Methods, uses and compositions

Abstract

To provide the production of transduction particles, such as phages, as well as compositions comprising the particles and use thereof.SOLUTION: Provided is a method of producing transduction particles, wherein the particles are capable of recognizing a receptor on bacterial target cells for transduction of the cells, the method comprising producing the particles in bacterial producer cells, wherein the producer cells do not express the receptor on their surface. Also provided is use of producer cells for improving the production yield of transduction particles.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the production of transducing particles, such as phages, as well as compositions comprising the particles and The particles deliver a toxic payload to target bacteria for antibacterial action. The present invention is particularly useful for detecting wild-type ("natural") phages and various types of phages. Also useful are the production of phage cocktails containing phages. This allows for the production of compositions of such particles for use in food production situations. [Background technology]

[0002] Helper phages for packaging phagemid DNA into phage virus particles. The use of ribosomal RNA in Escherichia coli host cells is known. For example, the M13 derivative Other examples include R408 and CM There is 13.

[0003] Bacteriophages (phages) are a phylum of viruses that infect bacteria, animals and plants. They are distinct from viruses, which are organisms that live in a "lytic" life cycle. They may have a "lysogenic" life cycle, which can be either lytic or nonlytic. Phages that replicate through a bacterial cycle are naturally assimilated by the host as a normal part of their life cycle. They cause the lysis of target bacterial cells. Phages that replicate through a lysogenic cycle are mild phages. They replicate by a lytic life cycle and cause the lysis of the host bacterium. or their DNA can be integrated into the DNA of the host bacterium to produce a non-infectious profile. Bacteriophages can only infect their specific bacterial host. Host specificity is generally at the strain level, species level, or more rarely are found at the genus level. This specificity allows phages to be used to target dangerous bacteria. Adsorption of bacteriophages to host cells can occur in a few rare cases. All except for are necessary for the initiation of the infection process.

[0004] The natural ability of phages to infect and kill bacteria is due to interactions between phages and bacteria. This is the fundamental phenomenon on which the concept of phage therapy is built, together with specificity of use. Therefore, phages with a lytic life cycle are suitable candidates for phage therapy. The use of phages in food production has recently been demonstrated for the biological control of unwanted pathogens. It has become a novel method of choice for the food industry, especially for the production of fresh and ready-to-eat food products. It increases safety.

[0005] PCT / EP2018 / 082053 and USSN15 / 985,658 are non-copyrighted. PCT / EP2018 / 071454 discloses the production of transducing particles. A method for the propagation of injectable particles is disclosed.

[0006] International patent application WO00 / 69269 discloses Enterococcus faecium Certain anti-vancomycin drugs for the treatment of infections caused by vancomycin-susceptible and -resistant strains The International Patent Application WO01 / 93904 discloses the use of phage strains such as Clostr and / or other antimicrobials for the prevention or treatment of gastrointestinal disease associated with idium species. The use of bacteriophages in combination with biological tools is disclosed.

[0007] US Patent Application 2001 / 0026795 describes a method for preventing the delayed inactivation of the host's defense system. The phage has been modified to extend the period during which it is active in killing bacteria. The authors describe methods for producing such bacteriophages.

[0008] U.S. Patent Application 2002 / 0001590 discloses a method for the treatment of multidrug-resistant bacteria, specifically methicillin-resistant The use of phage therapy against Staphylococcus aureus has been disclosed. International Patent Application WO02 / 07742 discloses bacteriophages having multiple host ranges. The company has disclosed the development of:

[0009] The use of phage therapy for the treatment of certain bacterial infectious diseases is described, for example, in U.S. Pat. 2 / 0044922, 2002 / 0058027, and International Patent Application WO01 / 939 This is disclosed in 04.

[0010] US 20160333348 uses phages as vectors to target host bacterial cells describe the use of CRISPR / Cas systems delivered to

[0011] Commercial-scale production of bacteriophage compositions for therapeutic use remains limited Current methods produce low titers of phage compositions, typically at 10 9 ~10 11 p fu / ml, 10 on a commercial scale 7 ~10 9 whereas for phage therapy Typically the titer required is 10 12 pfu / ml. Furthermore, to reach the desired titer, , a very large amount of liquid is required.

[0012] The use of transducing particles (e.g., bacteriophages) in industrial and medical settings has become widespread. As the need for such particles grows, there is a need for commercial quantities of such particles. Production of transducing particles that provide good yield titers and / or reduce production volumes There is a need for a method for doing so. Summary of the Invention

[0013] The present invention provides the following:

[0014] In the first configuration A method for producing transduction particles, the particles being capable of being introduced into bacterial target cells for the transduction of the cells. that the method produces particles within bacterial producer cells that can recognize the receptor. wherein the producer cell does not express the receptor on its surface.

[0015] In the second configuration Use of producer cells to improve production yields of transducing particles.

[0016] In the third configuration Compositions containing transducing particles obtained or obtainable by the method or use of the present invention A composition (optionally a pharmaceutical composition).

[0017] In the fourth configuration A method of killing bacterial target cells comprising contacting the cells with a composition according to the invention. The transduction particles infect cells and deliver therein a nucleic acid or nucleotide sequence of interest (NS I), wherein the NSI contains or codes for an antimicrobial agent that kills the target cells; or wherein the NSI comprises or codes for such a pharmaceutical component.

[0018] In the fifth configuration For use in a method of treating or preventing a disease or condition in a human or animal subject. The composition according to the invention for use in treating a disease or condition mediated by a bacterial target cell, The method includes administering a composition to a subject and contacting a target cell with the composition. thereby killing or inhibiting the growth or proliferation of the target cells, A composition for treating or preventing a disease or condition.

[0019] In the sixth configuration A plurality of transducing particles obtainable by the method.

[0020] A "transduction particle" may be a phage or may be smaller than a phage and may carry a nucleic acid (e.g. The particles are capable of transducing a specific antibody (e.g., encoding an antibiotic or a component thereof) into a target cell. Examples of transduction particles include phages or particles containing phage capsids. In one embodiment, each particle is a replication-defective phage particle. The gene is a genomic island (e.g., a pathogenicity island such as SaPI) or a modified version thereof. These are particles produced from the fusion. [Brief description of the drawings]

[0021] [Figure 1] Schematic diagram of the LPS core biosynthetic pathway in E. coli (www.ecocyc.org). Numbers indicate the order of synthesis. Genes involved in the pathway were individually knocked out and P2 phage growth was tested in each mutant. Genes essential for P2 plaque formation are marked with an asterisk. [Figure 2a]Figure 1. Transduction of E. coli C1a cells with lysates prepared from wild-type (WT) and phage receptor mutant (ΔrfaD) cells. (A) Production yield was assayed by transduction of the spectinomycin marker into C1a cells. The lysates were serially diluted and mixed with a fixed number of recipient cells. [Figure 2b] Transduction of E. coli C1a cells with lysates prepared from wild-type (WT) and phage receptor mutant (ΔrfaD) cells. (B) Transduction efficiency, defined as the number of spectinomycin-resistant colonies obtained per ml of lysate, was calculated from six independent lysates for both strains. Error bars indicate standard deviation from the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention relates to the production of transducing particles, such as phages, as well as compositions comprising the particles and The particles deliver a toxic payload to target bacteria for antibacterial action. The present invention is particularly useful for detecting wild-type ("natural") phages and various types of phages. Also useful are the production of phage cocktails containing phages. This allows for the production of compositions of such particles for use in food production situations. The transducing particles, e.g., phages, can be usefully used in the manufacture of pharmaceuticals, herbicides, and other therapeutic agents. The present invention can be used in compositions such as those for the treatment of cancer and other diseases. and embodiments are provided.

[0023] section 1. A method for producing transduction particles, the particles being capable of binding to bacterial targets for the transduction of cells. and wherein the method comprises the step of: detecting the presence of the particle in a bacterial producer cell; wherein said producer cell does not express said receptor on its surface. ,method. The particles can attach to receptors when surface expressed on the target cell. The particles attach to receptors on the target cells and transduce DNA into the target cells. In one example, the particle is phage-like or a phage. For example, The particle contains a phage capsid in which DNA is packaged. In one example, the producer and target cells are bacterial cells. It is a bacterial cell. The producer cells are different from the target cells. At a minimum, they are The receptor is not expressed (or is not substantially expressed) on the surface of target cells. The difference is that 2. The method of clause 1, wherein the producer cell and target cell are cells of the same species. Optionally, the producer and target cells may be combined with one another, with the exception that the target cells surface express the receptor. The target cells are cells of the same line. 3. The method of any preceding clause, wherein the producer cell is an E. coli cell. . In one example, the cells are E. coli Nissle or K-12 (e.g., K-12 MG 1655) cells. In one example, each producer cell contains a nucleic acid sequence of interest (NSI), a phage vector, Packaging sequences (e.g., pac or cos, or their homologs), and In one embodiment, the nucleic acid is a plasmid phagemid. The acid is packaged into the producer cells to produce the particles of the invention. In this embodiment, the producer cell comprises a nucleic acid packaged and Helper viruses used to provide basic functionality necessary for the execution and / or replication of The genome of a helper virus or a phage is well known to those skilled in the art. You are probably familiar with the phage. In one example, each producer cell contains (i) the genome of a helper phage, and (ii) a nucleic acid sequence of interest (NSI), a phage packaging sequence (e.g., pac or or cos), an origin of replication, and optionally a helper gene for activating the helper phage. - one or more nucleotide sequences each encoding a phage transactivator In one embodiment, component (i) is comprised in nucleic acid (ii). In the above, the nucleic acid (ii) is a plasmid phagemid. In one embodiment, the NSI is a protein of interest (POI), e.g., an enzyme, nuclease , antibiotic, antigen binding site, marker (e.g., antibiotic marker or fluorescent marker), if The term "generic name" or "generic drug" may be used to code for a gene or a pharmaceutical agent, or a component thereof. 4. The transduction particle contains a phage capsid, and the capsid is used for transduction of the target bacterial cell. The method of any preceding clause, further comprising administering to said subject a nucleic acid of interest (NSI) for . In one example, the NSI is contained in a phagemid. In one example, the NSI is contained in a plasmid. do. 5. The NSI contains or encodes an antimicrobial agent that kills target cells, or The method of claim 4, wherein the NSI comprises or encodes a component of such a drug. 6. The NSI is a CRISPR / Cas-based guide RNA (optionally a single guide RNA The method of claim 5, comprising a nucleotide sequence encoding A). Optionally, the guide hybridizes to a protospacer sequence contained in the target cell. In one embodiment, the target cell comprises a spacer sequence that can modify the sequence (e.g., Cas is then coupled to an associated protospacer to selectively induce cleavage (e.g., cleavage) and, optionally, kill the target cell. - an endogenously active Cas (e.g., In another embodiment, the Cas may alternatively be or. In addition, the DNA transduced into the target cell by the particle of the present invention may be encoded will be done. 7. The method of any preceding clause, wherein the transduction particle is a phage. 8. The method of any preceding clause, wherein the transduction particle is non-self-replicating. 9. The genome of each producer cell is adapted to synthesize and / or express said receptor. The method of any preceding clause, comprising a genetic modification that disrupts its expression as a facial receptor. In one example, each producer cell is an rfa mutant, e.g., a producer In one embodiment, the cell is an E. coli cell. In one embodiment, the rfa is rfaC. In one embodiment, rfa is rfaD. In one embodiment, rfa is rfaF. In one embodiment, rfa is rfaG. In one embodiment, rfa is rfaI. In this embodiment, rfa is rfaJ. See, e.g., FIG. 1. Optionally, an rfa gene in the genome (e.g., chromosome) of the producer cell, e.g. rfaC, rfaD, rfaE, rfaF, rfaG, rfaP, rfaQ, rfaY, Disruption of rfaB, rfaI, or rfaJ is present. Optionally, disruption is of rfaD and and / or rfaE. The cells are E. coli cells. Disruption can be achieved by knocking out a gene, introducing a foreign nucleotide into a gene, or Insertion of the nucleotide sequence, gene expression in the receptor, receptor component, or receptor in the producer cell. Mutations (e.g., deletions, substitutions, deletion ... or addition, or any combination in a gene. Standard molecular biology techniques will be apparent to those skilled in the art. 10. The method of clause 9, wherein said modification is a modification of the lipopolysaccharide (LPS) synthesis pathway. 11. The method of any preceding clause, wherein the receptor comprises LPS. In one example, the receptor is any receptor described in Tables 1-3 or elsewhere herein. In one example, the particle comprises a P2 phage capsid and the receptor is a P2 receptor. 12. A method as defined in any of the preceding clauses for improving the production yield of transducing particles. Use of the producer cells described in. 13. The method of claim 1, wherein the transduction particle is as defined in any one of clauses 1 to 11. 12. Use according to claim 12. 14. The yield is reduced compared to production in producer cells that surface express the receptor. 14. The use according to clause 12 or 13, to increase the expression level of the compound by at least 10-fold. 15. The use according to clause 14, wherein the increase is at least 100-fold. 16. The use according to clause 14, wherein the increase is between 10 and 1000 fold. 17. The method of any preceding clause, comprising isolating the transduction particles from cellular material. Method or use. 18. Transduction obtained or obtainable by the method or use according to paragraph 17 A composition (optionally a pharmaceutical composition) comprising the particles. Optionally, the composition includes an antibiotic that kills or is toxic to the target cells. nothing. 19. The composition of clause 18, which does not contain LPS from producer cells. 20. A method for killing bacterial target cells, comprising: infecting the cells with a composition according to paragraph 18 or 19. and contacting the cells with a composition, wherein the transducing particles infect the cells and introduce the NSI therein. and the NSI contains or encodes an antimicrobial agent that kills the target cells; or The method wherein the NSI comprises or encodes a component of such a drug. Optionally, any method or use of the compositions, populations, or particles of the invention may be performed in vivo. Optionally, the method or use is in vitro or ex vivo. The method or use is not carried out in or on a human or animal. is produced in vitro in or on human or animal tissue, cell, or serum samples. It will be implemented. 21. For use in methods of treating or preventing a disease or condition in a human or animal subject 20. The composition of claim 18 or 19 for use in treating a disease or condition comprising administering to a patient a therapeutically effective amount of a therapeutically effective amount of a bacterial target cell. and the method comprises administering to the subject the composition and administering to the subject a therapeutically effective amount of the composition to the subject. contacting a target cell with the composition of clause 18 or 19, thereby The target cells are killed or the growth or proliferation of the target cells is inhibited, thereby preventing or treating the disease. A composition for treating or preventing a disease or condition. 22. The transduction particles contained in the composition infect the target cells and induce NSI therein. the NSI contains or codes for an antimicrobial agent that kills the target cells; or 22. The composition according to claim 21, wherein the NSI comprises or codes for such a pharmaceutical component. . 23. The target cell is Escherichia, Klebsiella, Clostridium 23. The composition of claim 21 or 22, which is a Bacillus subtilis or Pseudomonas cell. . 24. The target cells are E. coli, K. pneumoniae, C. dificile 23. The composition of clause 21 or 22, wherein the composition is a P. aeruginosa cell.

