containing the mutation is downstream of the fimS promoter. In still other embodiments, both the upstream and downstream recombination sites are mutated. Exemplary mutations at the upstream (left) recombination site are shown in SEQ ID NOs: 4-7. SEQ ID NO: 4 shows the wild-type sequence of the fimS site of Escherichia coli (E. coli) strain CFT073 in the "ON" direction; SEQ ID NO: 5 shows a version of this fimS site containing a mutation in the left inverted repeat site that locks the promoter in the "ON" direction. SEQ ID NO: 6 shows the wild-type sequence of the fimS site of Escherichia coli (E. coli) strain MG1655 in the "ON" direction; SEQ ID NO: 7 shows a version of this fimS site containing a mutation in the left inverted repeat site that locks the promoter in the "ON" direction. In other aspects, the parental cell comprises a constitutive promoter operably linked to a component of T1P (such as the fim operon) and directing its constitutive expression (such as the J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23108, J23109, J23110, J23111, J23112, J23113, J23114, J23115, J23116, J23117, J23118, J23119, J23150 or J23151 promoter). Exemplary promoter sequences are provided, for example, by the iGEM Registry of Standard Biological Parts. In some aspects, the endogenous fimS promoter has been replaced by a constitutive promoter.

[0080] In other aspects, the present disclosure features a plurality of T1P-ADAS, where the ADAS is derived from a parental bacterial cell that can be induced to express components of T1P. In some aspects, the parental cell comprises an inducible promoter operably linked to a component of T1P (e.g., the fim operon) and directing its inducible expression (e.g., the pTac, pTrp, Para, RLac, pTet, or pRha promoter). In some aspects, the endogenous fimS promoter has been replaced by an inducible promoter.

[0081] In some embodiments, the parental cell is a bacterium that is Escherichia coli (E. coli). In some embodiments, the bacterium that is Escherichia coli (E. coli) is Escherichia coli (E. coli) CFT073.

[0082] In some embodiments, the parental cell expresses components of T1P at a level that is at least 1.1-fold higher than the level observed in an unmodified parental cell (e.g., at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold or more higher than the level observed in an unmodified parental cell; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold, or 100-fold or more higher than the level observed in an unmodified parental cell). In some embodiments, the parental cell expresses components of T1P at a level that is at least 1.5-fold higher than the level observed in an unmodified parental cell.

[0083] In some embodiments, the ADAS contains T1P at a level that is at least 1.1-fold higher than the level observed in a plurality of ADAS produced from control (e.g., unmodified) parental cells (e.g., at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or 100-fold or more higher than the level observed in a plurality of ADAS produced from control parental cells; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold or more higher than the level observed in a plurality of ADAS produced from control parental cells). In some embodiments, the ADAS contains T1P at a level that is at least 1.5-fold higher than the level observed in a plurality of ADAS produced from control parental cells.

[0084] In some embodiments, the proportion of the plurality of ADAS containing T1P is increased compared to the plurality of ADAS produced by parental cells that do not constitutively express the components of T1P; e.g., increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% (e.g., increased by 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100% or more than 100%).

[0085] In some embodiments, the present disclosure provides an ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being produced by a process comprising: (a) providing a parental cell modified to express a component of the native T1P at a level at least 1.5-fold higher than the level observed in the unmodified parental cell (e.g., at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold or more higher than the level observed in the unmodified parental cell; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold higher than the level observed in the unmodified parental cell); and (b) producing the ADAS from the parental bacterial cell, the ADAS comprising the T1P that is native to the parental cell. In some embodiments, the comparison between the T1P-ADAS and the unmodified parental cell is made under conditions where the unmodified parental cell does not produce T1P or produces T1P at a low level. In unmodified bacterial cells, the native promoter for type 1 pili (T1P), fimS, is phase variable and can switch between the "ON" and "OFF" directions in response to a number of signals including temperature, pH, osmolarity, and stress and stringent responses. Growing Escherichia coli (E. coli) in LB broth at 37°C with shaking (standard growth conditions for E. coli) in the laboratory promotes maintaining the fimS switch in the "OFF" direction. Switching growth to 37°C, static (not shaking), promotes maintaining the fimS switch in the "ON" direction.

[0086] Accordingly, in some embodiments, the present disclosure provides an ADS comprising a T1P derived from a parental bacterial cell that has not been exposed to culture conditions that promote the expression of the fim operon (e.g., has not been exposed to such conditions prior to the production of the ADAS or during the production of the ADAS), wherein the ADAS is produced by a process comprising: (a) providing a parental cell modified to express a component of natural T1P at a level that is at least 1.5-fold higher than the level observed in an unmodified parental cell (e.g., at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or 100-fold or more higher than the level observed in an unmodified parental cell; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold higher than the level observed in an unmodified parental cell); and (b) producing the ADAS from the parental cell, wherein the ADAS comprises a T1P that is native to the parental cell.

[0087] In some embodiments, the culture conditions are temperature, pH, osmolarity, agitation or activation of a stress response or stringent response. In some embodiments, the culture conditions that promote the expression of the fim operon are growth at 37°C under static (rather than agitated) conditions. Conditions that affect the expression of the fim operon are further described in Zhang et al., Proc Natl Acad Sci USA, 113(15):4182-4187, 2016.

[0088] ii. An ADAS derived from a parental cell comprising a heterologous T1P component In some embodiments, the plurality of ADASs described above are derived from a parental cell comprising one or more heterologous nucleotide sequences encoding a component of T1P. In some embodiments, the one or more heterologous nucleotide sequences comprise the fim operon. In some embodiments, the fim operon is the fim operon of Escherichia coli (E. coli) CFT073.

[0089] In some embodiments, the one or more heterologous nucleotide sequences include sequences having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1, the E. coli CFT073 fim operon sequence (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more than 99% identity to the nucleotide sequence of SEQ ID NO: 1, e.g., sequences having 90%-92%, 92%-94%, 94%-96%, 96%-98% or 98%-100% identity). In some embodiments, the one or more heterologous nucleotide sequences include sequences having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more heterologous nucleotide sequences include the nucleotide sequence of SEQ ID NO: 1.

[0090] In some embodiments, the one or more heterologous nucleotide sequences include sequences having at least 90% identity to the nucleotide sequence of SEQ ID NO: 3, the E. coli MG1655 fim operon sequence (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more than 99% identity to the nucleotide sequence of SEQ ID NO: 3, e.g., sequences having 90%-92%, 92%-94%, 94%-96%, 96%-98% or 98%-100% identity). In some embodiments, the one or more heterologous nucleotide sequences include sequences having at least 95% identity to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the one or more heterologous nucleotide sequences include the nucleotide sequence of SEQ ID NO: 3.

[0091] In some embodiments, one or more heterologous nucleotide sequences are operably linked to a constitutive promoter. In some embodiments, one or more heterologous nucleotide sequences further comprise a constitutive promoter operably linked to the fim operon. In some embodiments, the constitutive promoter is a modified fimS promoter that contains a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site (e.g., contains a mutation at a restriction site upstream of the fimS promoter; contains a mutation at a restriction site downstream of the fimS promoter; or contains mutations at both the upstream and downstream recombination sites).

[0092] In some embodiments, one or more nucleotide sequences encoding components of T1P are carried on a vector. In some embodiments, the parental bacterial cell is transiently transformed by the vector. In other embodiments, the parental bacterial cell is stably transformed by the vector.

[0093] In some embodiments, the parental cell contains a functional endogenous fim operon.

[0094] In some embodiments where the parental cell contains a functional endogenous fim operon, the parental cell expresses components of T1P at a level that is at least 1.1-fold higher than the level observed in an unmodified parental cell (e.g., at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or 100-fold or more higher than the level observed in an unmodified parental cell; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold or more higher than the level observed in an unmodified parental cell). In some embodiments, the parental cell expresses components of T1P at a level that is at least 1.5-fold higher than the level observed in an unmodified parental cell.

[0095] In some embodiments where the parental cell contains a functional endogenous fim operon, the ADAS contains T1P at a level that is at least 1.1-fold higher than the level observed in a plurality of ADAS produced from control (e.g., unmodified) parental cells (e.g., at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or 100-fold or more higher than the level observed in a plurality of ADAS produced from control parental cells; e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold or more higher than the level observed in a plurality of ADAS produced from control parental cells). In some embodiments, the ADAS contains T1P at a level that is at least 1.5-fold higher than the level observed in a plurality of ADAS produced from control parental cells.

[0096] In some embodiments where the parental cell contains a functional endogenous fim operon, the proportion of the plurality of ADAS containing T1P is increased compared to the plurality of ADAS produced by parental cells that do not constitutively express the components of T1P; e.g., increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% (e.g., increased by 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100% or more than 100%).

[0097] In some embodiments, the parental cell does not contain a complete endogenous fim operon.

[0098] In some embodiments, the present disclosure provides an ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being produced by a process comprising: (a) providing a parental cell modified to constitutively express a component of the T1P; and (b) producing the ADAS from the parental bacterial cell, the ADAS comprising the T1P.

[0099] In some embodiments, the present disclosure features an ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being produced by a process comprising: (a) providing a parental cell modified to express a component of a heterologous T1P; and (b) producing the ADAS from the parental bacterial cell, the ADAS comprising a T1P that is heterologous to the parental cell.

[0100] iii. Parental cell In some embodiments of the T1P-ADAS of Section IIA(i) or IIA(ii), the parental cell is a Gram-negative bacterial cell. The parental bacterium includes any suitable bacterial species in which the ADAS may be produced (e.g., a species that may be modified to produce the ADAS). Table 1 provides a non-limiting list of suitable Gram-negative genera from which the ADAS may be derived.

[0101] [Table 1]

[0102] In some embodiments, the Gram-negative bacterial cell is a bacterial cell of the genus Escherichia (e.g., Escherichia coli), Salmonella, Yersinia, Vibrio, Pseudomonas, Shigella, or Legionella. In some embodiments, the parental bacterial cell is a probiotic cell. In some embodiments, the parental bacterial cell is a mammalian pathogen or a mammalian commensal bacterium. In some examples, the mammalian commensal bacterium is of the genus Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus, or Actinomyces species or the mammalian pathogenic bacterium is Escherichia coli (EHEC), Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori.

[0103] In some embodiments, the parental bacterial cell is an auxotrophic parental bacterium, i.e., a parental bacterium that cannot synthesize the organic compounds required for growth. Such bacteria can grow only when the organic compounds are provided.

[0104] In some embodiments, the parental cell is a bacterium that is Escherichia coli. In some embodiments, the bacterium that is Escherichia coli is Escherichia coli CFT073.

[0105] In some embodiments, the parental cells have not been exposed to culture conditions that promote the expression of the fim operon, e.g., have not been exposed to such conditions before the production of ADAS or during the production of ADAS. In some embodiments, the culture conditions are temperature, pH, osmolarity, shaking, or activation of a stress response or a stringent response. In some embodiments, the culture conditions that promote the expression of the fim operon are growth at 37°C under static (rather than shaking) conditions. Conditions that affect the expression of the fim operon are further described in Zhang et al., Proc Natl Acad Sci USA, 113(15):4182-4187, 2016.

[0106] In some aspects, the disclosure features engineered bacteria that constitutively express components of T1P, wherein the engineered bacteria contain T1P at a level that is at least 1.1-fold higher compared to the level of T1P contained by non-engineered bacteria (e.g., at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold or more higher than the level of T1P contained by non-engineered bacteria, e.g., 1-fold to 2-fold, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 100-fold, or more than 100-fold higher compared to the level of T1P contained by non-engineered bacteria). In some embodiments, the engineered bacteria contain T1P at a level that is at least 1.5-fold higher compared to the level of T1P contained by non-engineered bacteria. In some embodiments, T1P is native T1P, e.g., T1P encoded by the fim operon that is endogenous to the bacteria. In some embodiments, T1P is heterologous T1P, e.g., T1P encoded by one or more nucleotide sequences (such as the fim operon) that are heterologous to the bacteria. In some embodiments, the bacteria are modified to produce ADAS.

[0107] In some embodiments, the disclosure features engineered bacteria that constitutively express components of T1P, which are modified to produce ADAS.

[0108] In some embodiments, the disclosure features ADAS produced by a method that includes: (a) providing engineered bacteria that constitutively express components of T1P; and (b) producing ADAS from the bacteria.

[0109] B. ADAS Containing Cargo In some embodiments, the ADAS (e.g., T1P-ADAS) provided by the present invention includes cargo, e.g., cargo contained within the ADAS. The cargo can be any moiety disposed within the ADAS (e.g., encapsulated within the ADAS) or conjugated to the surface of the ADAS. In some embodiments, the cargo includes nucleic acids, plasmids, polypeptides, proteins, enzymes, amino acids, small molecules, gene editing systems, hormones, immunomodulatory agents, carbohydrates, lipids, organic particles, inorganic particles, or ribonucleoprotein complexes (RNPs), or combinations thereof. In some embodiments, the cargo is delivered by a secretion apparatus (e.g., T3SS) included by the ADAS. In other embodiments, the cargo is not delivered by the T3SS.

[0110] In some embodiments, the nucleic acid is DNA, RNA, or a plasmid. In some embodiments, the nucleic acid (e.g., DNA, RNA (e.g., mRNA, ASO, circular RNA (circRNA), siRNA, shRNA, tRNA, dsRNA, or combinations thereof), or a plasmid) encodes a protein. In some embodiments, the protein is transcribed and / or translated in the ADAS. In some embodiments, the nucleic acid inhibits translation of a protein or polypeptide, e.g., siRNA or an antisense oligonucleotide (ASO).

[0111] In some embodiments, the cargo is an agent capable of modulating the microbiome of a target organism (e.g., the human microbiome or a non - human mammalian microbiome), such as a polysaccharide, an amino acid, an antibacterial agent (e.g., an anti - infective or antibacterial peptide, protein and / or natural product), a short - chain fatty acid, or a combination thereof. In some examples, the agent capable of modulating the host microbiome is a probiotic agent.

[0112] In some embodiments, the cargo is an enzyme. In some embodiments, the enzyme produces a target product by changing a substrate. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or environment to which the ADAS is delivered.

[0113] In certain embodiments, the cargo is modified to have improved stability compared to the unmodified version of the cargo. The "stability" of the cargo is the ratio, without units, of the half - life of the unmodified cargo to the half - life of the modified cargo when measured under the same environmental conditions. In some embodiments, the environment is experimentally controlled, e.g., a simulated body fluid, RNase - free water, cytoplasm, extracellular space, or "ADAS plasma" (i.e., the contents of the internal volume of the ADAS, e.g., after lysis). In other embodiments, the environment is an actual or simulated: mammalian intestine, mammalian skin, mammalian genital system, mammalian airway, mammalian bloodstream, or mammalian extracellular space. In certain embodiments, the ADAS does not substantially degrade the cargo.

[0114] In certain embodiments, the cargo comprises a protein. In certain embodiments, the protein has a stability higher than about 1.01, 1.1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 in the cytoplasm or other environment. The protein can be any protein including growth factors; enzymes; hormones; immunomodulatory proteins; antibiotic proteins such as antibacterial, antifungal or antiviral proteins; and targeting agents such as antibodies or nanobodies. In some embodiments, the protein is a hormone, for example, a paracrine, endocrine or autocrine hormone.

[0115] In some embodiments, the cargo is an anti-inflammatory agent, for example, a cytokine (e.g., a heterologously expressed anti-inflammatory cytokine or mutein thereof (e.g., IL-10, TGF-β, IL-22, IL-2)) or an antibody (e.g., an antibody or antibody fragment targeting tumor necrosis factor (TNF) (e.g., an anti-TNF antibody); an antibody or antibody fragment targeting IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment targeting IL-23 (e.g., an anti-IL-23 antibody)).

[0116] In certain embodiments, the cargo is an immunomodulatory agent. Examples of immunomodulatory agents include, for example, immunostimulants; checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4); chemotherapeutic agents; immunosuppressive factors; antigens; superantigens; and small molecules (e.g., cyclosporin A, cyclic dinucleotides (CDNs), or STING agonists (e.g., MK-1454)). In some embodiments, the immunomodulatory agent is a moiety that induces tolerance in a subject, such as an allergen, self-antigen (e.g., disease-associated self-antigen), or microbial-specific antigen. In some embodiments, the immunomodulatory agent is a vaccine, such as an antigen from a pathogen (e.g., a virus (e.g., a viral envelope protein) or a bacterium). In some embodiments, the antigen is a cancer neoantigen. In some embodiments, the pathogen is a coronavirus, such as SARS-CoV-2. In some embodiments, the cargo, an adjuvant, such as a molecule that alters the compartmentalization, presentation, or profile of one or more costimulatory molecules associated with an immunomodulatory molecule or vaccine antigen. In some examples, the adjuvant is an activator of an immune pathway upstream of the desired immune response (e.g., an activator of the innate immune pathway upstream of the adaptive immune response). In other examples, the adjuvant enhances the presentation of an antigen in immune cells or immune compartments (e.g., MHC class 1) of a target organism. In some examples, the adjuvant is listeriolysin O (LLO). In some embodiments, the ADAS comprises an antigen and one or more adjuvants.

[0117] In some embodiments, the cargo is an agent for treating or preventing cancer, such as an agent that reduces the likelihood that a patient will develop cancer or an agent that treats cancer (e.g., an agent that increases progression-free survival and / or overall survival of an individual having cancer).

[0118] Examples of agents for cancer prevention include, but are not limited to, anti-inflammatory agents and growth inhibitors. Examples of agents for treating cancer (e.g., solid tumor cancer) include, but are not limited to, anti-inflammatory agents, growth inhibitors, chemotherapeutic agents, immunotherapeutic agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments targeting cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as listeriolysin o), cytotoxins, inflammasome inhibitors, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0119] In some embodiments, the cargo is an enzyme. The enzyme may be an enzyme that exhibits catalytic activity in a target cell or organism (e.g., in a human or non-human mammal). In some embodiments, the catalytic activity is extracellular matrix (ECM) digestion (e.g., the enzyme is hyaluronidase and the catalytic activity is ECM digestion) or toxin removal. In some embodiments, the enzyme is enzyme replacement therapy, e.g., phenylalanine hydroxylase. In some embodiments, the enzyme is UDP-glucuronosyltransferase. In some embodiments, the enzyme has hepatic enzyme activity (e.g., porphobilinogen deaminase (PBGD), e.g., human PBGD (hPBGD)). In some embodiments, the enzyme is a protease, an oxidoreductase, or a combination thereof.

[0120] In some embodiments, the enzyme produces a target product by changing a substrate. In some embodiments, the substrate is present in the ADAS and the target product is produced in the ADAS. In other embodiments, the substrate is present in the target cell or environment to which the ADAS is delivered. In some embodiments, the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic di-AMP.

[0121] In some embodiments, the enzyme is conjugated chemically to the ADAS membrane and optionally to the outer membrane via a linker.

[0122] Alternatively, the cargo may be a nucleic acid encoding any of the enzymes described herein.

[0123] In some embodiments, the cargo is an agent that activates or inhibits the autophagy process (e.g., an activator such as listeriolysin o or an inhibitor such as IcsB).

[0124] In some embodiments, the cargo is an anti-infective agent, e.g., an antibacterial agent, e.g., an anti-infective or antibacterial peptide, protein, and / or natural product.

[0125] In some embodiments, the cargo is a protein that regulates the 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. In some embodiments, the transcription factor is a human transcription factor.

[0126] Regarding ADAS containing cargo, in some embodiments, the cargo is RNA such as circular RNA, mRNA, siRNA, shRNA, ASO, tRNA, dsRNA, or combinations thereof. In certain embodiments, the RNA has a stability higher than, for example, about 1.01, 1.1, 10, 100, 1000, 10000, 100000, 100000, or 10000000 in the ADAS plasma. The RNA cargo can be stabilized in certain embodiments, for example, by the addition of a step-loop structure such as a tRNA scaffold (e.g., non-human tRNALys3 and E. coli tRNAMet (Nat. Methods, Ponchon 2007). Both are well-characterized and recombinantly expressed. However, various other types such as aptamers, lncRNAs, ribozymes, etc. may also be used similarly. The RNA can also be stabilized when one or more ribonucleases obtain ADAS from a null (or hypomorphic) parental strain.

[0127] In some detailed embodiments, the RNA is protein-coding mRNA. In more detailed embodiments, the protein-coding mRNA encodes an enzyme (e.g., an enzyme that confers hepatic enzyme activity such as human PBGD (hPBGD) mRNA) or an antigen, for example, an antigen that elicits an immune response (such as an antigen that elicits a strong and persistent neutralizing antibody titer), such as mRNA encoding CMV glycoprotein gB and / or the pentameric complex (PC). In certain detailed embodiments, the RNA is a small non-coding RNA such as shRNA, ASO, tRNA, dsRNA, or combinations thereof.

[0128] In certain embodiments, the ADAS provided by the present invention includes a cargo comprising at least one component of a gene editing system. The components of a "gene editing system" include, along with suitable associated nucleic acids, proteins (or nucleic acids encoding said proteins) that can modify a DNA sequence of interest, such as genomic DNA sequences, for example, by insertion or deletion of the sequence of interest or by changing the methylation state of the sequence of interest, and nucleic acids related to the function of such proteins, such as guide RNAs (or encoding them). Exemplary gene editing systems include those based on the Cas system, such as the Cas9, Cpf1 or other RNA-based targeting systems with their accompanying RNAs (e.g., sequence complementary CRISPR guide RNAs), as well as zinc finger nucleases and TAL effectors conjugated to nucleases.

[0129] Other embodiments of the ADAS provided by the present invention include DNA as cargo, including plasmids, and optionally this DNA contains a protein coding sequence. Exemplary DNA cargo includes, in certain embodiments, a plasmid encoding a target RNA sequence (see the above examples), which may, for example, have tRNA inserts flanking both sides. Various DNA cargoes are encompassed by the present invention, including those that produce ADAS (e.g., drive FTZ overexpression, exonuclease that degrades the genome); long-lived plasmids (ATP synthase expression, rhodopsin expression); those that express stabilized non-coding RNA, tRNA, lncRNA; those that express a secretion apparatus tag protein, NleE2 effector domain, and localization tag; secretion apparatus T3 / 4SS, T5SS, T6SS; logic circuits, secretion apparatus that are conditionally expressed; and combinations thereof. In some embodiments, the logic circuit includes an IPTG-inducible Plac promoter and the hrpR portion of an AND gate, and inducible expression or repression cassettes such as, for example, the heat-inducible promoter pL (derived from λ phage, which is normally repressed by a heat-labile protein) and the hrpS portion of an AND gate. To operate an OR gate, the system described by Rosado et al., PLoS Genetics, 2018 can be used. Briefly, a cis-repressed mRNA encoding RFP can be used under a constitutive promoter. Next, this repression can be removed in the presence of RAJ11 sRNA. Next, a plasmid containing both the IPTG-inducible promoter PLac and the heat-inducible promoter pL that induce the expression of both RAJ11 sRNA can be used. The output is then likely to be RFP expression, which is seen in response to either input. These systems can be adapted to various sensor-type functions.

[0130] In some embodiments, the ADAS provided by the present invention includes a transporter in the membrane. In certain embodiments, the transporter is specific for glucose, sodium, potassium, metal ions, anionic solutes, cationic solutes, or water.

[0131] In some embodiments, the ADAS membrane provided by the present invention contains an enzyme. In a detailed embodiment, the enzyme is a protease, an oxidoreductase, or a combination thereof. In some embodiments, the enzyme is conjugated chemically, optionally via a linker, to the outer membrane of the ADAS membrane.

[0132] C. ADAS Containing a Bacterial Secretion Apparatus In certain embodiments, the T1P-ADAS provided by the present invention contains a bacterial secretion apparatus (e.g., an endogenous bacterial secretion apparatus or a heterologous bacterial secretion apparatus). A "bacterial secretion apparatus" is a protein, or protein complex, that can export a cargo from the cytoplasm of a bacterial cell (or, e.g., an ADAS derived therefrom) to the extracellular space, the periplasmic space of a Gram-negative bacterium, or the intracellular space of another cell. In some embodiments, the bacterial secretion apparatus functions by an active (e.g., ATP-dependent or PMF-dependent) process, and in certain embodiments, the bacterial secretion apparatus includes a tube or spike that spans from a host cell (or ADAS) to a target cell. In other embodiments, the bacterial secretion apparatus is a transmembrane channel. Exemplary bacterial secretion apparatuses include the tube-containing structures T3SS and T4SS (and T3 / T4SS as defined below), where the cargo moves through the inside of a protein tube, and T6SS, which carries a cargo at the tip of a spike. Other exemplary bacterial secretion apparatuses include the transmembrane types T1SS, T2SS, T5SS, T7SS, Sec, and Tat.