[0024] Examples of receptors for use in the present invention are discussed below.

[0025] Proteinaceous receptors are primarily outer membrane proteins, with the sugar moieties located in the cell wall, pellicle, or tegument. The receptor of the present invention includes, for example, any of these. is selected from the following.

[0026] Bacteriophage adsorption initiates the infection process. ) binding proteins interact with receptors on the bacterial cell surface, allowing the virus to It probably recognizes a susceptible host and positions itself for release of DNA. Thus, phage adsorption is not only a critical step in the infection process but also plays a key role in the viral They represent the initial point of contact between the virus and the host and determine host range specificity.

[0027] Bacteriophage adsorption generally involves three steps: initial contact, reversible binding, and The first step consists of fusion, fusion, and irreversible attachment (Duckworth 1987). The mechanism of interaction between phages and hosts caused by Brownian motion, dispersion, diffusion, or flow is This includes random collisions (Kokjohn and Miller 1992). In the reverse step, binding to bacterial surface components is not final and the phage continues to host This process is consistent with the experimental observation of phage detachment after elution. First identified by Garen and Puck (1951), phages It may act to search for specific receptors and to hold the phage close to the cell surface ( Kokjohn and Miller 1992). Bacterial receptors and phage binding domains The specific linkage between the in and the nucleotide may be mediated by enzymatic cleavage. Induce conformational rearrangements in other phage molecules, which then transfer the genetic material to the host (For further details on the mechanism of phage genome release, see (For a review, see Molineux and Panja (2013)).

[0028] Numerous review studies have demonstrated the broad range of host-associated receptors that bacteriophages target during adsorption. It emphasizes the components (proteins, carbohydrates, and cell surface structures) of the cell (Lindberg 19 77;Schwartz 1980;Wright, McConnell and Ka negasaki 1980;Heller 1992;Frost 1993;Hen ning and Hashemolhosseini 1994;Vinga et al. al.2006;Rakhuba et al. 2010;Chaturongaku Lal and Ounjai 2014). The nature and location of the phage receptors vary greatly depending on the phage and the host. In fact, bacteriophages are Gram-positive (X ia et al. 2011) and Gram-negative (Marti et al. 2013). in the bacterial wall, in the bacterial capsule or slime layer (Fehmel et al. 1975 ), and in appendages [e.g., cilia ( Guerrero-Ferreira et al. .2011) and flagella (Shin et al.2012)]. This diversity in the receptors and structures involved has led to their The mechanisms that phages and hosts have developed to overcome the evolutionary strategies employed by the host This is evidence of the multiplicity of mechanisms. Phages that are believed to inhabit various environments on this planet. Given the diversity and sheer volume of data, it is not expected that you will encounter so many possibilities. This is not impossible (Clokie et al. 2011). Nevertheless, all In most cases, so far, adsorption has been associated with components or protruding structures of the bacterial cell wall. It has been shown that the receptor is It may be any such receptor mentioned in this paragraph or elsewhere in this disclosure.

[0029] Optionally, the receptor is a lipopolysaccharide (LPS), a heptose moiety, a glycosylated cellular component of the host. Wall teichoic acid (WTA), YueB, or phage tail fiber protein or It contains a receptor recognized by gp21 of the phage.

[0030] Receptors in the cell walls of gram-positive bacteria Peptidoglycan, or murein, is an important component of the bacterial cell wall and is responsible for the formation of Bacteriophages. This is often related to the adsorption of amino acids and sugar derivatives, i.e. N-acetylglucosamine. Polymers composed of multiple units of acetylglucosamine and N-acetylmuramic acid. These sugar moieties are linked through glycosidic bonds and are bound to each other by amino acid bridges. The glycan tetrapeptide sheets are linked together. The cross-links are A peptide bond between carboxylic acid (an amino acid analogue) and D-alanine, or a short interpeptide bond These bridges are more abundant in gram-positive bacteria and their It gives rise to a characteristically thick cell wall.

[0031] Another major component of the cell wall of gram-positive bacteria that may be involved in phage adsorption is teichoic acid, i.e. It is composed of glycerol phosphate or ribitol phosphate and amino acids. These are polysaccharides that are linked to the muramic acid of peptidoglycan. When bound to lipids in the plasma membrane, it is called lipoteichoic acid (LPA). For further details on the composition, see Tortora, Funke and Case (2007). Willey, Sherwood and Woolverton (2008), Pom Merville (2010) and Madigan et al. (2012) It can be put out.

[0032] The majority of receptors identified to date are associated with peptidoglycan or teichoic acid structures. Among the 30 phages targeting Gram-positive bacteria reported in Table 1, Of these 10 phages, 9 For reversible binding, teichoic acid (phage SPP1) or peptidoglycan (phage 5 , 13, c2, h, ml3, kh, L, and p2). These structures appear to play an important role in phage adsorption to Gram-positive bacteria. Emphasise the role.

[0033] Optionally, the receptor of the present invention is a peptide glycoprotein that is a peptide glycoprotein of peptidoglycan, murein, teichoic acid, or lipoprotein. Optionally, each transduction particle of the present invention comprises a first pharmacokinetic (ST) molecule. or comprises a capsid of a first phage, wherein the first phage is one of the phage listed in Table 1. phages of the above families (and optionally producer and / or target cells) is a host for a phage listed in Table 1 and / or the receptor is a phage listed in Table 1. In one embodiment, the producer and target cells are Gram-positive Optionally, the producer and / or target cells are of a species listed in Table 1. or strains (where the producer and target cells are of different or the same species). Preferably, when the producer cell is a Gram-positive bacterium, the receptor is peptidoglycan. Alternatively, if the producer cell is a Gram-positive bacterium In this case, the receptor is a teichoic acid.

[0034] Receptors in the cell walls of gram-negative bacteria In gram-negative bacteria, the peptidoglycan layer is relatively thin and is the major component of the cell wall. These two layers are bounded by the lipoprotein of Braun. The outer membrane is made up of a lipid bilayer decorated with proteins, polysaccharides, and lipids. It is a complex structure composed of polysaccharides and lipids that form the LPS layer. It is a complex consisting of three parts: lipid A, the core polysaccharide, and the O-polysaccharide. Core A is generally composed of a fatty acid linked to a glucosamine phosphate disaccharide. The polysaccharide is linked to lipid A through a ketodeoxyoctonate linker. and O-polysaccharides (O-chains or O-antigens) are a few units of sugar that extend outward toward the outer membrane. Cells that contain all three components of LPS are designated smooth (S) type, Cells lacking the O-polysaccharide portion are classified as rough (R) type. The monosaccharides that bind the polysaccharides are highly variable, whereas those of the core polysaccharide are more conserved among species. Therefore, phages specific only to S-type strains tend to target the O-polysaccharide, and thus Compared with strains that can adsorb to R-type cells, they generally have a narrower host range ( Rakhuba et al. 2010 ).

[0035] Table 2(a) shows the receptor-binding proteins (RBPs) that are located on the cell wall and interact with phages. Interestingly, in coliphages, have no preference for proteinaceous or polysaccharide receptors, and some phages bind to cell walls Some adsorb to the sugar moiety of the protein, while others require both structures for adsorption. The situation is not much different for almonella phages, some of which express proteins Some use the sugar moiety, and some use both types of receptors. Domonas phages generally adsorb to polysaccharide receptors. Although no definite conclusions can be drawn from the results, Pseudomonas has two LPS moieties. LPS may have short chains, designated A-band, and longer B-band LPS. Note that (Beveridge and Graham 1991).

[0036] Optionally, the receptor of the present invention is a host cell wall protein. Optionally, the receptor is a sugar Optionally, the receptor comprises an O-antigen, LPS lipid A, or LPS core polysaccharide. In one example, the receptor is smooth LPS or rough LPS. Optionally, the host cell is Optionally, the host cell is an S-type bacterium and the receptor comprises a host O-antigen. Optionally, the host cell is an R-type bacterium. The receptor contains the host LPS lipid A.

[0037] Optionally, the receptor is a host cell wall protein. Optionally, the receptor is a sugar. Optionally, the receptor comprises an O-antigen, LPS lipid A, or LPS core polysaccharide. Optionally, the host cell is an S-type bacterium. and the receptor comprises a host O-antigen. Optionally, the host cell is an R-type bacterium and the receptor The body contains the host's LPS lipid A.

[0038] In one example, the host is E. coli and the transducing particle is a coliphage (or coliphage capsids), where the receptor is a polysaccharide receptor and / or a host cell wall receptor. In one example, the producer cell is an E. coli polysaccharide receptor and and / or have been engineered not to express the E. coli cell wall protein receptor.

[0039] In one example, the host is Salmonella, where the receptor is a polysaccharide receptor and / or or a host cell wall protein receptor. In one example, the producer cell is a Salmonella Expresses the ella polysaccharide receptor and / or the Salmonella cell wall protein receptor It has been manipulated so that this does not happen.

[0040] In one example, the host is Klebsiella, where the receptor is a polysaccharide receptor and / or or a host cell wall protein receptor. In one example, the producer cell is Klebsi Expressing the ella polysaccharide receptor and / or the Klebsiella cell wall protein receptor It has been manipulated so that this does not happen.

[0041] In one example, the host is a Pseudomonas, where the receptor is a polysaccharide receptor. In one example, the second cell is engineered not to express the Pseudomonas polysaccharide receptor. is.

[0042] Optionally, each transduction particle of the invention is a first phage or a first wherein the first phage is a phage of a family listed in Table 2. (Optionally, the producer cell is host to a phage listed in Table 2, and and / or the receptor is a receptor for a phage listed in Table 2).

[0043] In one embodiment, the producer and target cells are gram-negative cells. Optionally, the producer cell is an E. coli cell. Alternatively, the target cells are of a species or strain listed in Table 2 (e.g., where the cell species are different). or are the same).

[0044] Table 2(b) shows that phages not only adsorb to the bacterial surface but also bind to the sugar moiety in the O-chain structure. All of these phages belong to the Podovirida Please note that it belongs to the e family.

[0045] Receptors in other structures of Gram-negative bacteria In this section, we will discuss the cellular components other than the cell wall that act as receptors for particles or phages. Discuss bacterial structures. These include structures such as flagella, cilia, and capsules. These can be found in both Gram stained species (see Table 3 for examples).

[0046] Optionally, the receptor of the present invention is a flagellar, cilia, or capsular component (e.g., in the listed species). Ingredients listed in Table 3 or ingredients found in a host that is a different species than the listed species. Optionally, the phage is of a family listed in Table 3 (and optionally The host is a host for a phage listed in Table 3, and / or the receptor is a host for a phage listed in Table 3. Optionally, the phage is a phage listed in Table 3 ( Also optionally, the host is a host for a phage listed in Table 3, and / or a receptor are receptors for the phages listed in Table 3).