[0133] In some embodiments, the ADAS (e.g., T1P-ADAS) provided by the present invention contains a heterologous bacterial secretion apparatus. In some embodiments, the heterologous bacterial secretion apparatus is a type 3 secretion apparatus (T3SS). In some embodiments, the cargo contains a component that directs export by the bacterial secretion apparatus.

[0134] In some embodiments, the T3SS is from the genera in Table 2.

[0135] In some embodiments, the parental bacterial cell does not contain an endogenous T3SS.

[0136] [Table 2]

[0137] In some embodiments, the T3SS is a Salmonella T3SS, a Vibrio T3SS, an Escherichia T3SS, a Yersinia T3SS, a Shigella T3SS, a Pseudomonas T3SS, or a Chlamydia T3SS. In some embodiments, the Salmonella T3SS is a Salmonella enterica T3SS. In some embodiments, the Vibrio T3SS is a Vibrio parahaemolyticus T3SS. In some embodiments, the Escherichia T3SS is an enteropathogenic Escherichia coli (EPEC) T3SS. In some embodiments, the Yersinia T3SS is a Yersinia enterocolitica T3SS. In some embodiments, the Shigella T3SS is a Shigella flexneri T3SS.

[0138] In some embodiments, the parental bacterial cell comprises one or more heterologous nucleotide sequences encoding components of the T3SS. In some embodiments, the one or more nucleotide sequences encoding components of the T3SS are carried on a vector. In some embodiments, the parental bacterial cell is transiently transformed by the vector. In some embodiments, the parental bacterial cell is stably transformed by the vector. In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS. In some embodiments, this moiety is a transcriptional activator of one or more heterologous nucleotide sequences encoding components of the T3SS.

[0139] In another aspect, the disclosure features a T1P-ADAS derived from a parental bacterial cell, the T1P-ADAS comprising a bacterial type III secretion apparatus (T3SS) that is endogenous to the parental bacterial cell, where the parental bacterial cell is modified such that the level of an endogenous protein or polypeptide having the ability to be secreted by the T3SS is reduced.

[0140] In some embodiments, the parental bacterial cell is modified by deleting a transcriptional activator of an endogenous protein or polypeptide having the ability to be secreted by the T3SS.

[0141] In some embodiments, the parental bacterial cell is a Gram-negative bacterial cell.

[0142] In some embodiments, the parental bacterial cell is from the genus of Table 2.

[0143] In some embodiments, the parental bacterial cell is of the species Salmonella, Vibrio, Escherichia, Yersinia, or Shigella. In some embodiments, the species Salmonella is Salmonella enterica. In some embodiments, the species Vibrio is Vibrio parahaemolyticus. In some embodiments, the species Escherichia is enteropathogenic Escherichia coli (EPEC). In some embodiments, the species Yersinia is Yersinia enterocolitica. In some embodiments, the species Shigella is Shigella flexneri.

[0144] In some embodiments, the parental bacterial cell further comprises a moiety that increases the level of the T3SS in the ADAS. In some embodiments, this moiety is a transcriptional activator of the nucleotide sequence encoding a component of the T3SS.

[0145] In some embodiments, the parental bacterial cell is modified such that the level of a negative regulator of a component of the T3SS is reduced. In some embodiments, the chromosomal locus encoding the negative regulator is deleted from the parental bacterial cell.

[0146] In some embodiments, the parental bacterial cell is modified such that the level of one or more of LPS; proteins not essential for metabolism; toxins not associated with the T3SS; endotoxin; flagella; and pili is reduced.

[0147] In some embodiments, the ADAS further comprises at least one cargo, wherein the T3SS has the ability to deliver that cargo to a target cell. In some embodiments, the delivery is delivery to the cytoplasm of the target cell.

[0148] In some embodiments, the cargo is a protein or polypeptide.

[0149] In some embodiments, the cargo is endogenously secreted by the T3SS.

[0150] In some embodiments, the ADAS or parental bacterial cell is modified such that the level of cargo in the ADAS is increased.

[0151] In some embodiments, the cargo is not endogenously secreted by the T3SS.

[0152] In some embodiments, the cargo is endogenously secreted by a T3SS of a species other than the ADAS T3SS species.

[0153] In some embodiments, the cargo is endogenously secreted by a type IV secretion apparatus (T4SS) or a type VI secretion apparatus (T6SS).

[0154] In some embodiments, the cargo is modified for delivery by the T3SS.

[0155] In some embodiments, the cargo is an enzyme, a DNA modifier, a chromatin remodeling agent, a gene editing agent, a nuclear targeting agent, a binder, an immunogenic agent, or a toxin. In some embodiments, the enzyme is a metabolic enzyme. In some embodiments, the gene editing agent is a component of the CRISPR system. In some embodiments, the nuclear targeting agent is a transcription factor. In some embodiments, the binder is an antibody or antibody fragment. In some embodiments, the binder is a VHH molecule. In some embodiments, the immunogenic agent is an immunostimulant. In some embodiments, the immunogenic agent is an immunosuppressant.

[0156] In some embodiments, the cargo is modified by the addition of a secretion signal. In some embodiments, the secretion signal is the sequence of Table 3.

[0157]

Table 3

[0158] In some embodiments, the ADAS includes a cargo, where the cargo includes a moiety that directs export by a bacterial secretion apparatus. In some embodiments this moiety is a Pho / D, Tat, or synthetic peptide signal.

[0159] In certain embodiments, the ADAS provided by the present invention is a double-membrane ADAS. In a more detailed embodiment, the double-membrane ADAS further includes a bacterial secretion apparatus. In an even more detailed embodiment, the bacterial secretion apparatus is selected from a T3SS, T4SS, T3 / 4SS, or T6SS, and optionally this T3SS, T4SS, T3 / 4SS, or T6SS has a weakened or non-functional effector that does not affect the fitness of the target cell.

[0160] In some embodiments, the bacterial secretion apparatus has the ability to export a cargo across the outer membrane of the ADAS into a target cell, such as a mammalian cell, such as a T3SS, T4SS, T3 / T4SS or T6SS.

[0161] In some embodiments, the bacterial secretion apparatus is a T3 / 4SS. "T3 / 4SS" is a secretion apparatus based on a T3SS or T4SS, including hybrid and unmodified versions, which forms a protein tube that connects these two between a bacterium (or ADAS, e.g., T1P-ADAS) and a target cell to deliver one or more effectors. In some embodiments, the target cell is a mammalian cell. In some embodiments, the T3 / 4SS includes an effector, which may be a modified effector. Examples of T3SS apparatuses include the Salmonella SPI-1 apparatus, the EHEC coli ETT1 apparatus, the Xanthomonas citri / campestri T3SS apparatus, and the Pseudomonas syringae T3SS apparatus. Examples of T4SS apparatuses include the Agrobacterium Ti plasmid apparatus, the Helicobacter pylori T4SS. In certain embodiments, the T3 / 4SS has a modified effector function, e.g., an effector selected from SopD2, SopE, Bop, Map, Tir, EspB, EspF, NleC, NleH2, or NleE2. In more detailed embodiments, the modified effector function is a function of intracellular targeting, such as translocation into the nucleus, Golgi, mitochondria, actin, microvilli, ZO-1, microtubules, or cytoplasm. In even more detailed embodiments, the modified effector function is nuclear targeting based on NleE2 derived from E. coli. In other detailed embodiments, the modified effector function is a function of filopodia formation, tight junction disruption, microvilli disappearance, or SGLT-1 inactivation.

[0162] In some embodiments, the ADAS provided by the present invention comprising a bacterial secretion apparatus comprises a T6SS. In some embodiments, the T6SS comprises effectors that target and kill bacteria in its natural host. In certain specific embodiments, the T6SS is derived from Pseudomonas putida K1-T6SS, and optionally, this effector comprises the amino acid sequence of Tke2 (accession number AUZ59427.1), or a functional fragment thereof. In other embodiments, the T6SS comprises effectors that target and kill fungi in its natural host. For example, the T6SS is derived from Serratia marcescens, and the effector comprises the amino acid sequence of Tfe1 (Genbank: SMDB11_RS05530) or Tfe2 (Genbank: SMDB11_RS05390).

[0163] In some embodiments of the ADAS provided by the present invention comprising a bacterial secretion apparatus, the bacterial secretion apparatus has the ability to translocate cargo extracellularly. In certain more specific embodiments, the bacterial secretion apparatus is a T1SS, T2SS, T5SS, T7SS, Sec, or Tat.

[0164] D. ADAS and highly active ADAS derived from parental bacteria lacking a cell division topology specificity factor In some aspects, the present invention provides a composition comprising a T1P-ADAS and / or a plurality of T1P-ADAS derived from a parental bacterium having a reduced level, activity or expression of a cell division topology specificity factor.

[0165] In some aspects, the present invention provides a composition comprising a plurality of T1P-ADAS that do not comprise a cell division topology specificity factor and that substantially do not contain viable bacterial cells.

[0166] In some embodiments, T1P-ADAS is produced by a process comprising: (a) making, providing, or obtaining a plurality of parental bacteria described herein having a reduced level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of minicells, thereby producing highly active ADAS; and (c) separating the ADAS from the parental bacteria, thereby producing a composition substantially free of viable bacterial cells.

[0167] In some embodiments of the above aspects, the cell division topology-specific factor is a polypeptide having an amino acid sequence that is at least 20% identical to the E. coli minE polypeptide (SEQ ID NO: 25), for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 25. In some embodiments, the cell division topology-specific factor comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cell division topology-specific factor is a minE polypeptide. Exemplary species having the minE polypeptide are provided in Table 1 and Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005.

[0168] In some embodiments, the parental bacteria are E. coli and the minE polypeptide is E. coli minE. In other embodiments, the parental bacteria are Salmonella typhimurium and the minE polypeptide is S. typhimurium minE. In still other embodiments, the parental bacteria are the bacteria of Table 1 and the cell division topology-specific factor is the endogenous minE or DivIVA of the parental bacteria.

[0169] In some embodiments of the above aspect, the cell division topology specificity factor is a polypeptide having an amino acid sequence with at least 20% identity to the Bacillus subtilis DivIVA polypeptide (SEQ ID NO: 28), for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 28. In some embodiments, the cell division topology specificity factor comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the cell division topology specificity factor is a DivIVA polypeptide. Exemplary species having the DivIVA polypeptide are provided in Table 1 and Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005. In some embodiments, the parent bacterium is Bacillus subtilis and the cell division topology specificity factor is B. subtilis DivIVA.

[0170] In some embodiments, the ADAS or parent bacterium having a decrease in the level or activity of the cell division topology specificity factor also has a decrease in the level of one or more Z-ring inhibitory proteins.

[0171] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence with at least 20% identity to the E. coli minC polypeptide (SEQ ID NO: 26), for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 26. In some embodiments, the Z-ring inhibitory protein comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Z-ring inhibitory protein is a minC polypeptide.

[0172] In some embodiments, the Z-ring inhibitory protein is a polypeptide having an amino acid sequence that is at least 20% identical to the E. coli minD polypeptide (SEQ ID NO: 27), for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 27. In some embodiments, the Z-ring inhibitory protein comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Z-ring inhibitory protein is a minD polypeptide.

[0173] In some embodiments, the ADAS or the parental bacterium has a decrease in the level, activity or expression of at least two Z-ring inhibitory proteins. In some embodiments, the ADAS or the parental bacterium has a decrease in the expression of the minC polypeptide and the minD polypeptide. In some embodiments, the ADAS or the parental bacterium has a decrease in the expression of the minC polypeptide, the minD polypeptide and the minE polypeptide, for example, a deletion of the minCDE operon (ΔminCDE).

[0174] A decrease in the level, activity or expression of a cell division topology-specific factor or a Z-ring inhibitory protein, for example, a decrease in an ADAS or a decrease in a parental bacterial cell, may be achieved using any suitable method. For example, in some embodiments, the decrease in level or activity occurs by a loss-of-function mutation, such as a gene deletion. In some embodiments, the loss-of-function mutation is an inducible loss-of-function mutation, and the loss of function is induced by exposing the parental cell to an inducing condition. For example, the inducible loss-of-function mutation is a temperature-sensitive mutation, and the inducing condition is a temperature condition.

[0175] In some embodiments, the parental cell has a deletion of the minCDE operon (ΔminCDE) or a deletion of a homologous operon.

[0176] An ADAS lacking E. protease, RNase and / or LPS In another aspect, the present invention provides a composition comprising a plurality of T1P-ADAS, wherein the ADAS has a reduced protease level or activity compared to an ADAS produced from wild-type parental bacteria. In some embodiments, the ADAS is produced from parental bacteria that have been modified such that the expression of at least one protease is reduced or eliminated.

[0177] In some embodiments, the ADAS has a reduced RNase level or activity compared to an ADAS produced from wild-type parental bacteria. In some embodiments, the ADAS is produced from parental bacteria that have been modified such that the expression of at least one RNase is reduced or eliminated. In some embodiments, the RNase is an endoribonuclease or an exoribonuclease.

[0178] In another aspect, the present invention provides a composition comprising a plurality of T1P-ADAS, wherein the ADAS is modified to have reduced lipopolysaccharide (LPS). In some embodiments, the modification is a mutation of lipid A biosynthetic myristoyltransferase (msbB).

[0179] In certain embodiments, the T1P-ADAS provided by the present invention lacks one or more metabolically non-essential proteins. "Metabolically non-essential proteins" include, but are not limited to, pili, flagella, unwanted secretion apparatuses, transposases, effectors, phage elements, or regulatory elements such as flhC or OmpA. In some embodiments, the ADAS provided by the present invention lacks one or more of RNase, protease, or combinations thereof, and in a specific embodiment, lacks one or more endoribonucleases (such as RNase A, RNase h, RNase III, RNase L, RNase PhyM, etc.) or exoribonucleases (such as RNase R, RNase PH, RNase D, etc.); or serine, cysteine, threonine, aspartic acid, glutamic acid, and metalloproteases; or combinations of any of the foregoing.

[0180] F. ADAS Containing Targeting Components In another embodiment, the T1P-ADAS contains a targeting component. In some embodiments, the targeting moiety is a nanobody, a lectin, or a tumor targeting peptide. In some embodiments, the targeting moiety is an endogenous surface ligand of the parental cell (e.g., a surface ligand genetically inherited by the ADAS). In other embodiments, the targeting moiety is an exogenous ligand (e.g., an exogenous tissue targeting ligand) added to the ADAS using any of the ADAS modification methods described herein. The targeting moiety may facilitate tissue-related targeting of the ADAS to a certain tissue type or cell type.

[0181] In certain embodiments, the nanobody is a nanobody directed against a tumor antigen such as HER2, PSMA, or VEGF-R. In other embodiments, the lectin is a lectin, such as mannose-binding lectin (MBL). In still other embodiments, the tumor targeting peptide is an RGD motif or a CendR peptide.

[0182] G. ADAS Containing Additional Moieties The T1P-ADAS provided by the present invention can contain various additional components, including, for example, optoelectrochemical pumps, retinoids and retinal-producing cassettes, metabolic enzymes, targeting agents, cargos, bacterial secretion apparatuses, and transporters, including the aforementioned combinations, including the following specific detailed embodiments. In certain embodiments, the ADAS lacks other elements, such as metabolically non-essential genes and / or certain nucleases or proteases.

[0183] The T1P-ADAS contains, in certain embodiments, a functional ATP synthase and, in some embodiments, a proton pump embedded in the membrane. In some embodiments, the highly active ADAS is at least: 10000 nm 2 per 1, 5000 nm 2 per 1, 3500 nm2 Per 1, 1000 nm 2 It has an ATP synthase concentration of 1 per. In certain embodiments, the ADAS provided by the present invention includes an ATP synthase optionally lacking a regulatory domain, such as lacking an ε domain. The deletion can be achieved by various means. In certain embodiments, the deletion is due to an inducible deletion of the native ε domain. In certain embodiments, the deletion can be achieved by the adjacency of LoxP sites and inducible Cre expression or CRISPR knockout or can be made inducible (e.g., placed under the tTa tet transactivator on a plasmid in an ATP synthase knockout strain).

[0184] TIP-ADAS can, in some embodiments, include an optoelectrogenic proton pump. In certain embodiments, the optoelectrogenic proton pump is proteorhodopsin. In a more detailed embodiment, the proteorhodopsin includes the amino acid sequence of proteorhodopsin from uncultured marine bacterium clade SAR86, GenBank accession number AAS73014.1. In other embodiments, the optoelectrogenic proton pump is a Gloeobacter rhodopsin. In certain embodiments, the optoelectrogenic proton pump is bacteriorhodopsin, deltarhodopsin or halorhodopsin from Halobium salinarum, Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica or Haloarcula marismortui.

[0185] In some embodiments, the T1P-ADAS provided by the present invention further includes retinal. In certain embodiments, the ADAS provided by the present invention further includes a retinal synthesis protein (or protein apparatus) or a nucleic acid encoding the same.

[0186] In certain embodiments, the T1P-ADAS provided by the present invention further comprises one or more glycolytic pathway proteins. In some embodiments, the glycolytic pathway protein is, for example, phosphofructokinase (Pfk-A) comprising the amino acid sequence of UniProt accession number P0A796 or a functional fragment thereof. In other embodiments, the glycolytic pathway protein is, for example, triosephosphate isomerase (tpi) comprising the amino acid sequence of UniProt accession number P0A858 or a functional fragment thereof.

[0187] H. Highly active ADAS In some embodiments, the present invention provides a highly active T1P-ADAS. A "highly active" ADAS is an ADAS with a high potential for function, for example, an ADAS having the ability to perform a large amount of useful functions. Function refers to metabolic functions under suitable conditions, for example, including chemical synthesis (for example, synthesis of proteins, nucleic acids, lipids, carbohydrates, polymers, or small molecules), chemical modification (for example, modification of proteins, nucleic acids, lipids, carbohydrates, polymers or small molecules), or transport (for example, intracellular translocation, extracellular translocation, or secretion). In some embodiments, the highly active ADAS begins with a large pool of energy in the form of energy, for example, adenosine triphosphate (ATP). In other embodiments, the ADAS has the ability to take up or generate energy (for example, ATP) from another source.

[0188] In some embodiments, the 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. The ATP concentration can be evaluated by various means, including, in certain embodiments, the BacTiter-Glo™ assay (Promega) for the dissolved ADAS.

[0189] In addition to or instead of, high activity may be determined as the rate of increase or the amount of increase in ATP concentration in the ADAS over time. In some embodiments, the ATP concentration of the ADAS increases by at least 50%, at least 60%, at least 75%, at least 100%, at least 150%, at least 200%, or more than 200% after incubation under suitable conditions, for example, after incubation at 37 °C for 12 hours. In certain embodiments, the highly active ADAS has an ATP production rate of about: 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2, 3, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 500, 1000, 10000 ATP / sec / nm 2 has a higher ATP production rate for at least about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, or 2 weeks.

[0190] In other aspects, high activity is determined as the rate of decrease in ATP concentration over time. In some embodiments, the ATP concentration of the highly active ADAS may not decrease as rapidly as that of the non-highly active ADAS. In some embodiments, the drop in ATP concentration in the ADAS or 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) when measured using, for example, the BacTiter-Glo™ assay (Promega).

[0191] In addition to or alternatively, high activity may be determined as a longevity index of the ADAS. The longevity index is calculated as the ratio of the GFP production rate at 24 hours to that at 30 minutes. In some embodiments, high activity ADAS has a longevity index of 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 higher. In more detailed embodiments, the longevity index is measured in ADAS containing a functional GFP plasmid together with a promoter appropriate for the species, where the GFP concentration is measured by a plate reader at 30 minutes and 24 hours with respect to the number of ADAS, the average number of plasmids per ADAS, and the solution volume.

[0192] In some embodiments, the ADAS produces a protein, such as a heterologous protein. In some embodiments, high activity is determined as the production rate, production amount, or production period of the protein or the rate of induction of protein expression (e.g., the responsiveness of the ADAS to a signal). For example, the ADAS may comprise a plasmid that includes an inducible promoter and a nucleotide sequence encoding a heterologous protein, where when the ADAS is contacted with an inducer of the inducible promoter under appropriate conditions, the heterologous protein will be produced. In some embodiments, the production of the heterologous protein is increased by at least 1.6-fold in the ADAS contacted with the inducer, such as the high-activity ADAS, compared to the ADAS not contacted with the inducer. For example, in some embodiments, 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 the ADAS contacted with the inducer, such as the high-activity ADAS. In some embodiments, the production rate of the heterologous protein by the high-activity ADAS reaches a target level within a specific period after contacting the ADAS with the inducer, such as within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or more than 3 hours. In some embodiments, the protein (e.g., heterologous protein) is produced at a rate of at least 0.1 femtograms per hour per high-activity ADAS, such as at least 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10, 25, 50, 100, 250, 500, 1000, 2000, 3000, or 3500 fg / hour per ADAS. In some embodiments, the high activity of the ADAS is determined as the period during which the protein is produced. The high-activity ADAS can produce a protein (e.g., heterologous protein) over a period of at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or more than 48 hours.

[0193] I. ADAS Compositions and Formulations The present invention provides, inter alia, a composition or preparation containing a T1P-ADAS provided by the present invention, which includes an ADAS derived from a parental cell constitutively expressing a T1P component and an ADAS derived from a parental cell containing a heterologous T1P component. In some embodiments, the ADAS preparation substantially does not contain viable cells. Collectively, these are referred to as "a plurality of compositions provided by the present invention" or "one composition provided by the present invention", etc., and may contain any ADAS provided by the present invention and any combination of the ADAS provided by the present invention.

[0194] In some embodiments, the composition provided by the present invention includes a plurality of ADASs, and about 60%, 65%, 70%, 75%, 80%, 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 100% of the ADASs contain T1P.

[0195] The composition provided by the present invention can be prepared into any suitable formulation. For example, the formulation may be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosol, or spray administration. In some embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.

[0196] In some embodiments, the ADAS composition described herein contains viable bacterial cells less than 100 colony forming units (CFU / mL), for example, less than 50 CFU / mL, less than 20 CFL / mL, less than 10 CFU / mL, less than 1 CFU / mL, or less than 0.1 CFU / mL of viable bacterial cells.

[0197] In some embodiments, the present invention provides an ADAS composition, where the ADAS is lyophilized and reconstituted, and the reconstituted ADAS has at least 90% of the T1P level of the non-lyophilized ADAS, for example, at least 95%, 98% of the T1P level of the non-lyophilized ADAS, or a T1P level equal to at least that (e.g., the amount of T1P on the surface of the ADAS or the proportion of the ADAS containing T1P).

[0198] In some embodiments, the present invention provides an ADAS composition, which is stored, for example, stored at 4°C. After storage, the ADAS has at least 90% of the T1P level of the non-stored ADAS, for example, at least 95%, 98%, or a T1P level equal to at least that of the non-stored ADAS. In some embodiments, the storage lasts for at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 6 months, or at least 1 year.

[0199] In some embodiments, the ADAS may be maintained or in some "rest" state and can then be rapidly activated.

[0200] In some embodiments, the ADAS composition is formulated for delivery to a mammal, for example, formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosol, or spray administration. In some embodiments, the composition is formulated for oral delivery.

[0201] In some embodiments, the composition is formulated as a liquid, solid, aerosol, paste, gel, or gas composition.

[0202] III. Method for manufacturing ADAS A. Preparation of ADAS In some embodiments, the present disclosure features a method for producing an ADAS, the method including: (a) providing engineered bacteria that constitutively express a component of T1P; and (b) producing the ADAS from the bacteria.

[0203] Parent bacteria include any suitable bacterial species from which an ADAS may be produced (e.g., species that may be modified to produce an ADAS using the methods described herein). Table 1 provides a non-limiting list of suitable genera from which an ADAS may be derived.

[0204] In some embodiments, the invention features a method for manufacturing any of the ADAS compositions described in Section I herein, e.g., a T1P-ADAS composition. For example, methods for making an ADAS derived from a parent cell that constitutively expresses a T1P component, methods for making an ADAS derived from a parent cell that includes a heterologous T1P component, and methods for making any of the ADASs referred to herein are provided herein, where the ADAS includes a cargo.