[0047] Flagella are long, thin spiral structures that confer motility to cells. They are divided into basal bodies, flagellar filaments, and The flagellar filament is composed of subunits of the flagellin protein. (Willey, Sherwood and Woolverton Table 3(a) reports the phages attached to flagellar proteins. The adhesion of phages to the cleavage structures is generally reversible, and the spiraling motion of the flagella allows the phages to move The phage moves along the bacterial wall until it reaches the wall. Then, near the base of the flagellum, Irreversible adsorption occurs to receptors located on the bacterial surface (Schade, Adler and Ris 1967;Lindberg 1973;Guerrero-Fer Reira et al. 2011). Interestingly, some phages (φCbK and φCb13), which protrude from the capsid and are involved in reversible binding to the host flagellum. The irreversible adsorption was observed when the phage tail was attached to a thin filament. This occurs only when the ciliary inlet on the cytoplasmic pole interacts with the cytoplasmic pole (Guerrero-Ferrei For these phages, the interaction with the flagellum Even if they do, irreversible adsorption occurs on the cilia, so they do not interact with receptors within the cilia. The phages and mating pair formation structures used are reported in Table 3(b).

[0048] Pili are rod-shaped, fibrous appendages used for bacterial conjugation (L Indberg 1973). These extend from the donor cell and attach to the wall of the recipient cell. Depolymerization of the cilium triggers its contraction, bringing both cells closer together. Further adhesion of cells is achieved by the binding of proteins on their surface. Genetic material is transferred by zygotic conjugation (Madigan et al. 201 2) Adsorption to pili structures has so far been a different phenomenon from that observed in Caudovirales. In fact, Frost (1993) reported that In other words, the Cystoviridae and Inoviridae families adsorb on the ciliary structures. Interestingly, phages bind to the piliated parts of the This is the case for F-type phages, whose adsorption occurs only at the tip of the pilus. (Click and Webster 1998). In other phages such as φ6, In the case of Daugelavicius, attachment occurs on the side (shaft) of the structure. et al. 2005).

[0049] The capsule is a flexible cementitious substance that extends radially from the cell wall. They act as binders between the matrix and / or between cells and the substrate (Beveridge et al., 2003). nd Graham 1991). The slime layer is similar to the capsule, but is more easily deformed. Both are made of sticky substances released by bacteria and share many common components. The saccharide or protein (Madigan et al. 2012). Adsorption of phages to the lime layer is mediated by enzymatic cleavage of the exopolysaccharides that compose the layer. Hydrolysis of the layer is a reversible step, whereas irreversible binding occurs between the phage and the cell. This is achieved through binding to receptors on the wall (Rakhuba et al. 2010). As seen in Table 3(c), only a few fungi were identified that possessed RBP-recognizing exopolysaccharides. The phages are mostly of the Podoviridae morphology.

[0050] In one example, the producer cell is a Salmonella (e.g., S. enterica Serovar Typhimurium cells, where the receptor is the flagellum and vitamin B1 2. The protein is selected from the group consisting of outer membrane protein, BtuB, and lipopolysaccharide-associated O-antigen. In the example, the receptor is flagella or BtuB and the first phage is Siphovirida In one example, the receptor is the O-antigen of LPS, and the first phage is the Pod oviridae phage. Optionally, the receptor is a FliC host receptor or a FliC host receptor. jB receptor.

[0051] Optionally, the producer cell is a S. enterica or P. aeruginosa cell. Optionally, the receptor is a host receptor listed in Table 4.

[0052] The O-antigen structure of Salmonella O66 has been established and reportedly The known O-antigen structure of Escherichia coli O166 is similar to that of the O -units) and differ only in O-acetylation. O-antigen gene classes of Salmonella O66 and E. coli O166 The only exception is Salmonella typhimurium, which has been found to have a similar organization. The wzy gene in O66 has been replaced by a non-coding region. The function of the wzy gene in E. coli O166 was investigated by deletion and transfection. This was confirmed by construction and analysis of complementation mutants. , and D1, located outside the O-antigen gene cluster, as previously reported. A functional wzy gene located in the cytoplasm is involved in the biosynthesis of Salmonella O66 O-antigen. It is proposed that the O -Sequence identity for corresponding genes between antigen gene clusters ranges from 64 to 70%. This suggests that they may have originated from a common ancestor. After that, Salmonella O66 was identified as a mutant of the wzy gene located in the O-antigen gene cluster. Gene inactivation and the identification of two new genes, both of which are outside the O-antigen gene cluster. By acquiring genes (wzy gene and prophage gene for O-acetyl modification) It is possible that the specific O-antigen form was acquired by

[0053] In one example, the producer cell is an E. coli cell, capable of expressing E. coli ( For example, Escherichia coli O166 does not contain the wzy gene.

[0054] Optionally, the receptor is a lipopolysaccharide, a teichoic acid (optionally containing 1-3 glycerol hydroxyl groups). The sulphate is attached to the C4 hydroxyl of the ManNAc residue, and glycerol or ribozyme is A ManNAc(β1→4)GlcNAc disaccharide followed by a long chain of repeating diol phosphates. ), proteins, and flagella.

[0055] Optionally, the receptor comprises the O-antigen of the producer cell type.

[0056] Optionally, the phage or particle of the invention may be When replicating in the producer cell, endolysins or holoproteins are released into the producer cell. This can then be purified from the cellular material to release the particles. , are useful in producing the compositions of the present invention.

[0057] In one embodiment, each particle is capable of infecting a target bacterium, and the particles are Nucleotide sequences of interest (NSIs) capable of expressing protein or RNA in In one embodiment, the NSI comprises a control element operable in the target bacterium. The target cell is then transformed into a chromosome or episome by recombination with the chromosome or episome contained in the target cell. Optionally, this can be achieved by modifying a chromosome or episome (e.g. For example, Cas, TALEN, zinc finger, or meganucleases are used as guides. at predetermined sites using a specific nuclease or restriction endonuclease, Simultaneously or sequentially, cells are infected with a particle containing a first DNA that contains an NSI, and the DNA is then transfected into the cells. The NSI or its sequence is introduced into the cell so that it is inserted into the chromosome or In one example, the first DNA is introduced into an episome. The present invention further comprises one or more components of a CRISPR / Cas system operable to perform a transcriptional regulation (e.g., A nucleotide sequence encoding a guide RNA or crRNA that targets the site to be cleaved. columns) and further includes NSI.

[0058] In one embodiment, the NSI or the protein encoded thereby in the target bacterium The presence of the protein or RNA results in the death of the target cell or downregulation of the growth or proliferation of the target cell. one or more RNAs that mediate regulation or are encoded by the genome of the target cell or mediate switching off or downregulation of protein expression.

[0059] In one embodiment, the NSI or the protein encoded thereby in the target bacterium The presence of the protein or RNA mediates upregulation of growth or proliferation of the target cell, or is a sequence of one or more RNAs or proteins encoded by the genome of a target cell. It mediates the switching on or upregulation of expression.

[0060] In one embodiment, the NSI is a component of the CRISPR / Cas system that is toxic to the target bacterium. Code the following:

[0061] In one embodiment, the first NSI is a vector (e.g., a plasmid or a shuttle vector). ) is included.

[0062] One embodiment is a method of treating an environment ex vivo, the method comprising administering to a subject the production exposing the environment to a population of transducing particles obtainable by the method, a particle (e.g., a phage or a particle containing a phage capsid) that is attached to the target bacterium In one embodiment, the agent further comprises administering a phage to the host. In one embodiment, the agent is a herbicide, insecticide, or pesticide. , plant fertilizer, or cleaning agent.

[0063] A method of treating an infection of a target bacteria in a human or animal subject, the method comprising: and exposing the bacteria to a population of transducing particles obtainable by the method, wherein the particles Methods are provided for infecting and killing target bacteria.

[0064] Optionally, the target bacteria herein are present in the microbiome of a subject, e.g., intestinal microbiome. The microbiome is contained in the skin, scalp, hair, eyes, and ears. , oral cavity, throat, lungs, blood, rectum, anus, vagina, scrotum, penis, nose, or tongue microbiome It is a room.

[0065] In one example, the subject is further administered a pharmaceutical agent either simultaneously or sequentially with administration of the transducing particles. In one example, the drug is an antibiotic, an antibody, an immune checkpoint inhibitor (e.g., anti-PD-1 , anti-PD-L1, or anti-CTLA4 antibodies), adoptive cell therapy (e.g., CAR-T therapy), Or a vaccine.

[0066] In one example, the present invention employs a helper phage to package the NSI. In one example, the helper phage packages DNA containing the NSI to induce the first transduction. The particles can be produced by the helper phage, which is different from the helper phage. It is not capable of producing helper phage particles by itself.

[0067] A composition comprising a population of transducing particles of the invention, the particles being capable of replication of the transducing particles. and optionally including a helper phage according to the immediately preceding paragraph in the composition. Total transducing particles were 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.2, or or less than 0.1% are such helper phage particles. In an example, the composition includes a helper phage, which comprises less than 1% of the total transducing particles in the composition. The helper phage particles are such helper phage particles. In one example, the composition includes a helper phage. and less than 0.5% of the total transducing particles in the composition are such helper phage particles. In one example, the composition includes a helper phage, and all transducing particles included in the composition Less than 0.1% of the particles are such helper phage particles. The composition contains perphage, and less than 0.01% of the total transducing particles in the composition are such hematogenous. In one example, the composition includes a helper phage. Less than 0.001% of the total transducing particles contained are such helper phage particles. In one example, the composition includes a helper phage, which is 0.0 of the total transducing particles included in the composition. In one embodiment, the composition comprises a helper phage. The composition may contain phages, and less than 0.00001% of the total transducing particles contained therein may be such transducing particles. In one embodiment, the composition comprises a helper phage. Less than 0.000001% of the total transducing particles are such helper phage particles. In one example, the composition includes a helper phage and all transducing particles included in the composition. Less than 0.0000001% of the total number of helper phage particles. The composition contains helper phage and is 0.0000000 of the total transducing particles contained in the composition. Less than 1% are such helper phage particles.

[0068] In one example, the composition or population is characterized by, for example, a transduction assay. 0 3 , 10 4 , 10 5 , or 10 6 In one example, the composition or The population is at least 10 3 transducing particles and e.g. 10 14 Contains no more than 1 particle In one example, the composition or population comprises at least 10 4 transducing particles and e.g. 0 14 In one example, the composition or population contains at least 10 5 pcs Transducing particles and e.g. 14 In one example, the population contains at least Friend 10 6 transducing particles and e.g. 10 14 Contains no more than 1 particle. To obtain a measure, for example, if the genome of the particle of the invention contains an antibiotic marker, standard In this case, the particles of the present invention can be used to carry out a transduction assay. The composition of the population in a 1 ml sample is at least 10 3 , 1 0 4 , 10 5 , or 10 6 It contains 10 transducing particles, which are capable of infecting susceptible bacteria. Infected at a severity of <0.1 and plated in vitro on selective agar plates corresponding to antibiotic markers. ro at 20-37 °C, e.g., at 20 °C or 37 °C, and determine the number of infected cells. This can be determined by:

[0069] Optionally, at least 99.9, 99.8, 99.9% of the total transducing particles in the composition. .7, 99.6, 99.5, 99.4, 99.3, 99.2, 99.1, 90, 85, 8 0, 70, 60, 50, or 40% are particles of the invention.

[0070] In one example, the genome of the particle of the invention comprises an f1 origin of replication.

[0071] In one example, the helper phage is an E. coli phage. are E coli, C dificile, Streptococcus, Klebsie lla, Pseudomonas, Acitenobacter, Enterobacter eracea, Firmicutes, or Bacteroidetes phages or comprises a capsid of such a genus, which capsid packages the NSI. In one example, the helper phage is an engineered M13 phage.

[0072] In one example, the genome of the particle of the invention comprises a phagemid, the phagemid being linked to a helper phag The protein contains a packaging signal for packaging the particle in the presence of a gene.

[0073] The particles of the invention contain a nucleotide sequence of interest (NSI), e.g., as defined herein. The CRISPR / Cas system can be engineered in a target bacterium that can be infected by the particle. Once inside the target bacterium, it may contain DNA, including the nucleic acid sequence encoding the component. The particle DNA is optionally packaged in the absence of helper phage to produce transducing particles. This is usefully because the genome of the particle of the invention and The activity of the product (protein and / or nucleic acid) encoded by the NSI and its encoded product in the environment containing the target bacteria exposed to particles This can be done, for example, to limit and control the dosage of a substance to a human or animal subject, a plant, or the like. Controlling environmental medical procedures in other environments (e.g. soil or foodstuffs or ingredients) It is useful for

[0074] In one embodiment, each particle of the invention comprises one or more phage structural proteins. Examples of phage structural proteins include: Phage coat proteins, collar proteins, and phage tail fiber proteins In one example, the particle comprises capsid and tail fiber proteins of a first type of phage. For example, phage types include E. coli, Klebsiella pneumoniae, e, Pseudomonas aeruginosa, Clostridium dif ficile, Helicobacter pylori, Staphylococcu aureus, Salmonella (e.g., Typhimurium), or C. It is an ampylobacter phage.

[0075] Optionally, at least 95% (e.g., 100%) of the transducing particles in the composition are , particles of the present invention.