[0205] In some embodiments, the ADAS (e.g., T1P-ADAS) is generated from a parental strain that is a human bacterium, such as a commensal human bacterium (e.g., Escherichia coli, Staphylococcus sp., Bifidobacterium sp., Micrococcus sp., Lactobacillus sp., or Actinomyces sp.) or a pathogenic human bacterium (e.g., Escherichia coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori), or an extremophilic microorganism.

[0206] In some embodiments, the ADAS and / or the parent strain are any of the functionalized derivatives described above, including, for example, a functional cassette that induces bacteria to perform one or more of antibacterial secretion, plastic digestion, toxin secretion, survival in extreme environments, nanoparticle creation, incorporation into other organisms, environmental response, and reporter signal generation.

[0207] The parent bacteria can include any of the functionalized derivatives described above, including, for example, a functional cassette that induces bacteria to perform one or more of secreting antimicrobial agents, digesting plastics, secreting toxins, surviving in extreme environments, creating nanoparticles, being incorporated into other organisms, responding to the environment, and generating reporter signals.

[0208] In some embodiments, the ADAS is derived from a parent strain engineered or induced to overexpress ATP synthase. In some more detailed embodiments, the ATP synthase is heterologous to the parent strain. In certain specific embodiments, the parent strain is modified to express functional F o F1 ATP synthase.

[0209] In certain embodiments, the ADAS provided by the present invention is obtained from a parent strain cultured under conditions selected from an applied voltage (e.g., 37 mV), a non-atmospheric oxygen concentration (e.g., 1 - 5% O2, 5 - 10% O2, 10 - 15% O2, 25 - 30% O2), a low pH (about: 4.5, 5.0, 5.5, 6.0, 6.5), or a combination thereof.

[0210] Thanks to the diversity of bacteria, the ADAS can be made with a membrane modified such that, for example, the in vivo distribution of the ADAS upon administration to target cells is improved. In certain embodiments, the membrane is modified to have reduced immunogenicity or immunostimulatory properties in mammals. For example, in certain embodiments, the ADAS is obtained from a parental strain where the immunostimulatory ability of the parental strain has been reduced or eliminated by detergents, post-production treatment with enzymes, or functionalization with PEG. In certain embodiments, the ADAS is created from a parental strain and the membrane is modified by knockout of the LPS synthesis pathway in the parental strain, for example by knocking out msbB. In other detailed embodiments, the ADAS is created from a parental strain that produces cell wall-deficient particles upon exposure to high osmotic pressure conditions.

[0211] In some embodiments, the method involves transforming the parental strain with an inducible DNase system such as exoI (NCBI GeneID:946529) and sbcD (NCBI GeneID:945049) nucleases, or I-CeuI (e.g., Swissprot:P32761.1) nuclease. In more detailed embodiments, the method involves using a single, double, triple, or quadruple auxotrophic strain and having a complementing gene on a plasmid encoding the inducible nuclease.

[0212] In some embodiments of the method provided by the present invention, the parental strain is cultured under conditions selected from an applied voltage (e.g., 37 mV), a non-atmospheric oxygen concentration (e.g., 1 - 5% O2, 5 - 10% O2, 10 - 15% O2, 25 - 30% O2), a low pH (4.5 - 6.5), or a combination thereof.

[0213] In certain embodiments of the method provided by the present invention, the parental strain lacks flagella and undesirable secretion apparatuses, and optionally the flagella and undesirable secretion apparatuses are removed using λRed recombinase.

[0214] In some embodiments, in the method provided by the present invention, a flagellar control component is excised from the parental strain genome by inserting a plasmid containing a CRISPR domain targeting flagellar control genes such as, for example, flhD and flhC.

[0215] In certain embodiments, the method provided by the present invention is a method for creating a highly active ADAS, where the ADAS containing a plasmid encoding a rhodopsin gene is cultured in the presence of light. In a more detailed embodiment, the rhodopsin is proteorhodopsin from SAR86 uncultured bacteria having the amino acid sequence of GenBank accession number AAS73014.1, or a functional fragment thereof. In an even more detailed embodiment, the culture is supplemented with retinal. In other more detailed embodiments, the rhodopsin is proteorhodopsin and the plasmid further contains a gene for synthesizing retinal (such a plasmid is the pACYC-RDS plasmid from Kim et al., Microb Cell Fact, 2012).

[0216] In certain detailed embodiments, the parental strain then contains a nucleic acid sequence encoding a nanobody that is expressed on the membrane of the ADAS.

[0217] In some embodiments of the method provided by the present invention, the parental strain contains a nucleic acid sequence encoding one or more bacterial secretion apparatus operons. Exemplary plasmids include the Salmonella SPI-1 T3SS, Shigella flexneri T3SS, Agrobacterium Ti plasmid, and Pseudomonas putida K1-T6SS apparatus.

[0218] In certain embodiments, the parental strain contains a cargo. In some embodiments, the parental strain contains a nucleic acid sequence encoding a set of genes for synthesizing a small molecule cargo.

[0219] IV. Purification of ADAS and ADAS Compositions In some embodiments of the methods and compositions provided herein, the ADAS is purified from a composition (e.g., a culture) comprising viable bacteria, such as parental bacteria. An exemplary method for producing a composition comprising a plurality of ADAS and substantially free of viable bacterial cells comprises: (a) creating, providing, or obtaining a plurality of parental bacteria that exhibit a decrease in the level or activity of a cell division topology-specific factor; (b) exposing the parental bacteria to conditions that permit the formation of minicells, thereby producing the ADAS; and (c) separating the ADAS from the parental bacteria, thereby producing a composition substantially free of viable bacterial cells.

[0220] Purification separates the ADAS from viable parental bacterial cells, which contain the genome and can be larger. Separation of highly active ADAS from parental bacteria can be carried out using several methods as described herein. Exemplary purification methods described herein include centrifugation, selective growth, and buffer exchange / concentration processes.

[0221] In some aspects, provided herein are ADAS compositions and methods of comparing such compositions that are substantially free of parental bacterial cells and / or viable bacterial cells, e.g., having 500 or fewer colony forming units (CFU) per mL, 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. In some embodiments, an ADAS composition substantially free of parental bacterial cells may be completely free of bacterial cells.

[0222] For the production of ADAS provided by the present invention, an auxotrophic parent strain can be used. As described in more detail below, such a production method is useful for the purification of ADAS. For example, after the production of ADAS, the parent bacterial cells may be removed by growing them in a medium lacking nutrients (e.g., amino acids) essential for the viability of the parent bacteria. In some embodiments, the ADAS provided by the present invention is derived from an auxotrophic parent strain with respect to at least 1, 2, 3, 4, or more of arginine (e.g., knockout of argA, strains JW2786-1 and NK5992, etc.), cysteine knockout of cysE (strains JW3582-2 and JM15, etc.), glutamine, e.g., knockout of glnA (strains JW3841-1 and M5004, etc.), glycine, e.g., knockout of glyA (strains JW2535-1 and AT2457, etc.), histidine, e.g., knockout of hisB (strains JW2004-1 and SB3930, etc.), isoleucine, e.g., knockout of ilvA (strains JW3745-2 and AB1255, etc.), leucine, e.g., knockout of leuB (strains JW5807-2 and CV514, etc.), lysine, e.g., knockout of lysA (strains JW2806-1 and KL334, etc.), methionine, e.g., knockout of metA (strains JW3973-1 and DL41, etc.), phenylalanine, e.g., knockout of pheA (strains JW2580-1 and KA197, etc.), proline, e.g., knockout of proA (strains JW0233-2 and NK5525, etc.), serine, e.g., knockout of serA (strains JW2880-1 and JC158, etc.), threonine, e.g., knockout of thrC (strains JW0003-2 and Gif 41, etc.), tryptophan, e.g., knockout of trpC (strains JW1254-2 and CAG18455, etc.), tyrosine, e.g., knockout of tyrA (strains JW2581-1 and N3087, etc.), valine / isoleucine / leucine, e.g., knockout of ilvd (strains JW5605-1 and CAG18431, etc.).

[0223] In certain embodiments, the method involves using a single, double, triple, or quadruple auxotrophic parental strain, optionally further comprising a plasmid expressing ftsZ.

[0224] Method of using V.ADAS Method of delivering A.ADAS In some aspects, the present disclosure features a method of delivering ADAS to cells, the method comprising: (a) providing a composition comprising a plurality of ADASs (e.g., T1P-ADAS) of the invention; and (b) contacting the cells with the composition of step (a).

[0225] In some embodiments, the delivery of ADAS to cells is increased by at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more) compared to ADAS derived from control (e.g., unmodified) parental cells (e.g., increased by 1% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, 90% - 100% or more compared to ADAS derived from control parental cells). In some aspects, the delivery of ADAS to cells is increased by at least 10% compared to ADAS derived from control parental cells.

[0226] In some embodiments, an effective amount of the ADAS is delivered to the cells at a dose that is at least 1% lower than the dose required for the ADAS derived from the control parental cells (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% (e.g., increased by 1% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90% or 90% - 99% lower than the dose required for the ADAS derived from the control parental cells). In some embodiments, an effective amount of the ADAS is delivered to the cells at a dose that is at least 10% lower than the dose required for the ADAS derived from the control parental cells.

[0227] In some embodiments, the cells are mammalian cells (e.g., human cells or non - human mammalian cells including cells of livestock animals, domestic animals or harmful animals). In some embodiments, the mammalian cells are intestinal cells, such as gut - associated lymphoid tissue (GALT) cells, Peyer's patch cells, M cells, lamina propria cells, small intestine cells or large intestine cells. In other embodiments, the mammalian cells are bladder cells, immune cells or blood - brain barrier cells. In some embodiments, the mammalian cells are mannosylated cells.

[0228] B. Method of Cargo Delivery In some aspects, the present disclosure features a method of delivering a cargo to a cell, the method comprising: (a) providing a composition comprising a plurality of the ADASs (e.g., T1P - ADAS) of the invention, wherein the ADASs further comprise the cargo; and (b) contacting the cells with the composition of step (a).

[0229] In some embodiments, the ADAS further comprises a heterologous bacterial secretion apparatus. In some embodiments, the heterologous bacterial secretion apparatus is a T3SS. In some embodiments, the delivery is delivery to the cytoplasm of the cell.

[0230] In some embodiments, the delivery of cargo to cells is increased by at least 1% compared to the ADAS derived from control (e.g., unmodified) parental cells (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% increase (e.g., 1% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, 90% - 100% or more than 100% increase compared to the ADAS derived from control parental cells). In some embodiments, the delivery of cargo to cells is increased by at least 10% compared to the ADAS derived from control parental cells.

[0231] In some embodiments, the effective amount of cargo is delivered to cells at a dose that is at least 1% lower than the dose required for the ADAS derived from control (e.g., unmodified) parental cells (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% lower (e.g., 1% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90% or 90% - 99% lower than the dose required for the ADAS derived from control parental cells). In some embodiments, the effective amount of cargo is delivered to cells at a dose that is at least 10% lower than the dose required for the ADAS derived from control parental cells.

[0232] In some embodiments, the cell is a mammalian cell (e.g., a human cell or a non-human mammalian cell including cells of livestock animals, domestic animals or harmful animals). In some embodiments, the mammalian cell is an intestinal cell, such as an intestinal associated lymphoid tissue (GALT) cell, Peyer's patch cell, M cell, lamina propria cell, small intestinal cell or large intestinal cell. In other embodiments, the mammalian cell is a bladder cell, immune cell or blood-brain barrier cell. In some embodiments, the mammalian cell is a mannosylated cell.

[0233] C. Methods for modulating cells In some aspects, the present disclosure features a method for modulating a cell, the method comprising: (a) providing a composition comprising a plurality of ADASs (e.g., T1P-ADAS) of the present invention; and (b) contacting the cell with the composition of step (a), whereby the cell is modulated.

[0234] In some embodiments, the cell is a mammalian cell (e.g., a human cell or a non-human mammalian cell including cells of livestock animals, domestic animals or harmful animals). In some embodiments, the mammalian cell is an intestinal cell, such as an intestinal associated lymphoid tissue (GALT) cell, Peyer's patch cell, M cell, lamina propria cell, small intestinal cell or large intestinal cell. In other embodiments, the mammalian cell is a bladder cell, immune cell or blood-brain barrier cell. In some embodiments, the mammalian cell is a mannosylated cell.