[0076] In another embodiment, the composition comprises a second transducing (e.g., phage) particle, The child is different from the first particle of the present invention (i.e. the particle recited in claim 1).

[0077] Optionally, the composition population comprises at least 10 3 , 10 4 , 10 5 , or 10 6 It contains phage particles.

[0078] Optionally, each particle of the invention comprises a vector for NSA, the vector being a promoter. For example, a vector can be a plasmid or a phagemid that can replicate in a host bacterium. The vector is a shuttle vector (e.g., a pUC vector) that can be used to

[0079] Optionally, the genome of each particle of the invention contains a packaging signal, e.g. c or cos sequence or a homolog thereof.

[0080] Optionally, the transducing particle of the invention is a gentle phage. The transduction particle is a lytic phage.

[0081] Optionally, the particles of the invention are capable of infecting a target bacterium, and the particles An eye capable of expressing protein or RNA (e.g. gRNA or crRNA) or the NSI is operable in the target bacterium. Contains a control element.

[0082] Optionally, the NSI or the protein encoded thereby in the target bacterium is also The presence of the target cell or RNA results in the death of the target cell or down-regulation of the growth or proliferation of the target cell. and one or more RNAs mediating or encoded by the genome of the target cell. or mediate the switching off or downregulation of protein expression.

[0083] Optionally, the NSI or the protein encoded thereby in the target bacterium is also or the presence of the RNA mediates upregulation of growth or proliferation of the target cell, or Expression of one or more RNAs or proteins encoded by the genome of the target cell They mediate the switching on or upregulation of the current gene.

[0084] Optionally, the particles of the invention are capable of infecting a target bacterium, and each particle is capable of infecting a target cell. The term "engineered" includes engineered antimicrobial means for killing bacteria. The linking means is introduced and is not naturally present in the phage or particle. For example, the means may be recombinant, artificial, or synthetic. It is.

[0085] In one example, each particle of the invention comprises a genomic island DNA or a pathogenicity island. (e.g., saPI) DNA, and optionally the DNA kills the NSI or target bacteria. The engineered antimicrobial means includes DNA that binds to Cas nucleases (e.g., Cas 9 or Cas3), and / or a primer for expression in the target bacterium. (encoding id RNA).

[0086] Optionally, the antimicrobial means comprises one or more components of a CRISPR / Cas system. Optionally, the component (i) encodes a guide RNA (e.g., a single guide RNA); is a DNA sequence comprising a CRISPR array for producing a guide RNA, (ii) a DNA sequence that allows the RNA to be targeted to the genome of a target bacterium; (iii) a DNA sequence encoding an ase, and / or (iv) one or more of the cascades In one example, the Cas in the present specification is Cas9. In one example, the Cas herein is Cas3. Cas is activated by the target bacterium. It may be identical to the encoded Cas.

[0087] Optionally, the antimicrobial means is a Cas nuclease, a TALEN, a zinc finger nuclease The nucleic acid encoding the nuclease or a guided nuclease, such as a meganuclease, .

[0088] Optionally, the composition, population, or transduction particle, respectively, is administered to a human or animal subject. or the composition is for use in medical practice in humans or animals. In one example, the pharmaceutical composition is for use in medical practice in an animal subject. are livestock or companion animals (e.g., cows, pigs, goats, sheep, horses, dogs, cats, In one example, the animal is an insect (any stage of its life cycle, e.g. In one example, the animal is a protozoan. In an example, the animal is a cephalopod.

[0089] Optionally, the composition is an herbicide, a pesticide, a food or beverage processing agent, a food or beverage additives, petrochemical or fuel treatment agents, water purification agents, cosmetic additives, detergent additives, is an environmental (e.g. soil) additive or cleaning agent.

[0090] In some embodiments, the particles of the invention are incapable of self-replicating and therefore do not require a helper phage The requirement for replication by a first phage, for example, involves a first phage that would otherwise be replicated. The composition is delivered to the site of action (e.g., the intestine) when exposed to the subject's urine and feces secretions. In some embodiments, the present invention provides a method for detecting and / or detecting a subject in a biological environment, and / or provides a method for containing the subject in an environmental manner. The inability of the helper phage to self-package prevents the formation of packaged particles. This limits the availability of elements that the particles need to produce, thus increasing the yield of the particles of the invention. This provides containment by limiting replication, for example through CRISPR / Cas transcription factors. It would be useful to confine the antimicrobial activity provided by particles such as antimicrobial principles.

[0091] In one example, when the subject is a human, the subject is not an embryo.

[0092] Optionally, the environment is a soil microbiome, i.e., a plant, a plant part (e.g., a leaf , fruits, vegetables, or flowers) or plant products (e.g. pulp), water, waterways, fluids, foodstuffs or its components, beverages or their components, medical devices, cosmetics, detergents, blood, body fluids, Medical equipment, industrial equipment, oil rigs, petrochemical processing, storage or transportation equipment, vehicles In one example, the environment is an ex vivo body fluid (e.g., urine, blood, blood products, etc.). , sweat, tears, sputum, or saliva), body solids (e.g., feces), or a human to whom the composition is administered. or tissue of an animal subject.

[0093] Optionally, the antimicrobial means comprises one or more components of a CRISPR / Cas system. For example, the component may (i) encode or guide a guide RNA (e.g., a single guide RNA). A DNA sequence comprising a CRISPR array for producing guide RNA, A is a DNA sequence capable of targeting the genome of a target bacterium; (ii) Cas (e.g., C as9, Cas3, Cpf1, CasX, or CasY) nuclease-encoding D and / or (iii) one or more components of a cascade (e.g., a Cas A) comprises a DNA sequence encoding the

[0094] Optionally, the antimicrobial means is a Cas nuclease, a TALEN, a zinc finger nuclease The nucleic acid encoding the nuclease or a guided nuclease, such as a meganuclease, .

[0095] In one example, the particles, populations, or compositions of the invention can be placed in a medical container, e.g., a syringe, vial, or the like. In one example, the present invention is The particles, populations, or compositions of the invention are contained in a sterile container. The particles, populations, or compositions of the invention are contained in a medically compatible container. or the composition is contained in a fermentation vessel, e.g., a metal, glass, or plastic vessel. do.

[0096] In one example, the particles, populations, or compositions of the invention can be administered, for example, orally, IV, subcutaneously, intranasally, intraocularly, or intravenously. Administering a drug to a human or animal subject by intravenous, vaginal, topical, rectal, or inhalation administration. In combination with instructions for use or packaging labeling that includes instructions for use in In one example, the particles, populations, or compositions of the invention are included in an oral pharmaceutical formulation. In an example, the particles, populations, or compositions of the invention are included in an intranasal or intraocular pharmaceutical formulation. In examples, the particles, populations, or compositions of the invention are used in personal hygiene compositions (e.g., shampoos, In one example, the particles, populations, or In one example, the particle, population, or composition of the present invention is included in a cleaning formulation. For example, in cleaning formulations for cleaning medical or industrial devices or equipment. In one example, the particles, populations, or compositions of the invention are added to a foodstuff, foodstuff ingredient, or foodstuff processing agent. In one example, the particles, populations, or compositions of the invention are incorporated into beverages, beverage components, or beverages. In one example, the particles, populations, or compositions of the present invention are included in a medical bandage. In one example, the particles, populations, or swabs of the present invention are Or the composition is included in a herbicide or insecticide. The composition is included in an insecticide.

[0097] In one example, each particle is a first phage particle or a first phage particle. psid (and optionally a tail fiber of a first phage), the first phage being C orticoviridae, Cystoviridae, Inoviridae, Le viviridae, Microviridae, Myoviridae, Podovi Ridae, Siphoviridae, or Tectiviridae viruses In one example, the helper phage is idae, Inoviridae, Leviviridae, Microviridae , Myoviridae, Podoviridae, Siphoviridae, or In one example, the helper phage is a filament virus. M13, Noviridae, tailed phages (e.g. Myoviridae, S iphoviridae, or Podoviridae), or non-tailed phages (e.g. Tectiviridae).

[0098] In one example, both of the first phages are Corticoviridae. In one example, the first phage is In In one example, the first phage is Leviviridae. In one example, the first phage is Microviridae. In one example, the first phage is Siphovirid ae. In one example, the first phage is Tectiviridae.

[0099] In one example, the CRISPR / Cas components are components of a type I CRISPR / Cas system. In some examples, the CRISPR / Cas components are components of a type II CRISPR / Cas system. In the example, the CRISPR / Cas components are components of a type III CRISPR / Cas system. In the present study, the CRISPR / Cas components are components of a type IV CRISPR / Cas system. In one embodiment, the CRISPR / Cas component is a component of a V-type CRISPR / Cas system. The CRISPR / Cas components include a nucleotide sequence encoding Cas9 (e.g., S pyogenes Cas9, S aureus Cas9, or S thermop hilus Cas9). In one example, the CRISPR / Cas component encodes Cas3. Nucleotide sequences (e.g., E. coli Cas3, C. difficile C as3, or Salmonella Cas3). In one example, The fragment comprises a nucleotide sequence encoding Cpf. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding CasX. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding CasY.

[0100] In one example, the genome of the particle is engineered to express CRISPR / Cas components or other gene products that can be manipulated within the target bacterium. The protein of interest is encoded from a nucleotide sequence that includes an operable promoter.

[0101] In one example, the host bacterium and / or target bacterium is E. coli. The bacterium and / or the target bacterium is C dificile (e.g., the vector is E. coli i and the host bacterium is C dificile). In one example, the host bacterium and / or target bacterium is Streptococcus, e.g., S. thermophilus (e.g., the vector is a shuttle that can be operated in E. coli). In one example, the host cell is a Streptococcus. The fungus and / or target bacterium is Pseudomonas, e.g., P. aeruginosa (e.g., the vector is a shuttle vector operable in E. coli and In one example, the host bacterium and / or the target bacterium are Klebsiella, e.g., K pneumoniae (e.g., the vector is E It is a shuttle vector that can be operated in coli and the host bacterium is Klebsiella. In one example, the host bacterium and / or the target bacterium is Salmonella, e.g., S. typhimurium (e.g., the vector is a shuttle vector operable in E. coli the vector and the host bacterium is Salmonella).

[0102] Optionally, each producer and / or target bacterium is a Gram-negative bacterium (e.g. Optionally, each producer and and / or the target bacterium is a Gram-positive bacterium. Optionally, each producer and and / or the target bacteria is Mycoplasma, Chlamydia, Spirochete, or Mycobacterium Optionally, each producer and / or target bacterium is a Streptococcus cerevisiae. eptococcus (e.g. pyogenes or thermophilus) Optionally, each producer and / or target bacterium is Staphylococcus aureus. coccus (e.g., aureus, e.g., MRSA). The producer and / or target bacteria are E. coli (e.g., O157:H7) hosts. For example, where Cas is encoded by a vector or is an endogenous host Ca Optionally, the nuclease activity of each of the producers and and / or the target bacterium is a Pseudomonas (e.g., aeruginosa). Optionally, each producer and / or target bacterium can be cultured in Vibro (e.g., olerae (e.g., 0139) or vulnificus). Each producer and / or target bacterium is a Neisseria (e.g., gonn orrhoeae or meningitidis. The inducer and / or target bacteria are Bordetella (e.g., pertussis Optionally, each producer and / or target bacterium is a Haem ophilus (e.g., influenzae). Optionally, The inducer and / or target bacterium is Shigella (e.g., dysenteriae). Optionally, each producer and / or target bacterium is Brucella. la (e.g. abortus). Optionally, each producer and / or Alternatively, the target bacterium is a Francisella host. The sensor and / or target bacterium is a Xanthomonas host. Each producer and / or target bacterium is an Agrobacterium host. Optionally, each producer and / or target bacterium is an Erwinia host. Optionally, each producer and / or target bacterium is Legion ella (e.g., pneumophila). Optionally, each producer The serogroup and / or target bacteria is Listeria (e.g., monocytogenes) Optionally, each producer and / or target bacterium is Campyl obacter (e.g., jejuni). Optionally, each producer and / or the target bacterium is Yersinia (e.g., pestis). , the respective producer and / or target bacteria are Borelia (e.g., burgdorferi), dorferi). Optionally, each producer and / or target cell The bacterium is Helicobacter (e.g., pylori). Optionally, each The producer and / or target bacteria may be Clostridium (e.g., Clostridium difici le or botulinum). Optionally, the respective producers and and / or the target bacterium is Erlichia (e.g., chaffeensis). In the selection, the respective producer and / or target bacteria are Salmonella (e.g. typhi or enterica, e.g. serovar typhimurium, e.g. D T104). Optionally, each producer and / or target bacterium is C hlamydia (e.g., pneumoniae). Optionally, The inducer and / or target bacteria are Parachlamydia hosts. The respective producer and / or target bacteria are Corynebacterium m (e.g., amycolatum). Optionally, each producer and and / or the target bacterium is Klebsiella (e.g., pneumoniae). Optionally, each producer and / or target bacterium is Enterococcus s (e.g. faecalis or faecim, e.g. linezolid resistant). In the selection, the respective producer and / or target bacteria are Acinetobacter r (e.g., baumannii, e.g., multidrug resistant).