[0235] The modulation may be any observable change in the state of a cell (e.g., transcriptome, proteome, epigenome, biological effect, or health or disease state), such as a mammalian cell, when measured using techniques and methods known in the art for such measurements, e.g., methods for measuring the level or expression of proteins, transcripts, epigenetic markers, or methods for measuring an increase or decrease in the activity of a biological pathway. In some embodiments, modulating the state of a cell involves increasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway). In other embodiments, modulating the state involves decreasing a parameter of the cell (e.g., the level or expression of a protein, transcript, or the activity of a biological pathway).

[0236] The cell may be isolated (e.g., in vitro) or in an organism (e.g., in vivo). The methods provided herein encompass enabling the ADAS provided by the present invention or the composition provided by the present invention to access the target cell in an effective amount. Access to the target cell may be direct (e.g., the target cell is directly regulated by the ADAS, such as by secretion in the immediate vicinity of the agent (e.g., the cargo of the ADAS) proximate to the target cell or injection of the agent (e.g., cargo) into the target cell) or indirect. Indirect regulation of the target cell may also be by targeting another cell, e.g., an adjacent cell that may be commensal or pathogenic to the target cell. The adjacent cell may be in vitro or in vivo, similar to the target cell (e.g., in an organism that may be commensal or pathogenic). These methods are collectively referred to as "methods of use provided by the present invention", etc. In related aspects, the present invention provides for the targeted use of the ADAS and composition provided by the present invention according to the methods of use provided by the present invention.

[0237] For example, in some embodiments, the present invention provides a method of modulating the state of mammalian cells by providing an effective amount of an ADAS provided by the present invention or a composition provided by the present invention with means for reaching mammalian cells. In certain embodiments, the ADAS or composition is provided with means for reaching mammalian cells in vivo in a mammal (e.g., a human). In some embodiments, the mammalian cells are exposed to bacteria in a healthy mammal. In more detailed embodiments, the mammalian cells are lung epithelial cells, immune cells, skin cells, oral epithelial cells, intestinal epithelial cells, genital epithelial cells, or urinary tract cells. In even more detailed embodiments, the mammalian cells are intestinal epithelial cells, such as intestinal epithelial cells from a human subject having an inflammatory bowel disease such as Crohn's disease or colitis. In even more detailed embodiments, the mammalian cells are intestinal epithelial cells from a subject having an inflammatory bowel disease, and the ADAS comprises a bacterial secretion apparatus and a cargo comprising an anti-inflammatory agent.

[0238] In other embodiments, the mammalian cells are exposed to bacteria in a diseased state. In certain embodiments, the mammalian cells are pathogenic, such as a tumor. In other embodiments, the mammalian cells are exposed to bacteria in a diseased state, such as a wound, ulcer, tumor, or inflammatory disorder.

[0239] In certain embodiments, the ADAS is derived from a mammalian commensal parent strain. In other embodiments, the ADAS is derived from a mammalian pathogenic parent strain.

[0240] In some embodiments, the state of mammalian cells is regulated by providing a means of access to bacterial or fungal cells in the vicinity of the mammalian cells to an effective amount of the ADAS provided by the present invention or the composition provided by the present invention. That is, these methods involve indirectly regulating the state of mammalian cells. In certain embodiments, the bacterial or fungal cells are pathogenic. In more detailed embodiments, the fitness of the pathogenic bacteria or fungal cells is reduced. In other certain embodiments, the bacterial or fungal cells are commensal. In more detailed embodiments, the fitness of the commensal bacteria or fungal cells is increased. In even more detailed embodiments, the fitness of the commensal bacterial or fungal strain is increased by a decrease in the fitness of competing bacteria or fungi that can be neutral, commensal, or pathogenic.

[0241] D. Methods for Treating Mammals In some aspects, the present disclosure features a method for treating a mammal (e.g., a human or non-human mammal, such as a farm animal, a domestic animal, or a pest animal) in need thereof, the method comprising: (a) providing a composition comprising a plurality of ADASs (e.g., T1P-ADAS) of the present invention; and (b) contacting the mammal with an effective amount of the composition of step (a), thereby treating the mammal.

[0242] In some embodiments, the therapeutic effect is achieved at a dose that is at least 1% lower than the dose required for the ADAS derived from the control parental cells (e.g., unmodified) (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (e.g., 1% - 5%, 5% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 99% lower than the dose required for the ADAS derived from the control parental cells). In some embodiments, the therapeutic effect is achieved at a dose that is at least 10% lower than the dose required for the ADAS derived from the control parental cells.

[0243] The mammal in need of treatment may have a disease, such as cancer. In some embodiments, the ADAS carries a chemotherapy cargo or an immunotherapy cargo.

[0244] Other embodiments The present invention is further described in the following numbered paragraphs. 1. A plurality of extrachromosomal dynamic activity systems (ADAS) comprising type 1 pili (T1P), wherein the ADAS is a plurality of extrachromosomal dynamic activity systems derived from a parental bacterial cell that constitutively expresses components of the T1P. 2. The plurality of ADAS according to paragraph 1, wherein the parental cell comprises a modified fimS promoter operably linked to a component of the T1P and directing its constitutive expression. 3. The plurality of ADAS according to paragraph 1 or 2, wherein the components of the T1P are encoded by the fim operon. 4. The plurality of ADAS according to paragraph 3, wherein the parental cell comprises a modified fimS promoter operably linked to the fim operon, and the modified fimS promoter comprises a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site. 5. The plurality of ADAS according to any one of paragraphs 1 to 4, wherein the parental cell expresses the components of the T1P at a level at least 1.5-fold higher than the level observed in the unmodified parental cell. 6. The plurality of ADAS according to any one of paragraphs 1 to 5, wherein the ADAS comprises the T1P at a level at least 1.5-fold higher than the level observed in a plurality of ADAS produced from unmodified parental cells. 7. The plurality of ADAS according to any one of paragraphs 1 to 6, wherein the proportion of the plurality of ADAS comprising the T1P is increased compared to a plurality of ADAS produced by parental cells that do not constitutively express the components of the T1P. 8. The plurality of ADAS according to any one of paragraphs 1 to 7, wherein the parental cell comprises an endogenous fim operon. 9. The plurality of ADAS according to any one of paragraphs 1 to 8, wherein the parental cell is a bacterium that is Escherichia coli (E. coli). 10. The bacterium that is Escherichia coli (E. coli) is the plurality of ADAS according to item 9, which is Escherichia coli (E. coli) CFT073. 11. The parent cell is the plurality of ADAS according to any one of items 1 to 10, which contains one or more heterologous nucleotide sequences encoding components of T1P. 12. The one or more heterologous nucleotide sequences are the plurality of ADAS according to item 11, which contain the fim operon. 13. The fim operon is the plurality of ADAS according to item 12, which is the fim operon of Escherichia coli (E. coli) CFT073. 14. The one or more heterologous nucleotide sequences are the plurality of ADAS according to item 13, which contain a sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1. 15. The one or more heterologous nucleotide sequences are the plurality of ADAS according to item 14, which contain a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1. 16. The one or more heterologous nucleotide sequences are the plurality of ADAS according to item 15, which contain the nucleotide sequence of SEQ ID NO: 1. 17. The one or more heterologous nucleotide sequences are the ADAS according to any one of items 12 to 16, which further contain a constitutive promoter operably linked to the fim operon. 18. The constitutive promoter is the plurality of ADAS according to item 17, which is a modified fimS promoter containing a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site. 19. The one or more nucleotide sequences encoding components of T1P are the plurality of ADAS according to any one of items 11 to 18, which are carried by a vector. 20. The parent bacterial cell is the plurality of ADAS according to item 19, which is transiently transformed by a vector. 21. The parent bacterial cell is the plurality of ADAS according to item 19, which is stably transformed by a vector. 22. The parent cell is the plurality of ADAS according to any one of items 10 to 21, which is a Gram-negative bacterial cell. 23. The Gram-negative bacterial cells are bacterial cells of Escherichia coli, Salmonella, Yersinia, Vibrio, Pseudomonas, Shigella, or Legionella, and the plurality of ADAS according to item 22. 24. The parent cells do not contain a complete endogenous fim operon, and the plurality of ADAS according to any one of items 11 to 23. 25. The parent cells have not been exposed to culture conditions that promote the expression of the fim operon, and the plurality of ADAS according to any one of items 3, 4, and 12 to 24. 26. The culture conditions are temperature, pH, osmolarity, shaking, or activation of a stress response or a stringent response, and the plurality of ADAS according to item 25. 27. The ADAS contains a cargo, and the plurality of ADAS according to any one of items 1 to 26. 28. The cargo is a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulatory agent, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP), and the plurality of ADAS according to item 27. 29. The cargo is encapsulated in the ADAS, and the plurality of ADAS according to item 27 or 28. 30. The cargo is attached to the surface of the ADAS, and the plurality of ADAS according to item 27 or 28. 31. The ADAS contains a heterologous bacterial secretion apparatus, and the plurality of ADAS according to any one of items 1 to 30. 32. The heterologous bacterial secretion apparatus is a type III secretion apparatus (T3SS), and the plurality of ADAS according to item 31. 33. The cargo contains a component that directs export by the bacterial secretion apparatus, and the plurality of ADAS according to item 31 or 32. 34. A composition comprising the plurality of ADAS according to any one of items 1 to 33. 35. The composition is formulated for delivery to a mammal, and the composition according to item 34. 36. The composition is formulated for oral delivery, and the composition according to item 35. 37. A method for delivering ADAS to a cell, comprising: (a) providing a composition comprising a plurality of ADAS according to any one of items 1 to 36; and (b) contacting the composition of step (a) with the cell A method comprising. 38. The method according to item 37, wherein the delivery of ADAS to the cell is increased by at least 10% compared to ADAS derived from unmodified parental cells. 39. The method according to item 37 or 38, wherein the effective amount of ADAS is delivered to the cell at a dose that is at least 10% lower than the dose required for ADAS derived from unmodified parental cells. 40. A method for delivering a cargo to a cell, comprising: (a) providing a composition comprising a plurality of ADAS according to any one of items 1 to 36, wherein the ADAS further comprises a cargo; and (b) contacting the composition of step (a) with the cell A method comprising. 41. The method according to any one of items 37 to 40, wherein the ADAS further comprises a heterologous bacterial secretion apparatus. 42. The method according to item 41, wherein the heterologous bacterial secretion apparatus is a T3SS. 43. The method according to any one of items 37 to 42, wherein the delivery is delivery to the cytoplasm of the cell. 44. The method according to any one of items 40 to 43, wherein the delivery of the cargo to the cell is increased by at least 10% compared to ADAS derived from unmodified parental cells. 45. The method according to any one of items 40 to 43, wherein the effective amount of the cargo is delivered to the cell at a dose that is at least 10% lower than the dose required for ADAS derived from unmodified parental cells. 46. A method for regulating a cell, comprising: (a) providing a composition comprising a plurality of ADAS according to any one of items 1 to 36; and (b) contacting the composition of step (a) with the cell, whereby the cell is regulated Method. 47. The method according to any one of items 37 to 46, wherein the cell is a mammalian cell. 48. The method according to item 47, wherein the mammalian cell is an intestinal cell. 49. The method according to item 48, wherein the intestinal cell is a gut-associated lymphoid tissue (GALT) cell, a Peyer's patch cell, an M cell, a lamina propria cell, a small intestinal cell or a large intestinal cell. 50. The method according to item 47, wherein the mammalian cell is a bladder cell. 51. The method according to item 47, wherein the mammalian cell is an immune cell. 52. The method according to item 47, wherein the mammalian cell is a blood-brain barrier cell. 53. The method according to any one of items 47 to 52, wherein the mammalian cell is a mannosylated cell. 54. A method for treating a mammal in need thereof, comprising: (a) providing a composition comprising a plurality of ADAS according to any one of items 1 to 36; and (b) contacting the mammal with an effective amount of the composition of step (a) to thereby treat the mammal. The method comprising. 55. The method according to item 54, wherein the therapeutic effect is achieved at a dose that is at least 10% lower than the dose required for ADAS derived from unmodified parental cells. 56. An ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being (a) providing a parental cell modified to constitutively express a component of T1P; and (b) producing an ADAS from the parental bacterial cell, the ADAS comprising T1P. An ADAS produced by a process comprising. 57. An ADAS comprising a T1P derived from a parental bacterial cell, the ADAS being (a) providing a parental cell modified to express a component of natural T1P at a level that is at least 1.5-fold higher than the level observed in an unmodified parental cell; and (b) producing an ADAS from the parental bacterial cell, the ADAS comprising T1P that is natural to the parental cell. An ADAS produced by a process comprising 58. An ADAS comprising a T1P derived from a parental bacterial cell, the ADAS comprising: (a) providing a parental cell modified to express components of a heterologous T1P; and (b) producing the ADAS from the parental bacterial cell, the ADAS comprising a T1P that is heterologous to the parental cell An ADAS produced by a process comprising 59. An engineered bacterium that constitutively expresses components of a T1P, the engineered bacterium comprising the T1P at a level at least 1.5-fold higher compared to the level of T1P contained by a non-engineered bacterium. 60. The bacterium of paragraph 59, wherein the T1P is a native T1P. 61. The bacterium of paragraph 59, wherein the T1P is a heterologous T1P. 62. An engineered bacterium that constitutively expresses components of a T1P, the bacterium being modified to produce an ADAS. 63. A method for producing an ADAS, comprising: (a) providing an engineered bacterium that constitutively expresses components of a T1P; and (b) producing the ADAS from the bacterium A method comprising 64. An ADAS produced according to the method of paragraph 63.

[0245] The present invention is further described in the following numbered paragraphs. 1. A preparation comprising a plurality of extrachromosomal dynamic activity systems (ADAS) derived from a parental bacterial cell genetically engineered to constitutively express type 1 pili (T1P), the plurality of ADAS binding to target cells via the T1P. 2. The preparation of paragraph 1, wherein the parental bacterial cell comprises a modified fimS promoter operably linked to a component of the T1P and directing its constitutive expression. 3. The preparation of paragraph 1 or 2, wherein the components of the T1P are encoded by the fim operon. 4. The parent bacterial cell comprises a modified fimS promoter operably linked to the fim operon, the modified fimS promoter comprising a mutation at the recombinase cleavage site that prevents recombination of the fimS promoter in the "OFF" direction, the preparation according to item 3. 5. The parent bacterial cell expresses the components of T1P at a level at least 1.5-fold higher than the level observed in the unmodified parent bacterial cell, the preparation according to any one of items 1 to 4. 6. The plurality of ADASs comprises T1P at a level at least 1.5-fold higher than the level observed in the plurality of ADASs produced from unmodified parent bacterial cells, the preparation according to any one of items 1 to 5. 7. The proportion of the plurality of ADASs comprising T1P is increased compared to the plurality of ADASs produced by parent bacterial cells that do not constitutively express the components of T1P, the preparation according to any one of items 1 to 6. 8. The parent bacterial cell comprises an endogenous fim operon, the preparation according to any one of items 1 to 7. 9. The parent bacterial cell is a bacterium that is Escherichia coli (E. coli), the preparation according to any one of items 1 to 8. 10. The bacterium that is Escherichia coli (E. coli) is Escherichia coli (E. coli) CFT073, the preparation according to item 9. 11. The parent bacterial cell comprises one or more heterologous nucleotide sequences encoding the components of T1P, the preparation according to any one of items 1 to 10. 12. The one or more heterologous nucleotide sequences comprise a fim operon, the preparation according to item 11. 13. The fim operon is the fim operon of Escherichia coli (E. coli) CFT073, the preparation according to item 12. 14. The one or more heterologous nucleotide sequences comprise a sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1, the preparation according to item 13. 15. The one or more heterologous nucleotide sequences comprise a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1, the preparation according to item 14. 16. The preparation according to item 15, wherein one or more heterologous nucleotide sequences comprise the nucleotide sequence of SEQ ID NO: 1. 17. The preparation according to any one of items 12 - 16, wherein one or more heterologous nucleotide sequences further comprise a constitutive promoter operably linked to the fim operon. 18. The preparation according to item 17, wherein the constitutive promoter is a modified fimS promoter that contains a mutation preventing recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site. 19. The preparation according to any one of items 11 - 18, wherein one or more nucleotide sequences encoding components of T1P are carried by a vector. 20. The preparation according to item 19, wherein the parental bacterial cell is transiently transformed by the vector. 21. The preparation according to item 19, wherein the parental bacterial cell is stably transformed by the vector. 22. The preparation according to any one of items 10 - 21, wherein the parental bacterial cell is a Gram - negative bacterial cell. 23. The preparation according to item 22, wherein the Gram - negative bacterial cell is a bacterial cell of Escherichia coli, Salmonella, Yersinia, Vibrio, Pseudomonas, Shigella or Legionella. 24. The preparation according to any one of items 11 - 23, wherein the parental bacterial cell does not contain a complete endogenous fim operon. 25. The preparation according to any one of items 3, 4 and 12 - 24, wherein the parental bacterial cell has not been exposed to culture conditions that promote the expression of the fim operon. 26. The preparation according to item 25, wherein the culture conditions are temperature, pH, osmolarity, shaking or activation of a stress response or stringent response. 27. The preparation according to any one of items 1 - 26, wherein the ADAS contains a cargo. 28. The preparation according to item 27, wherein the cargo is nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulatory agent, carbohydrate, lipid, organic particle, inorganic particle or ribonucleoprotein complex (RNP). 29. The preparation according to item 27 or 28, wherein the cargo is encapsulated in the ADAS. 30. The preparation according to item 27 or 28, wherein the cargo is added to the surface of the ADAS. 31. The preparation according to any one of items 1 to 30, wherein the ADAS comprises a heterologous bacterial secretion apparatus. 32. The preparation according to item 31, wherein the heterologous bacterial secretion apparatus is a type III secretion apparatus (T3SS) or a type VI secretion apparatus (T6SS). 33. The preparation according to item 31 or 32, wherein the cargo comprises a component that directs export by the bacterial secretion apparatus. 34. A composition comprising the preparation of a plurality of ADAS according to any one of items 1 to 33. 35. The composition according to item 34, wherein the composition is formulated for delivery to a mammal. 36. The composition according to item 35, wherein the composition is formulated for oral delivery. 37. A method for delivering an ADAS to a cell, the method comprising contacting a cell with a composition comprising the preparation of a plurality of ADAS according to any one of items 1 to 36. 38. The method according to item 37, wherein the delivery of the ADAS to the cell is increased by at least 10% compared to the ADAS derived from unmodified parental bacterial cells. 39. The method according to item 37 or 38, wherein the effective amount of the ADAS is delivered to the cell at a dose that is at least 10% lower than the dose required for the ADAS derived from unmodified parental bacterial cells. 40. A method for delivering a cargo to a cell, the method comprising contacting a cell with a composition comprising the preparation of a plurality of ADAS according to any one of items 1 to 36, wherein the ADAS further comprises the cargo. 41. The method according to any one of items 37 to 40, wherein the ADAS further comprises a heterologous bacterial secretion apparatus. 42. The method according to item 41, wherein the heterologous bacterial secretion apparatus is T3SS or T6SS. 43. The method according to any one of paragraphs 37 to 42, wherein the delivery is delivery to the cytoplasm of the cell. 44. The method according to any one of paragraphs 40 to 43, wherein the delivery of the cargo to the cell is increased by at least 10% compared to the ADAS derived from the unmodified parental bacterial cell. 45. The method according to any one of paragraphs 40 to 43, wherein the effective amount of the cargo is delivered to the cell at a dose that is at least 10% lower than the dose required for the ADAS derived from the unmodified parental bacterial cell. 46. A method for modulating a cell, comprising contacting the cell with a composition comprising a preparation of a plurality of ADAS according to any one of paragraphs 1 to 36, whereby the cell is modulated. 47. The method according to any one of paragraphs 37 to 46, wherein the cell is a mammalian cell. 48. The method according to paragraph 47, wherein the mammalian cell is an intestinal cell. 49. The method according to paragraph 48, wherein the intestinal cell is an intestinal associated lymphoid tissue (GALT) cell, a Peyer's patch cell, an M cell, a lamina propria cell, a small intestinal cell or a large intestinal cell. 50. The method according to paragraph 47, wherein the mammalian cell is a bladder cell. 51. The method according to paragraph 47, wherein the mammalian cell is an immune cell. 52. The method according to paragraph 47, wherein the mammalian cell is a blood-brain barrier cell. 53. The method according to any one of paragraphs 47 to 52, wherein the mammalian cell is a mannosylated cell. 54. An ADAS derived from a parental bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS (a) providing a parental bacterial cell modified to constitutively express a component of T1P; and (b) producing an ADAS from the parental bacterial cell, wherein the ADAS comprises T1P produced by a process comprising. 55. An ADAS derived from a parental bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS (a) Providing a parental bacterial cell modified to express components of a native T1P at a level at least 1.5-fold higher than the level observed in an unmodified parental bacterial cell; and (b) Producing an ADAS from the parental bacterial cell, wherein the ADAS comprises a T1P that is native to the parental bacterial cell An ADAS produced by a process comprising. 56. An ADAS derived from a parental bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS (a) Providing a parental bacterial cell modified to express components of a heterologous T1P; and (b) Producing an ADAS from the parental bacterial cell, wherein the ADAS comprises a T1P that is heterologous to the parental bacterial cell An ADAS produced by a process comprising. 57. A genetically engineered bacterium that constitutively expresses components of T1P, wherein the engineered bacterium comprises T1P at a level at least 1.5-fold higher compared to the level of T1P contained by an unengineered bacterium. 58. The bacterium according to item 57, wherein the T1P is a native T1P. 59. The bacterium according to item 57, wherein the T1P is a heterologous T1P. 60. A genetically engineered bacterium that constitutively expresses components of T1P, wherein the bacterium is modified to produce an ADAS. 61. A method for producing an ADAS, the method comprising producing an ADAS from a bacterium genetically engineered to constitutively express components of T1P. 62. An ADAS produced according to the method of item 61.

Example

[0246] Example 1: Method for Producing an ADAS that Expresses an Endogenous or Heterologous Type 1 Pili (T1P-ADAS) A. ADAS that Expresses Endogenous T1P The natural promoter for type 1 pili (T1P), fimS, is phase variable. A phase-variable promoter is part of the DNA that can reverse after cleavage by a site-specific recombinase. The recombinases fimB and fimE bind to inverted repeat sites (IR sites; shown as black boxes in FIG. 1) adjacent to fimS, cut, and can switch the promoter to the "ON" direction (the promoter faces the operon; the fim operon is expressed) or the "OFF" direction (the promoter faces away from the operon, the fim operon is not expressed) (FIG. 1). The fimS expression state is hereditary but also reversible, and the reversal is affected by a number of signals including temperature, pH, osmolarity, and stress and stringent responses. In the laboratory, growing Escherichia coli (E. coli) in LB broth at 37°C with shaking (standard growth conditions for E. coli) promotes maintaining the fimS switch in the "OFF" direction. Switching growth to 37°C, static (not shaking), promotes maintaining the fimS switch in the "ON" direction.

[0247] The recombinase cleavage site upstream of the promoter (green box in FIG. 1) was mutated using Lambda-RED recombineering to lock the promoter in the "ON" direction ("locked on" ("LON") operon), and thus generate bacteria that constitutively express the fim operon (i.e., constitutively express T1P). This was done in two E. coli strains: a laboratory-adapted strain (BW25113) and a pathogenic strain (CFT073). The bacteria were further engineered to produce ADAS by removing the minCDE locus using Lambda-RED recombineering as described in WO 2020 / 123569 pamphlet.

[0248] The wild-type sequences (5→3) of the fimS sites in Escherichia coli (E. coli) strains CFT073 and MG1655 with the fimS promoter in the 「ON」 direction are shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The left inverted repeat site is underlined and italicized. SEQ ID NO: 5 and SEQ ID NO: 7 shown below indicate the mutations made to the left inverted repeat site. The mutations are indicated by capital letters. Although very similar, the fimS operons of CFT073 and MG1655 have some differences in the promoter sequence. The mutations made to lock the inverted repeat site sequence and the promoter to 「on」 are the same.

[0249] CFT073 fimS native 「on」 direction (SEQ ID NO: 4)

Chem.

Chem.

Chem.

Chem.

[0250] Bacterial strains containing the LON operon expressed T1P when grown with shaking at 37°C. Unmodified strains (e.g., wild-type bacteria without mutant fimS) grown under these growth conditions would not have produced T1P.

[0251] The presence of T1P on the surface of ADAS produced from engineered BW25113 and CFT073 E. coli strains containing the lock-on operon was confirmed using a transmission electron microscope as shown in Figure 2.