[0103] Further aspects of targeting cells and targeting antibiotic resistance in such cells using the present invention. An example is as follows: 1. Optionally, the target bacteria is treated with antibacterial agents, such as methicillin, vancomycin, linezolid, daptomycin, mycin, quinupristin, dalfopristin, and teicoplanin. These are antibiotic-resistant Staphylococcus aureus cells. 2. Optionally, the target bacteria is a cephalosporin (e.g., ceftazidime), Bapenems (e.g. imipenem or meropenem), fluoroquinolones, aminoglycosides and an anti-inflammatory drug selected from the group consisting of gentamicin, gentamicin or tobramycin, and colistin. Pseudomonas aeruginosa cells resistant to bioagents. 3. Optionally, the target bacteria is, for example, a carbapenem-resistant Klebsiella (e.g. Bapneumoniae cells. 4. Optionally, the target bacteria is a medicament such as, for example, erythromycin, clindamycin, beta-lamycin, Choose from methadone, macrolides, amoxicillin, azithromycin, and penicillin Streptococcus resistant to commonly used antibiotics (e.g., thermophilus, pneumoniae, or pyogenes cells. 5. Optionally, the target bacteria is treated with, for example, ceftriaxone, azithromycin, and cytomegalovirus. Salmonella resistant to antibiotics selected from rofloxacin (e.g., serovar Ty phi) cells. 6. Optionally, the target bacterium is selected from, for example, ciprofloxacin and azithromycin. These are Shigella cells that are resistant to antibiotics and are selected for use in the treatment of bacterial infections. 7. Optionally, the target bacteria is treated with, for example, isoniazid (INH), rifampicin (RMP), ), fluoroquinolones, amikacin, kanamycin, and capreomycin, and Mycobacterium t resistant to antibiotics selected from resistance to zithromycin uberculosis cells. 8. Optionally, the target bacterium is, for example, an Enterococcus resistant to vancomycin. It is a cell. 9. Optionally, the target bacterium is selected from, for example, cephalosporins and carbapenems. These are Enterobacteriaceae cells that are resistant to antibiotics. 10. Optionally, the target bacteria is treated with, for example, trimethoprim, itrofurantoin, cephalosporin, and amoxicillin. 11. Optionally, the target bacteria is treated with, for example, fluoroquinolone antibiotics and carbapenems. Clostridium (e.g., dificile) cells that are resistant to the selected antibiotic. do. 12. Optionally, the target bacteria is treated with, for example, cefixime (e.g., an oral cephalosporin), Ceftriaxone (an injectable cephalosporin), azithromycin, and tetracycline Neisseria gonnorrhoea cells resistant to selected antibiotics from be. 13. Optionally, the target bacteria is treated with antibacterial agents such as beta-lactams, meropenem, and carbapenem. Acinetobacter [Acinetoebacter] b resistant to antibiotics selected from Nem aumannii cells. 14. Optionally, the target bacteria is selected from, for example, ciprofloxacin and azithromycin. Campylobacter cells resistant to the antibiotic of choice. 15. Optionally, the target cell produces beta (β)-lactamase. 16. Optionally, the target cells are cells resistant to an antibiotic recited in any one of Examples 1 to 14. It is a bacterial cell.

[0104] Mobile genetic elements, genomic islands, pathogenicity islands, and more Genetic variation in bacteria and archaea is due to mutations, rearrangements, and horizontal gene transfer. This can be achieved by transfer and recombination. These proteins are essential for the regulation of basic metabolic activities, information processing, and housekeeping, such as structural and regulatory components of bacteria. In addition to the core genes that code for pooling functions, genes that are essential for adaptation and survival under certain environmental conditions Many accessory genes encoding antimicrobial resistance, toxins, and enzymes that contribute to the It has been demonstrated that it was acquired by horizontal introgression of mobile genetic elements (MGEs). Mobile genetic elements include plasmids, bacteriophages, genomic islands, Infections including chromosomal cassettes, pathogenicity islands, and integrative and complex elements A group of homogeneous molecules. Genomic islands are flanked by direct repeats of 16–20 bp. Relatively large fragments ranging from 10 to 200 kb are often integrated into tRNA gene clusters. These are segments of DNA that are can be different from other parts of the chromosome, and thus may be an aberrant part acquired by horizontal gene transfer. They are recognized as discrete DNA segments.

[0105] Pathogenicity islands (PTIs) are horizontally transferred genes known as genomic islands. Staphylococcus aureus is a subset of the genetic elements found in In the 1990s, there were highly expressed genes that were induced to excise and replicate by certain resident prophages. There exists a specific family of mobile PTIs. These PTIs are small-headed phage-like particles. The phage is packaged in the phage and transferred at a frequency proportional to the plaque-forming titer of the phage. This process is called the SaPI excision replication-packaging (ERP) cycle, and occurs frequently. SaPI transfer is performed using the S SaPI is a bacteriophage specific transfer pathway (SPST) that is involved in the assembly and Highly conserved and distinct from all other horizontally acquired genomic islands It has a genetic organization similar to that of SaPI1 and SaPIbov2. The integrase is used for both excision and integration of the corresponding element and is similar to other SaPIs. The same is assumed to be true for phage 80α, SapI1, SapI2. , and SapIbov1, while several different SaPIs can be induced. , φ11 can induce SapIbov1 but not either of the other two SapIs. .

[0106] “Staphylococcal pathogenicity island DN A packaging system involving cos-site pa ckaging and phage-encoded HNH endonuclea ses”, Quiles-Puchalt et al, PNAS April 22 ,2014.111(16)6016-6021. The SaPI pathogenicity island of the human sarcoma is highly mobile and is a potent pathogenic agent for superantigens and other pathogens. SaPI carries and disseminates sexual genes using two unrelated, complementary mechanisms. It has been reported that it hijacks the packaging machinery of the phage it preys upon. The packaging of the phage is called "pac" or "cos" depending on the type of phage. The SaPI strategy is initiated by the recognition of a specific sequence in the phage DNA. -Packaging of SaPI into full-sized phage particles depends on the phage machinery Helper phage pac- or cos-like sequences in the SaPI genome ensure binding These strategies interfere with phage reproduction, which ultimately leads to the transfer of phage particles. Reducing the number of offspring confers a significant benefit to the bacterial population.

[0107] Staphylococcus pathogenicity islands (SaPIs) are intra- and inter-species pathogenicity islands. Chromosomally located mobility that contributes substantially to gene transfer, host adaptation, and virulence It is the prototypic member of a widespread family of genetic elements. The characteristics are induction of SaPI excision and replication by certain helper phages and phages. Most SaPIs are abundant packages that efficiently encapsidate into infectious particles. The SapI packaging mechanism recognizes the SapI-specific pac site and initiates SapI packaging. It encodes a homologue of the small terminase subunit (TerS), which determines specificity. Some of the known SaPIs do not encode recognizable TerS homologues, but Regardless of the gene, it is efficiently packaged by helper phage and transferred at high frequency. Quiles-Puchalt et al. First, it reports on SaPIbov5, which is two different undocumented packages. We found that SaPIbov5 uses a typical pac-type helper by phages or by cos-type phages into full-sized phage-like particles That is, it has both pac and cos properties and is packaged in a fungal form. It uses two different TerS sequences encoded by the cos phage. This is an example of SaPI packaging in which Escherichia coli It is similar to the P4 plasmid of Staphylococcus aureus. Cos site packaging in Escherichia coli requires a large target for cleavage and melting of the cos site. HNH nuclei carried exclusively by cos phages in addition to the amino acid sequence subunits. It is even more unique in that it requires

[0108] Characterization of phage-induced SaPI and some of its helper phages It has been established that the pac (or abundant) mechanism is used for encapsidation. In keeping with this concept, several SaPIs encode homologues of TerS. , which is complexed with the phage-encoded large terminase subunit TerL. The SaPI DNA is then packaged in an infectious particle composed of phage proteins. These are small capsid forms that match the small SaPI genome. The first characterized SaPI sequence also contains the morphogenesis (cpm) module, which initiates the synthesis of the nucleosome. Among the columns, there were several sequences that contained neither a TerS nor a cpm homologue, and Several SaPIs and many phages from other species have been subsequently identified from the source. The same is true for inducible chromosomal islands. In this regard, these are deletion derivatives of the elements that originally carried these genes. or the terS and cpm genes were present but not recognized by homology. It was assumed that

[0109] Quiles-Puchalt et al. The key feature of caging is required for HNH nuclease, which in turn activates φSLT We observed that the HNH domain-carrying protein is encoded by the terminase module. Proteins are widespread in nature and are present in organisms from all kingdoms of life. It is a small, degenerate nucleic acid binding and cleavage module of about 30-40 amino acid residues, and is a single The HNH motif is involved in the killing of bacteria, Many different processes, including DNA repair, replication, and recombination, as well as RNA-related processes, are involved. HNH has been found to play an important role in several cellular processes. Endonuclease is a cos site enzyme that binds many cos-site bacteriophages of gram-positive and gram-negative bacteria. and always encodes terminase and other morphogenetic proteins. Quiles-Puchalt et al. HNH nuclease, encoded by φSLT, which is related to β-terminal nuclease, is a nonspecific nuclease. It has enzyme activity and is required for packaging of these phages and SaPIbov5. Quiles-Puchalt et al. L is required jointly for cleavage of the cos site. reported that only cos phages of Gram-negative and Gram-positive bacteria can express HNH genes. Cos cleavage is the opposite of pac site cleavage, which produces blunt-end products. We also observed that HNH nuclease activity was low, consistent with a specific requirement for site cleavage. It is essential for some cos phages but not others. Evidence suggests that there are differences between the TerL proteins of the two types of phages. That is, one can cleave both strands, the other can generate double-strand breaks. It requires a second protein to function.

[0110] Alternatively, instead of bacteria, the respective producer and / or target cells may be Archaeal cells, and there are viruses that can infect archaeal cells instead of phages. Each particle comprises the capsid (and optionally the tail) of such a virus. (including maintenance).

[0111] Optionally, the transduction particle is a non-self-replicating particle. "Non-self-replicating transduction particle" can deliver nucleic acid molecules encoding antibacterial agents or components to bacterial cells, Particles that do not package their own replicated genome into the transduction particle (e.g., A plasmid or phage-like particle, or a genomic island (e.g., SaPI) or a modified version thereof. Alternatively, phage refers to a particle produced from a modified version of the phage. Instead, viruses that infect animal, human, plant, or yeast target cells are used. is packaged, for example, an adenovirus when the target cell is a human cell.

[0112] Optionally, the genome of each particle contains genes encoding phage structural proteins. Instead, these are supplied by helper phages during production. Optionally, the genome of each particle can contain one or more phage genes, It lacks nA, terS, and terL.

[0113] Optionally, the genomic islands are expressed in target and / or producer bacterial cells. Also optionally, in the particles of the invention, the genomic island DNA is a naturally occurring In one example, the genomic islands are SaPI, SaPI1, Sa Selected from the group consisting of SaPI2, SaPIbov1, and SaPibov2 genomic islands. For example, the island is a modified pathogenicity island. The sex islands are those naturally found in the target and / or producer bacterial cells. Land, e.g. Staphylococcus SaPI or Vibro PLE or , P. aeruginosa pathogenicity islands (e.g., PAPI or PAGI, e.g. PAPI-1, PAGI-5, PAGI-6, PAGI-7, PAGI-8, PAGI- Optionally, the pathogenicity island is SapI (S aureus pathogenicity island). Optionally, in this case Helper phages are used during production, where helper phages are φ11, 80α, φ 12, or φSLT. Staphylococcus phage 80α is the In one example, the genome of each particle is modified. Optionally, the pathogenic island is included in the vector, and the helper phage is 80α. The phage-inducible chromosomal island-like element (PLE) of V. cholerae and optionally the first phage is ICP1. Optionally, the pathogenicity island is E coli PLE.

[0114] Optionally, the genome of each particle is composed of P4 DNA, e.g., at least one P4 package. The particles contain a P4 packaging signal and an NSI or antibody coding sequence. In one embodiment, the particles may contain helper filaments to produce the particles. The helper phage is P2 phage, or any other phage that is replication-deficient and can The modified P2 is present as a prophage in the genome of the producer cell at will. It is a phage.

[0115] Optionally, transcription of the particle nucleic acid results in the creation of copies of the antimicrobial agent or component or NSI in the target cell. Optionally, the target cell is killed. In some cases, such as in medical settings, constitutive transcription and production in target cells is of use. It is possible.

[0116] Optionally, transcription of the particle nucleic acid results in the creation of copies of the antimicrobial agent or component or NSI in the target cell. This is under the control of an inducible promoter for transcription of the target cell. in the environment (e.g. soil or water) or in industrial cultivation or fermentation vessels, It would be useful to control the switching on of antibacterial activity against target bacterial cells. The target cells are useful in industrial processes (e.g., fermentation in the brewing or dairy industries) and The induction is to control the process (e.g., stop) by using antimicrobial agents against the target bacteria. It may be possible to (reduce or eliminate)

[0117] If the agent contains multiple components, e.g., the agent is a CRISPR / Cas system, or CRISPR arrays that encode crRNAs that can operate with Cas in target cells When the nucleic acid is a nucleic acid encoding a guide RNA (e.g., a single guide RNA), the crRNA or The gRNA guides Cas to a target sequence in the cell to modify the target (e.g., cut it). Optionally, the gene is integrated into the chromosome and and / or contained in one or more cellular episomes.

[0118] Optionally, the agent is a guided nuclease system or a component thereof, and the agent is They are capable of recognizing and cleaving cellular DNA (e.g., chromosomal DNA). In examples, such cleavage results in one or more of the following: (i) The target cells are killed by the antibacterial agent. (ii) the growth or proliferation of the target cells is reduced; and / or (iii) The target cells are sensitized to the antibiotic, so that the antibiotic becomes toxic to the cells. It is sexuality.

[0119] Optionally, the guided nuclease system is a CRISPR / Cas system, a TALEN system, Optionally, the system is selected from a meganuclease system, or a zinc finger system. The ISPR / Cas system consists of two particle genomes: (a) the CR gene encoding the crRNA; (b) a guide RNA (gRNA, e.g., a single guide RNA) The crRNA or gRNA encodes a nucleic acid that encodes a target gene, and the crRNA or gRNA operates together with Cas in the target cell. The crRNA or gRNA can guide Cas to a target nucleic acid sequence in a target cell. Optionally, the target sequence is modified (e.g., cleaved or inhibited from transcription). Alternatively, the Cas is a Cas encoded by a functional endogenous nucleic acid of the target cell. For example, Optionally, the system comprises a CRISPR / C As system, each particle genome contains Cas (e.g., The nucleic acid sequence encoding the target nucleic acid sequence is encoded by a gene encoding a target nucleic acid sequence (e.g., a Cas nuclease) that modifies a target nucleic acid sequence contained in a target cell.

[0120] Any Cas herein refers to Cas3, Cas9, Cas13, CasX, Ca sY, or Cpf1.

[0121] Optionally, the system is a CRISPR / Cas system and each particle genome comprises one or It encodes multiple cascade Cas (e.g., Cas A, B, C, D, and E).

[0122] Optionally, each particle genome is operable in a target bacterial cell with a cascade of Cas. The vector further encodes an actinically operable Cas3.

[0123] Optionally, the producer and / or target cells are cells of a first species or strain. , the first species or strain is a gram-positive species or strain.

[0124] Optionally, the producer and / or target cells are cells of a first species or strain. , the first species or strain is a gram-negative species or strain.

[0125] Optionally, the first species or strain is selected from Table 6. For example, the first species or strain is Sh igella, E coli, Salmonella, Serratia, Klebsi ella, Yersinia, Pseudomonas, and Enterobacter r. These are the species that the P2 phage can infect: In one embodiment, the particle genome comprises one or more P4 sequences (e.g., P4 packaging sequences). sequence) and the genome is packaged by the P2 phage capsid. The genome is then packaged by the P2 structural protein and the resulting transducing particle expresses a wide range of A wide spectrum of species, namely Shigella, E. coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter can be usefully infected with two or more of the following: provides a broadly applicable delivery platform, and the antimicrobial specificity of the target cells is ella, E coli, Salmonella, Serratia, Klebsiel la, Yersinia, Pseudomonas, and Enterobacter A guide RNA that specifically targets one or more given species in the group is loaded onto the particle genome. This can be achieved by coding.

[0126] "Non-replicating" or "replicating deficient" means that the particle cannot replicate itself. For example, the particle genome may contain the proteins (e.g., constructs) required to produce the transducing particle. It lacks one or more nucleotide sequences encoding a specific polypeptide (protein).

[0127] In one example, the reduction in growth or proliferation of the target cells is at least 50, 60, 70, 80, 90, 100, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 0, or 95%.

[0128] In one example, each producer cell and / or target cell is ococcal, Vibrio, Pseudomonas, Clostridium, E coli, Helicobacter, Klebsiella, and Salmonella lla cells.

[0129] Optionally, each particle comprises: a. A nucleotide sequence encoding an antibacterial agent or component thereof for expression in a target bacterial cell. Arrangement, b. a constitutive promoter for controlling expression of the agent or component; c. an optional terS nucleotide sequence; d. origin of replication (ori), and e. phage packaging sequences (optionally pac, cos, or homologs thereof); And f. A plasmid lacking: g. Encode the phage structural proteins necessary for the production of transducing particles (optionally phages). A primer that lacks all nucleotide sequences that code for the nucleotide sequence of ... Sumido, and h. Optionally, terL The plasmid comprises:

[0130] Optionally, the antimicrobial agent is a CRISPR / Cas system and the plasmid is a Cas targeting nucleic acid. with Cas in the target cell guiding it to a nucleotide sequence and modifying (e.g., cleaving) the sequence Encodes an operable crRNA or guide RNA (e.g., a single gRNA) the law of nature (a) Target cells are killed by the antimicrobial agent, (b) the growth or proliferation of the target cells is reduced; or (c) The target cells are sensitized to the antibiotic, so that the antibiotic is toxic to the cells. be.

[0131] Optionally, the antimicrobial agent is a CRISPR / Cas system and the plasmid is a Cas targeting nucleic acid. A crRNA or a gene in a target cell that guides the gene to a nucleotide sequence and modifies (e.g., cleaves) the sequence. The gene encodes a Cas that can be manipulated with a guide RNA (e.g., a single gRNA) to the law of nature (a) Target cells are killed by the antimicrobial agent, (b) the growth or proliferation of the target cells is reduced; or (c) The target cells are sensitized to the antibiotic, so that the antibiotic is toxic to the cells. be.

[0132] Optionally, the plasmid further encodes said crRNA or gRNA.

[0133] The transduction particles obtainable by the method of the present invention can be used for medical use. For example, to treat or prevent infection of a human or animal subject by a target bacterial cell. The transduction particles are then used to infect target cells and kill the cells using an antimicrobial agent. , and is administered to the subject.

[0134] Optionally, the particles are for administration to humans or animals for medical use.

[0135] Further aspects of the invention are as follows. The present invention is optionally for industrial or domestic use or for such use. For example, this can be used in methods for the production of food, oil and petroleum products, and in the manufacture of food products. Or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry industry, aerospace industry, automotive industry, biotechnology industry, medical industry, Healthcare industry, Dental industry, Energy industry, Consumer products industry, Pharmaceutical industry, Mining industry, Cre Cleaning industry, forestry industry, fisheries industry, leisure industry, recycling industry, cosmetics industry, plastics industry industry, pulp or paper industry, textile industry, clothing industry, leather or suede or for or used in the animal leather, tobacco, or steel industries It is used.

[0136] The invention is optionally for use in industry or the environment is an industrial environment. The industries are medical and healthcare, pharmaceuticals, human food, feed, plant fertilizer, beverages, dairy, food Meat processing, agriculture, livestock farming, poultry farming, seafood, veterinary medicine, oil, gas, petrochemicals, water treatment, sewage Water treatment, packaging, electronics and computers, personal healthcare and toiletries, chemical Cosmetics, Dentistry, Non-medical Dentistry, Ophthalmology, Non-medical Ophthalmology, Mining and Mineral Processing, Metal Mining and Gold Metal processing, quarrying, aviation, automotive, rail, transportation, space, environment, soil treatment, pulp and paper, Clothing manufacturing, dyes, printing, adhesives, air conditioning, solvents, biological defense, vitamin supplementation, low temperature storage, textiles Soaking and production, Biotechnology, Chemicals, Industrial cleaning products, Household cleaning products, Soap and detergents, consumer products, forestry, fisheries, leisure, recycling, plastics and leather. , Leather and suede, Waste disposal, Funeral and contracting, Fuel, Construction, Energy, Steel , and the tobacco industry.

[0137] In one example, each particle genome encodes a respective guide RNA that targets a target bacterium. The array contains a CRISPR array that is loaded into the target bacterial genome. or two or more spacers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 , 20, 30, 40, 50 or more spacers).

[0138] In one example, the target bacteria is contained in the environment as follows: In one example, the environment is a human microorganism Biome, e.g., oral microbiome or gut microbiome, or bloodstream In one example, the environment is not an environment in or on a human. In one example, the environment is a non-human In one embodiment, the environment is not the environment in or on the animal. In one embodiment, the environment is an air environment. In an embodiment, the environment is an agricultural environment. In one embodiment, the environment is an oil or petroleum recovery environment. For example, an oil or petroleum field or well. In one example, the environment is a biological or chemical environment that is contaminated by humans or non-human animals. The environment is in or on food or beverages intended for consumption by a mammal.

[0139] In one example, the environment is the human or animal microbiome (e.g., the gut, vagina, scalp, axilla, In one example, the target bacteria is a human or animal microbiome (e.g. gut, vaginal, scalp, axillary, skin, or oral microbiome) It is included in the group.

[0140] In one example, the particles, populations, or compositions of the invention can be administered intranasally, topically, or orally to humans. or is intended for administration to a human or non-human animal. Those skilled in the art who wish to treat the microbiome of an animal should be aware of the following: For example, if the microbiome is in the gut microbiome, the best route of administration can be determined. In the case of the chromobiome, administration may be intranasal or oral. Where the microbiome is the scalp or axillary microbiome, administration may be local. Where the microbiome is the mouth or pharynx, administration may be oral.

[0141] In any of the uses or methods herein, in one embodiment, the particles of the invention is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 9 5, 100, 110, 120, 130, 140, 150, 160, 170, 180, 19 at a multiplicity of infection (MOI) of 0, 200, 300, 400, 500, 600, or 700. The target bacteria can be contacted with the medium of infection (MOI) of 20-200, 20-100, 50-20, etc. 0, 50-100, 75-150, 100 or about 100, or 200 or about 2 In one example, this is a microbiome sample containing the target bacteria (e.g., Obtain samples of the elephant's waterway or gut microbiome and measure CFU / ml or m Determine the g number and use this to determine the environment to which the microbiome should be exposed or treated. or by titrating the dose of phage at a desired MOI to be administered to a subject. It can be determined.

[0142] In one example, the environment is drinking water or water (e.g., waterways or drinking water for human consumption) or is contained in soil. Water is optionally in heating, cooling, or industrial systems. , or in a drinking water storage container.

[0143] In one example, the producer and / or target bacteria is Anaerotruncus, A. cetanaerobacterium, Acetitomaculum, Acetiv ibrio, Anaerococcus, Anaerofilum, Anaerosin us, Anaerostipes, Anaerovorax, Butyrivibrio , Clostridium, Capracoccus, Dehalobacter, Di alister, Dorea, Enterococcus, Ethanoligenen s, Faecalibacterium, Fusobacterium, Gracili bacter, Guggenheimella, Hesperia, Lachnoba cterium, Lachnospira, Lactobacillus, Leucon ostoc, Megamonas, Moryella, Mitsuokella, Ori bacterium, Oxobacter, Papillibacter, Propri onispira, Pseudobutyrivibrio, Pseudoramiba cter, Roseburia, Ruminococcus, Sarcina, Sein onella, Shuttleworthia, Sporobacter, Sporob acterium, Streptococcus, Subdoligranulum, S yntrophococcus, Thermobacillus, Turibacter and Weisella.

[0144] In one example, the particle, population, composition, use, or method is for reducing pathogenic infection or In humans or animals, for example, to rebalance the microbial population of the gut or oral cavity. For example, the particles, populations, compositions, The use or method comprises the steps of: detecting Clostridium d in the human or animal gut microbiome; ificile or to knock down E. coli bacteria.

[0145] In one example, the disease or condition is cancer, an inflammatory or autoimmune disease or condition, e.g. Obesity, diabetes, IBD (e.g. target cells are E. coli or Klebsiella cells ), GI tract conditions or oral conditions.

[0146] Optionally, the environment or the target bacteria is a gut microbiome, a skin microbiome, an oral microbiome, Throat microbiome, hair microbiome, axillary microbiome, vaginal microbiome, rectal microbiome, anal microbiome , eye microorganisms, nasal microorganisms, tongue microorganisms, lung microorganisms, liver microorganisms, renal microorganisms, reproductive organs Microbiome, Penile Microbiome, Scrotal Microbiome, Mammary Microbiome, Ear Microbiome, Urethral Microbiome, Optionally, the environment is comprised in the lip microbiome, organ microbiome, or dental microbiome; The target bacteria may be present on the surface of plants (e.g., tobacco, crops, fruit trees, vegetables, or tobacco), e.g. on or in plants) or in the environment (e.g. soil or water or watercourses or aqueous liquids body).