[0252] B. ADAS Expressing Heterologous T1P Heterologous expression of T1P was achieved by cloning the CFT073 fim operon (ATCC 700928; SEQ ID NO: 1) containing the genes FimA, FimI, FimC, FimD, FimF, FimG, and FimH into a plasmid containing a constitutive promoter (thus generating pT1P) and expressing the plasmid in bacteria. The operon was taken from the pathogenic E. coli strain CFT073 and expressed in the K12 E. coli BW25113. The CFT073 version of the fim operon was chosen because previous studies had shown the superiority of the type 1 fimbriae of this strain in ligand binding. The promoter was J23117 (SEQ ID NO: 2). The bacteria were further engineered to produce ADAS by removing the minCDE locus using Lambda-RED recombination as described in WO 2020 / 123569 pamphlet.

[0253] Example 2: Endogenous and Heterologous T1P-ADAS Agglutinate Red Blood Cells To determine whether the engineered bacteria of Example 1 and the ADAS produced therefrom contain T1P at detectable levels, the bacteria and ADAS were evaluated using a red blood cell (RBC) agglutination assay.

[0254] The RBC agglutination assay is a standard assay in the art for demonstrating the expression of functional T1P. T1P binds to mannosylated residues, and guinea pig RBCs (gpRBCs) are highly mannosylated. The assay involves a dilution series of the test substance (e.g., the engineered bacteria of Example 1 or the ADAS produced therefrom) (2 ×) was incubated in PBS or PBS + mannose with a fixed number of gpRBC, and performed by determining the dilution at which the test substance no longer agglutinated RBCs (read as HA titer). Since T1P agglutination is mannose-sensitive, PBS + mannose was included as a functional control.

[0255] ADAS expressing T1P from endogenous (LON T1P; Example 1A) and heterologous (pT1P; Example 1B) sources was found to agglutinate RBCs (Figure 3). This binding can be inhibited by mannose, which serves as proof of T1P-dependent agglutination. Furthermore, functional expression was shown in T1P-ADAS derived from two different Escherichia coli (E. coli) strains (Figure 3).

[0256] Example 3: Endogenous T1P-ADAS shows enhanced binding to intestinal cells Previous investigations have shown that T1P promotes tropism to areas within the intestinal tract (e.g., Peyer's patches) as well as other body sites and cell types (Martinez et al., The EMBO Journal, 19:2803-2812, 2000; Carvalho et al.; J Exp Med., 206(10):2179-2189, 2009; Hase et al., Nature, 462(7270):226-230, 2009; Avalos et al., Sci Rep., 6:18109, 2016; Sheikh et al., PLoS Negl Trop Dis., 11(5):e00055862017, 2017; Spaulding et al., Nature, 546(7659):528-532, 2017; Le Guennec et al., Cellular Microbiology, 22:e13132, 2019).

[0257] This example demonstrates that the ADAS (T1P-ADAS) produced according to Example 1A exhibits enhanced binding to the human colorectal cancer cell line HT-29 compared to non-T1P-ADAS (i.e., ADAS produced from bacteria that were not engineered to contain the lock-on fim operon according to Example 1A). The assay was performed by incubating HT-29 cells with ADAS for 1 hour, removing unbound ADAS, fixing the remaining bound ADAS, and staining with a fluorescent antibody against LPS. A fluorescence microscope (Figure 4) was used to count the number of ADAS per HT-29 cell nucleus. The enhanced binding of T1P-ADAS was also demonstrated to be mannose-sensitive.

[0258] The foregoing invention has been described in some detail by way of figures and examples for purposes of clarity of understanding, but the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby incorporated by reference in their entirety. Other embodiments are within the scope of the claims.

Claims

**Claim 1** A preparation comprising a plurality of extrachromosomal dynamic activity systems (ADAS) derived from genetically engineered parental bacterial cells that constitutively express type 1 pili (T1P), wherein the plurality of ADAS bind to target cells via the T1P. **Claim 2** The preparation according to claim 1, wherein the parental bacterial cells comprise a modified fimS promoter operably linked to a component of the T1P and directing its constitutive expression. **Claim 3** The preparation according to claim 1 or 2, wherein the component of the T1P is encoded by the fim operon. **Claim 4** The preparation according to claim 3, wherein the parental bacterial cells comprise a modified fimS promoter operably linked to the fim operon, and the modified fimS promoter comprises a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site. **Claim 5** The preparation according to any one of claims 1 to 4, wherein the parental bacterial cells express the component of the T1P at a level at least 1.5-fold higher than the level observed in unmodified parental bacterial cells. **Claim 6** The preparation according to any one of claims 1 to 5, wherein the plurality of ADAS comprises the T1P at a level at least 1.5-fold higher than the level observed in a plurality of ADAS produced from unmodified parental bacterial cells. **Claim 7** The preparation according to any one of claims 1 to 6, wherein the proportion of the plurality of ADAS comprising the T1P is increased compared to a plurality of ADAS produced by parental bacterial cells that do not constitutively express the component of the T1P. **Claim 8** The preparation according to any one of claims 1 to 7, wherein the parental bacterial cells comprise an endogenous fim operon. **Claim 9** The preparation according to any one of claims 1 to 8, wherein the parental bacterial cells are bacteria of Escherichia coli (E. coli). **Claim 10** The preparation according to claim 9, wherein the bacteria of Escherichia coli (E. coli) are Escherichia coli (E. coli) CFT073. **Claim 11** The preparation according to any one of claims 1 to 10, wherein the parental bacterial cells comprise one or more heterologous nucleotide sequences encoding the component of the T1P. **Claim 12** The preparation according to claim 11, wherein the one or more heterologous nucleotide sequences comprise the fim operon. **Claim 13** The preparation according to claim 12, wherein the fim operon is the fim operon of Escherichia coli (E. coli) CFT073. **Claim 14** The preparation according to claim 13, wherein the one or more heterologous nucleotide sequences comprise a sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

1.

15. The preparation according to claim 14, wherein the one or more heterologous nucleotide sequences comprise a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:

1.

16. The preparation according to claim 15, wherein the one or more heterologous nucleotide sequences comprise the nucleotide sequence of SEQ ID NO:

1.

17. The preparation according to any one of claims 12 to 16, wherein the one or more heterologous nucleotide sequences further comprise a constitutive promoter operably linked to the fim operon.

18. The preparation according to claim 17, wherein the constitutive promoter is a modified fimS promoter comprising a mutation that prevents recombination of the fimS promoter in the "OFF" direction at the recombinase cleavage site.

19. The preparation according to any one of claims 11 to 18, wherein the one or more nucleotide sequences encoding the components of the T1P are carried on a vector.

20. The preparation according to claim 19, wherein the parental bacterial cell is transiently transformed by the vector.

21. The preparation according to claim 19, wherein the parental bacterial cell is stably transformed by the vector.

22. The preparation according to any one of claims 10 to 21, wherein the parental bacterial cell is a Gram-negative bacterial cell.

23. The preparation according to claim 22, wherein the Gram-negative bacterial cell is a bacterial cell of Escherichia coli, Salmonella, Yersinia, Vibrio, Pseudomonas, Shigella or Legionella.

24. The preparation according to any one of claims 11 to 23, wherein the parental bacterial cell does not contain a complete endogenous fim operon.

25. The preparation according to any one of claims 3, 4 and 12 to 24, wherein the parental bacterial cell has not been exposed to culture conditions that promote expression of the fim operon.

26. The preparation according to claim 25, wherein the culture conditions are temperature, pH, osmolarity, shaking or activation of a stress response or stringent response.

27. The ADAS is a preparation according to any one of claims 1 to 26, including cargo.

28. The preparation according to claim 27, wherein the cargo is a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulatory drug, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP).

29. The preparation according to claim 27 or 28, wherein the cargo is encapsulated in the ADAS.

30. The preparation according to claim 27 or 28, wherein the cargo is added to the surface of the ADAS.

31. The ADAS is a preparation according to any one of claims 1 to 30, including a heterologous bacterial secretion apparatus.

32. The preparation according to claim 31, wherein the heterologous bacterial secretion apparatus is a type III secretion system (T3SS) or a type VI secretion system (T6SS).

33. The preparation according to claim 31 or 32, wherein the cargo comprises a component that directs export by the bacterial secretion apparatus.

34. A composition comprising a preparation of a plurality of ADAS according to any one of claims 1 to 33.

35. The composition according to claim 34, which is formulated for delivery to a mammal.

36. The composition according to claim 35, which is formulated for oral delivery.

37. A method for delivering ADAS to a cell, comprising contacting a cell with a composition comprising a preparation of a plurality of ADAS according to any one of claims 1 to 36.

38. The method according to claim 37, wherein the delivery of the ADAS to the cell is increased by at least 10% compared to ADAS derived from unmodified parental bacterial cells.

39. The method according to claim 37 or 38, wherein the effective amount of the ADAS is delivered to the cell at a dose that is at least 10% lower than the dose required for ADAS derived from unmodified parental bacterial cells.

40. A method for delivering cargo to a cell, comprising contacting a cell with a composition comprising a preparation of a plurality of ADAS according to any one of claims 1 to 36, wherein the ADAS further comprises cargo.

41. The method according to any one of claims 37 to 40, wherein the ADAS further comprises a heterologous bacterial secretion apparatus.

42. The method according to claim 41, wherein the heterologous bacterial secretion apparatus is T3SS or T6SS.

43. The method according to any one of claims 37 to 42, wherein the delivery is delivery to the cytoplasm of the cell.

44. The method according to any one of claims 40 to 43, wherein the delivery of the cargo to the cell is increased by at least 10% compared to an ADAS derived from an unmodified parental bacterial cell.

45. The method according to any one of claims 40 to 43, wherein the effective amount of the cargo is delivered to the cell at a dose that is at least 10% lower than the dose required for an ADAS derived from an unmodified parental bacterial cell.

46. A method for modulating a cell, comprising contacting the cell with a composition comprising a preparation of a plurality of ADASes according to any one of claims 1 to 36, whereby the cell is modulated.

47. The method according to any one of claims 37 to 46, wherein the cell is a mammalian cell.

48. The method according to claim 47, wherein the mammalian cell is an intestinal cell.

49. The method according to claim 48, wherein the intestinal cell is a gut-associated lymphoid tissue (GALT) cell, a Peyer's patch cell, an M cell, a lamina propria cell, a small intestinal cell or a large intestinal cell.

50. The method according to claim 47, wherein the mammalian cell is a bladder cell.

51. The method according to claim 47, wherein the mammalian cell is an immune cell.

52. The method according to claim 47, wherein the mammalian cell is a blood-brain barrier cell.

53. The method according to any one of claims 47 to 52, wherein the mammalian cell is a mannosylated cell.

54. An ADAS derived from a parental bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS (a) providing a parental bacterial cell modified to constitutively express components of T1P; and (b) producing an ADAS from the parental bacterial cell, wherein the ADAS comprises the T1P An ADAS produced by a process comprising.

55. An ADAS derived from a parental bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS (a) providing a parental bacterial cell modified to express components of natural T1P at a level that is at least 1.5-fold higher than the level observed in an unmodified parental bacterial cell; and (b) producing an ADAS from the parental bacterial cell, wherein the ADAS comprises the T1P that is natural to the parental bacterial cell An ADAS produced by a process comprising **Claim 56** An ADAS derived from a parent bacterial cell genetically engineered to constitutively express T1P, wherein the ADAS comprises: (a) providing a parent bacterial cell modified to express components of a heterologous T1P; and (b) producing the ADAS from the parent bacterial cell, the ADAS comprising the T1P that is heterologous to the parent bacterial cell An ADAS produced by a process comprising **Claim 57** A genetically engineered bacterium that constitutively expresses components of T1P, wherein the engineered bacterium comprises the T1P at a level that is at least 1.5-fold higher compared to the level of T1P contained by a non-engineered bacterium. **Claim 58** The bacterium according to claim 57, wherein the T1P is a natural T1P. **Claim 59** The bacterium according to claim 57, wherein the T1P is a heterologous T1P. **Claim 60** A genetically engineered bacterium that constitutively expresses components of T1P, wherein the bacterium is modified to produce an ADAS. **Claim 61** A method for producing an ADAS, the method comprising producing the ADAS from a bacterium genetically engineered to constitutively express components of T1P. **Claim 62** An ADAS produced according to the method of claim 61.

Images