[0147] Optionally, the disease or condition of the human or animal subject is: (a) a neurodegenerative disease or condition, (b) a brain disease or condition; (c) a disease or condition of the CNS; (d) memory loss or damage; (e) a cardiac or cardiovascular disease or condition, such as a heart attack, stroke, or atrial fibrillation; (f) liver disease or condition; (g) kidney disease or condition, e.g., chronic kidney disease (CKD); (h) a disease or condition of the pancreas; (i) a pulmonary disease or condition, such as cystic fibrosis or COPD; (j) gastrointestinal disease or condition; (k) any disease or condition of the throat or mouth; (l) eye disease or condition; (m) a disease or condition of the reproductive organs, such as a disease or condition of the vagina, labia, penis, or scrotum; (n) Sexually transmitted diseases or conditions, such as gonorrhea, HIV infection, syphilis, or chlamydia Dye, (o) ear disease or condition; (p) skin diseases or conditions; (q) a heart disease or condition; (r) nasal diseases or conditions; (s) a blood disease or condition, such as anemia, e.g. anemia of chronic disease or cancer; (t) viral infection, (u) Pathogenic bacterial infection; (v) cancer, (w) an autoimmune disease or condition, e.g., SLE; (x) Inflammatory diseases or conditions, such as rheumatoid arthritis, psoriasis, dermatitis, asthma, ulcerative colitis, Colitis, colitis, Crohn's disease, or IBD, (y) autism; (z) ADHD, (aa) bipolar disorder, (bb) ALS (amyotrophic lateral sclerosis), (cc) osteoarthritis, (dd) congenital or developmental defects or conditions; (ee) miscarriage, (ff) blood coagulation status; (gg) bronchitis, (hh) dry or wet AMD, (ii) neovascularization (e.g., in tumors or the eye); (jj) the common cold, (kk) epilepsy, (ll) fibrosis, e.g. liver or lung fibrosis, (mm) fungal diseases or conditions, such as thrush; (nn) a metabolic disease or condition, e.g., obesity, anorexia, diabetes, type I or type II diabetes, (oo) ulcers, e.g. gastric or skin ulcers, (pp) dry skin, (qq) Sjögren's syndrome, (rr) Cytokine storm, (ss) deafness, loss of or damage to hearing; (tt) Slow or accelerated metabolism (i.e., slower than average for the subject's weight, sex, and age) (extension or enhancement) (uu) Fertility disorders, e.g. infertility or subfertility; (vv) Jaundice; (lol) Skin rash, (xx) Kawasaki disease, (yy) Lyme disease, (zz) allergies, e.g. to nuts, grass, pollen, dust mites, cat or dog fur or is allergic to dander, (aaa) malaria, typhoid fever, tuberculosis, or cholera; (bbb) Depression, (ccc) Mental retardation, (ddd)microcephaly, (eee) Malnutrition, (fff) conjunctivitis, (ggg) pneumonia, (hhh) Pulmonary embolism, (iii) pulmonary hypertension, (jjj) Bone disorders, (kkk) Sepsis or septic shock, (lll) sinusitis, (mmm) stress (e.g. occupational stress), (nnn) thalassemia, anemia, von Willebrand disease, or hemophilia, (ooo) Shingles or herpes, (ppp) Menstruation, (qqq) Decrease in sperm count is selected from.

[0148] Neurodegenerative or CNS Diseases or Conditions for Treatment or Prevention According to the Invention In one example, the neurodegenerative or CNS disease or condition is Alzheimer's disease, geriatric psychiatric Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinson's syndrome, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetes From the group consisting of diabetic neuropathy, and Creutzfeldt-Creutzfeldt-Jakob disease For example, the disease is Alzheimer's disease. For example, the disease is Parkinson's syndrome. It is a group.

[0149] The methods of the invention may be used in humans or animals to treat CNS or neurodegenerative diseases or conditions. In examples where the method is performed on a human subject, the method induces downregulation of Treg cells in the subject. This induces systemic monocyte-derived macrophages and / or Treg cells in the choroid plexus. and facilitating entry across the endothelial cell wall into the subject's brain, thereby preventing or treating a disease or condition (e.g., amyloidosis). In one embodiment, the method treats, prevents, or reduces the progression of chronic myelopathy (such as rheumatoid arthritis). The method includes inducing IFN- In one example, the method restores nerve fibers and / or enhances nerve cord circulation. In one example, the method restores nerve myelin and / or In one embodiment, the method of the present invention reduces the progression of nerve myelin damage. 41, and / or the method is directed to treating or preventing a disease or condition disclosed in WO20 15136541 (the disclosures herein may be used in conjunction with any of the methods disclosed therein). provides, for example, treatment and / or prevention of CNS and neurodegenerative diseases and conditions. Such methods, diseases, conditions, and potential treatments that can be administered to a subject for therapeutic agents, such as immune checkpoint inhibitors, such as anti-PD-1, anti-PD-L1, anti-TIM 3, or other antibodies and other agents disclosed herein. (which is incorporated herein in its entirety).

[0150] Cancer for treatment or prevention by the method Cancers that may be treated include non-vascularized or substantially non-vascularized tumors, such as Cancer includes non-solid tumors (hematological tumors, e.g., leukemia and lymphomas) and vascularized tumors. Cancer types to be treated according to the present invention may include idiopathic, myeloma, and the like, or may include solid tumors. These include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and and malignant tumors and diseases, such as sarcomas, carcinomas, and melanomas. These include, but are not limited to, adult tumors / cancers and pediatric tumors / cancers.

[0151] Hematologic cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include acute Myeloid leukemia (acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic leukemia) myeloid, promyelocytic, myelomonocytic, monocytic, and erythroleukemias), chronic leukemias (chronic myeloid (granulocyte Leukemias, including myeloid leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia; Polycythemia vera, Lymphoma, Hodgkin's disease, Non-Hodgkin's lymphoma (inactive and high grade morphology), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, bone marrow Dysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0152] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. They can be benign or malignant. The names of the various types of solid tumors refer to the type of cells that form them. Examples of solid tumors, such as sarcomas and carcinomas, include , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, Mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer , breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, Sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytic lipocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma , bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor , seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (brain stem gliomas and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma , CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyogioma, Ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma angioma, neuroblastoma, retinoblastoma, and brain metastases).

[0153] Autoimmune Diseases for Treatment or Prevention by the Method 1. Acute disseminated encephalomyelitis (ADEM) 2. Acute necrotizing hemorrhagic leukoencephalitis 3. Addison's disease 4. Agammaglobulinemia 5. Alopecia areata 6. Amyloidosis 7. Ankylosing spinal cord disease 8. Anti-GBM / anti-TBM nephritis 9. Antiphospholipid syndrome (APS) 10. Autoimmune angioedema 11. Autoimmune aplastic anemia 12. Autoimmune autonomic neuropathy 13. Autoimmune hepatitis 14. Autoimmune hyperlipidemia 15. Autoimmune immunodeficiency 16.Autoimmune inner ear disease (AIED) 17. Autoimmune myocarditis 18. Autoimmune oophoritis 19. Autoimmune pancreatitis 20. Autoimmune retinitis 21. Autoimmune thrombocytopenic purpura (ATP) 22. Autoimmune thyroid disease 23. Autoimmune urticaria 24. Axonal and neuronal neuropathy 25. Barrow's disease 26. Behcet's disease 27. Bullous pemphigoid 28. Cardiac myopathy 29. Castleman's disease 30. Celiac disease 31. Chagas Disease 32. Chronic Fatigue Syndrome 33. Chronic inflammatory demyelinating polyneuropathy (CIDP) 34. Chronic recurrent multifocal ostomyelitis (CRMO) 35. Churg-Strauss syndrome 36. Cicatricial pemphigoid / benign mucous membrane pemphigoid 37. Crohn's disease 38. Kogans Syndrome 39.Cold agglutinin disease 40. Congenital heart block 41. Coxsackie myocarditis 42.CREST disease 43. Essential mixed cryoglobulinemia 44. Demyelinating neuropathy 45. Dermatitis herpetiformis 46. ​​Dermatomyositis 47. Devic's disease (neuromyelitis optica) 48. Discoid Loops 49. Dressler syndrome 50. Endometriosis 51. Eosinophilic esophagitis 52. Eosinophilic fasciitis 53. Erythema nodosum 54. Experimental allergic encephalomyelitis 55. Evans syndrome 56.Fibromyalgia 57. Fibrinating alveolitis 58.Giant cell arteritis (temporal arteritis) 59. Giant cell myocarditis 60. Glomerulonephritis 61. Goodpasture syndrome 62. Polyangiitis endoblastomatosis (GPA) (previously called Wegener's endoblastomatosis) Ta) 63. Graves' disease 64. Guillain-Barre syndrome 65. Hashimoto's encephalitis 66. Hashimoto's thyroiditis 67.Hemolytic anemia 68. Henoch-Schonlein purpura 69. Herpes gestationis 70. Hypogammaglobulinemia 71. Idiopathic thrombocytopenic purpura (ITP) 72. IgA nephropathy 73. IgG4-related sclerosing disease 74. Immunomodulatory Lipoproteins 75. Inclusion body myositis 76.Interstitial cystitis 77. Juvenile arthritis 78. Juvenile diabetes (type 1 diabetes) 79. Juvenile myositis 80. Kawasaki syndrome 81. Lambert-Eaton Syndrome 82.Leukocytoclastic vasculitis 83. Lichen planus 84. Lichen sclerosus 85.Ligny conjunctivitis 86. Linear IgA disease (LAD) 87. Lupus (SLE) 88. Lyme disease, chronic 89. Meniere's disease 90. Microscopic polyangiitis 91.Mixed connective tissue disease (MCTD) 92. Mooren's ulcer 93. Mucca-Habermann Disease 94. Multiple sclerosis 95.Myasthenia gravis 96. Myositis 97. Narcolepsy 98. Neuromyelitis Optica (Debicchi's) 99. Neutropenia 100. Ocular cicatricial pemphigoid 101.Optic neuritis 102. Relapsing rheumatism 103. PANDAS (Pediatric autoimmune neuropsychiatric disorders associated with streptococcus) 104. Paraneoplastic cerebellar degeneration 105. Paroxysmal nocturnal hemoglobinuria (PNH) 106. Parry-Romberg syndrome 107. Parsonage-Turner syndrome 108. Pars planitis (peripheral uveitis) 109. Pemphigus 110. Peripheral neuropathy 111. Perivenous encephalomyelitis 112. Pernicious anemia 113. POEMS syndrome 114. Polyarteritis nodosa 115. Autoimmune polyglandular syndromes types I, II, and III 116. Rheumatic polymyalgia 117. Polymyositis 118. Post-myocardial infarction syndrome 119. Postpericardiotomy syndrome 120. Progesterone dermatitis 121. Primary biliary cirrhosis 122. Primary sclerosing cholangitis 123. Psoriasis 124. Psoriatic arthritis 125. Idiopathic pulmonary fibrosis 126. Pyoderma gangrenosum 127. Erythroblastic aplasia 128. Raynaud's phenomenon 129. Reactive arthritis 130. Reflex sympathetic dystrophy 131. Reiter's syndrome 132. Relapsing polychondritis 133.Restless legs syndrome 134. Retroperitoneal fibrosis 135. Rheumatic fever 136. Rheumatoid arthritis 137. Sarcoidosis 138. Schmidt syndrome 139.Scleritis 140. Scleroderma 141. Sjögren's syndrome 142. Sperm and testicular autoimmunity 143. Stiff person syndrome 144. Subacute bacterial endocarditis (SBE) 145. Susac syndrome 146.Sympathetic ophthalmia 147.Takayasu arteritis 148. Temporal arteritis / giant cell arteritis 149. Thrombocytopenic purpura (TTP) 150. Tolosa-Hunt Syndrome 151. Transverse myelitis 152.1 diabetes 153.Ulcerative colitis 154.Undifferentiated Connective Tissue Disease (UCTD) 155. Uveitis 156. Vasculitis 157. Vesicular bullous skin disease 158. Vitiligo 159. Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)) )

[0154] Inflammatory Diseases for Treatment or Prevention by the Method 1. Alzheimer's Disease 2. Ankylosing spondylitis 3. Arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) 4. Asthma 5. Atherosclerosis 6. Crohn's disease 7. Colitis 8. Dermatitis 9.Diverticulitis 10. Fibromyalgia 11.Hepatitis 12.Irritable Bowel Syndrome (IBS) 13. Systemic lupus erythematosus (SLE) 14. Nephritis 15. Parkinson's disease 16. Ulcerative colitis

[0155] In one example, any composition of the present invention may be used, for example, when the composition is in a fluid (e.g., liquid) or solid form. When present, there should be at least 1 × 10 per ml or mg. 3 The inventive traits of In one example, any of the compositions of the present invention may contain at least 1×10 4 In one example, any composition of the invention comprises 1 At least 1 × 10 per ml or mg 5 In one embodiment, the transduction particle of the present invention comprises Any composition of the present invention may contain at least 1×10 6 The present invention In one example, any of the compositions of the invention may contain less than 1000 mg of transducing particles per ml or mg. At least 1×10 7 In one example, any composition of the invention comprises a transduction particle of , at least 1 × 10 per ml or mg 8 The transduction particles of the invention include Any composition of the present invention may contain at least 1×10 9 The original publication In one example, any of the compositions of the present invention contains At least 1×10 10 In one embodiment, the transduction particles of the present invention include any of the combinations of the present invention. The composition should be at least 1 × 10 per ml or mg. 11 The transduction particles of the present invention In one example, any composition of the invention contains at least 1×10 12 In one example, any composition of the invention contains 1 ml or At least 1 × 10 per mg 13 In one embodiment, the transduction particle of the present invention comprises Any composition of the present invention may contain at least 1×10 14 The present invention Including inclusion particles.

[0156] In one example, any composition of the present invention may be used, for example, when the composition is in a fluid (e.g., liquid) or solid form. When present, 1 × 10 per ml or mg 14 Transduction of the present invention up to In one example, any composition of the invention contains 1×10 particles per ml or mg. 1 3 In one example, any composition of the invention may contain up to 1 ml of transducing particles. is 1×10 per mg 12 In one embodiment, the transduction particles of the present invention include any of The composition of interest is 1 x 10 per ml or mg. 11 The transduction particles of the present invention include In one example, any composition of the invention may contain 1×10 10 Up to In one example, any composition of the invention may be administered in a volume of 1 ml or 1 mg. 1 x 10 per 9 The transducing particles of the present invention include up to

[0157] In one example, any composition of the present invention may be used, for example, when the composition is in a fluid (e.g., liquid) or solid form. When present, there should be at least 1 × 10 per ml or mg. 3 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 Transduction of the present invention In one example, any composition of the present invention contains at least 1×10 4 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1×1 0 14 In one example, any composition of the invention contains 1 ml or is at least 1 × 10 per mg 5 ~1×10 10 , 1×10 11 , 1×10 12 , 1 ×10 13 , or 1 × 10 14 In one embodiment, the transduction particle of the present invention comprises any of the transduction particles of the present invention. The composition of interest has a concentration of at least 1 x 10 per ml or mg. 6 ~1×10 10 , 1×1 0 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 The transduction particles of the present invention In one example, any composition of the invention contains at least 1×10 7 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 of In one example, any composition of the invention may be administered in a volume of 1 ml or 1 mg. At least 1×10 per 8 ~1×1010 , 1×10 11 , 1×10 12 , 1×10 1 3 , or 1 × 10 14 In one example, any of the compositions of the present invention may be used. The substance should have a concentration of at least 1 × 10 per ml or mg. 9 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 The transduction particles of the invention include Any composition of the present invention may contain at least 1×10 10 ~1 ×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 The present invention In one example, any composition of the invention contains At least 1×10 11 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1×10 14 In one example, any composition of the invention comprises a transduction particle of , at least 1 × 10 per ml or mg 12 ~1×10 10 , 1×10 11 , 1 ×10 12 , 1×10 13 , or 1 × 10 14 In one embodiment, the transduction particle of the present invention comprises Any composition of the present invention may contain at least 1 x 10 13 ~1× 10 10 , 1×10 11 , 1×1012 , 1×10 13 , or 1 × 10 14 The present invention In one example, any of the compositions of the invention may contain less than 1000 mg of transducing particles per ml or mg. At least 1×10 14 ~1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 ,Ma or 1×10 14 The transduction particle of the present invention comprises

[0158] In one example, the composition is for administration to a subject for medical use, e.g., to treat a disease in a subject. Use of one or more of the transducing particles of the invention to treat or prevent a disease or condition. In one example, the composition comprises a single dose. In one example, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 doses (or In one example, each dose contains (or at least contains) the phage. At least) 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 1 The doses are 0, 125, 200, or 250 mg or ml (i.e., the dose is and including the phage and, for example, an excipient, diluent, or carrier.

[0159] In one example, the composition is for administration to a subject for non-medical use, e.g., for agricultural use. In one example, the composition comprises a single dose of the transducing particles of the invention. In one example, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30 doses (or at least). Each dose may contain (or at least) 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200, 250, 500 , 750, 1000, 2000, 3000, 4000, 5000, 10000, 5000 0, 100,000 mg or ml (i.e., the dose is the amount described above, The dose is determined by the amount of the agent that is contacted with the target bacteria. The composition may be dissolved or diluted in a solvent (e.g., an aqueous solvent or water) prior to use for the preparation of the composition. One imperial gallon is used for agricultural purposes, such as spraying crops, or for drinking purposes. The present invention also includes a dose of a transducing particle of the invention for use in animals or livestock, such as for use in veterinary medicine.

[0160] Optionally, the NSI is contained in a high copy number plasmid. Optionally, the NSI is contained in a medium copy number plasmid. Low, medium, and high copy number ori and plasmids The meanings are known to those skilled in the art and are terms of the art. The number refers to the average number of plasmid copies per cell. For example, Midi is a plasmid that exists in 1-10 copies per bacterial cell that contains the plasmid. Medium copy number plasmids are 11-50 (e.g., 11-40 or 2) per cell. 0–30 or 40) copies per cell, whereas high copy numbers are >50 (e.g., 100, 200, 250, 300, 400, 500, 600, or 700) copies. In one example, the plasmid or vector comprising the first DNA is a medium copy number plasmid or In one example, the plasmid or vector containing the first DNA is a high copy Examples of common ori and plasmids are shown in Table 8. vinegar. EXAMPLES

[0161] [Example 1] Enhancement of transducing particle production by inhibiting phage reuptake. Background technology This work concerns the production of transducing particles containing the DNA sequence of interest. The particles then infect target bacteria, introduce DNA, and express this DNA in the target bacteria. These particles can be advantageously used to enhance their DNA. A was then injected into the same set of bacterial strains to produce the original phagocytosis that this particle was designed on. Binding of phage particles to host cells requires the presence of a single Or multiple specific molecules must be present to provide phage receptors. The inventors have surprisingly observed a very large increase in particle production yield. While not wishing to be bound by any theory, it is believed that phage reception from the surface of producer cells The body's elimination can prevent the reabsorption of the produced particles, thus greatly increasing the production yield. Make it big.

[0162] Methods and Results The present inventors have demonstrated that the well-studied P2 phage / Escherichia coli The KE i system was used as a model. To identify P2 receptor mutants, we IO Collection ("Construction of Escherichia coli" li K-12 in-frame, single-gene knockout m utants: the Keio collection”, Tomoya Bab a et al, DOI 10.1038 / msb4100050, Molecule ar Systems Biology (2006) 2, 2006.0008) A set of single knockout mutants was analyzed in a standard soft agar overlay spot assay. The formation of P2 plaques was tested. LB agar was prepared in a petri dish and 100 ul Cover with 3 ml of soft agar overlay (LB + 0.6% agar) containing overnight cell culture. The P2 vir phage lysate was spotted onto the plate and incubated overnight at 37°C. After incubation, the plates were checked for plaque formation. We found that a set of rfa mutants involved in S core biogenesis did not form plaques ( Figure 1).

[0163] To construct receptor deletion mutants for further study, we used Lambd Using the a Red system, the rfaD gene was amplified in the E. coli C1a P2 lysogen. was replaced with a Zeocin resistance marker ("One-step inactivation of chromosomal genes in Escherichia col i K-12 using PCR products”, Kirill A. Da tsenko and Barry L. Wanner, PNAS June 6, 2000 97 (12) 6640-6645; https: / / doi.org / 10.1073 / pnas.120163297). Plasmid pEM7 / zeo(I The Zeocin marker from Nvitrogen was amplified using primer rfaDupR (SEQ ID NO: 1). and rfaDdnR (SEQ ID NO: 2) were used for PCR amplification. P2 lysogen cells were transformed with plasmid pKD46 (Gen The transformants were transformed with 100ug / ml of Grown to mid-log phase at 30°C in LB containing ampicillin and 0.4% Arabidopsis. The cells were induced with 20% glycerol for 2 h. The recombinants were electroporated with the PCR fragment. Selection was performed at 7°C to eliminate the pKD46 plasmid, which also has temperature-sensitive replication. Correct replacement of the rfaD gene with the eo marker was sequence verified.

[0164] of the parental strain (C1a P2 lysogen) and the receptor mutant (C1a P2 lysogen ΔrfaD). Both were expressed as (i) the arabinose-inducible P4 phage transactivation region (P2 helper function (ii) a P4 packaging site; and (iii) spectinomycin resistance. and (iv) a plasmid containing the CloDF13 origin of replication. Ta.

[0165] To compare the yield of transducing particles obtained with the two strains, overnight cell cultures were incubated for 50 μl. It contained 100 µg / ml spectinomycin, 10 mM MgSO4, and 5 mM CaCl2. The cells were diluted 1:25 in LB medium containing 0.8% aramid. After shaking at 37°C for 90 min, the cells were incubated in 0.8% aramid. Binose was added to the culture. Due to induction of chromosomal P2 phage, the cells Cell debris was removed by centrifugation, and the lysate was extracted with chloroform. Any remaining cells were removed.

[0166] Production yield was quantified by measuring transduction of the spectinomycin marker. LB containing 10 mM MgSO4 and 5 mM CaCl2 in a volume of 100 μl The lysate was serially diluted (10-fold steps) in medium and the dilutions were added to 100 μl of E. After 30 min at 37°C, 10 μl of each sample was added to the oliC1a overnight cell culture. The colonies were then spotted onto LB spectinomycin plates and incubated overnight. The results are shown in Figure 2.

[0167] result: Removal of the phage receptor surprisingly increased the yield of transducing particles by more than 100-fold. Thus, the present invention significantly reduces the production costs of transducing particles or phages. It is possible.

[0168] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0169] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

[0170]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

[0171]

Table 4-1

Table 4-2

[0172]

Table 5-1

Table 5-2

[0173]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

[0174]

Table 7

[0175]

Table 8

Claims

1. A method for producing transduction particles, the particles being capable of inducing a bacterial target cell for the transduction of cells. and wherein the particle is capable of recognizing a receptor on a bacterial producer cell, the method comprising: wherein the producer cell does not express the receptor on its surface. method.

2. 10. The method of claim 1, wherein the producer cell and the target cell are cells of the same species. 。

3. The method of any preceding claim, wherein the producer cell is an E. coli cell. Law.

4. The transduction particle comprises a phage capsid, the capsid being capable of transducing a target bacterial cell. The method according to any preceding claim, further comprising the step of: The method described.

5. The NSI contains or encodes an antimicrobial agent that kills target cells, or The method of claim 4, wherein the SI comprises or encodes a component of such a drug.

6. The NSI is a guide RNA (optionally a single guide RNA) for a CRISPR / Cas system. The method of claim 5, comprising a nucleotide sequence encoding

7. 10. The method of any preceding claim, wherein the transduction particle is a phage.

8. 10. The method of any preceding claim, wherein the transduction particles are non-self-replicating.

9. The genome of each producer cell is responsible for the synthesis and / or cell surface The method of any preceding claim, comprising a genetic modification that prevents its expression as a receptor. Law.

10. The method of claim 9 , wherein the modification is a modification of the lipopolysaccharide (LPS) synthesis pathway.

11. 10. The method of any preceding claim, wherein the receptor comprises LPS.

12. For improving the production yield of transducing particles, Use of producer cells.

13. The transduction particle is as defined in any one of claims 1 to 11. The use according to claim 12.

14. At least one of the above-mentioned methods is to produce a low yield compared to production in a producer cell that surface expresses the receptor.

14. The use according to claim 12 or 13, for increasing the concentration of .alpha.-aminobutyric acid in a subject by 10-fold.

15. 15. The use according to claim 14, wherein the increase is at least 100-fold.

16. The use according to claim 14, wherein the increase is between 10 and 1000 fold.

17. The method according to any preceding claim, further comprising isolating the transduction particles from cellular material. Method or use.

18. Transduction obtained or obtainable by the method or use according to claim 17 A composition (optionally a pharmaceutical composition) comprising the particles.

19. The composition of claim 18 , which does not contain LPS from a producer cell.

20. A method of killing bacterial target cells, comprising: subjecting said cells to the composition of claim 18 or 19. and contacting the cells with a composition, wherein the transducing particles infect the cells and introduce the NSI therein. and the NSI comprises or encodes an antimicrobial agent that kills the target cells; or The method wherein said NSI comprises or encodes a component of such a drug.

21. For use in a method of treating or preventing a disease or condition in a human or animal subject.

20. The composition of claim 18 or 19 for use in treating a disease or condition comprising a bacterial target cell. and the method comprises administering to the subject the composition and administering to the subject a therapeutically effective amount of the composition to the subject.

20. The method of claim 18, further comprising contacting a target cell with the composition of claim 18 or 19, thereby The cells are killed or the growth or proliferation of the target cells is inhibited, thereby preventing the disease. A composition for treating or preventing a disease or condition.

22. The transduction particles contained in the composition infect the target cells and introduce the NSI therein. the NSI contains or encodes an antimicrobial agent that kills the target cells, 22. The composition of claim 21, wherein the NSI comprises or encodes a component of such a drug. 。

23. The target cells are Escherichia, Klebsiella, Clostridia um, or a Pseudomonas cell. 。

24. The target cell is E. coli, K. pneumoniae, C. difficile, or or P. aeruginosa cells.

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