JP2022542742A - Phage and transduction particles - Google Patents

Abstract

The present invention relates to the production of phage and transduction particles using DNA (e.g., plasmids and helper phages, mobile genetic elements (MGEs), or plasmids with chromosomally integrated helper phage genes), and phages with them, Helper phages, kits, compositions and methods. Particles are particularly useful for delivering toxic payloads to target bacteria for antimicrobial action. Embodiments allow the production of highly pure compositions of such particles for medical or environmental use and for encapsulation of particles, which can be useful for encapsulating antimicrobial activity.

Description

The present invention relates to the production of phages and transduction particles containing phage proteins using DNA (e.g., plasmids and helper phages, or plasmids with chromosomally integrated helper phage genes), as well as phages, helper phages, kits with these. , compositions and methods. Particles are particularly useful for delivering toxic payloads to target bacteria for antimicrobial action. Embodiments enable the production of high-purity compositions of such particles for medical or environmental use and for particle containment, which can contain antimicrobial effects, control dosing, or eliminate unwanted exogenous genes. can be useful for reducing the risk of acquiring

The use of helper phages to package phagemid DNA into phage virus particles is known. For example, E. There is a M13KO7 helper phage, a derivative of M13 used in E. coli host cells. Other examples are R408 and CM13.

The present invention relates to the production of phage and transduction particles and provides the following.

In a first configuration,
a) a first DNA and b) one or more second DNAs
A kit comprising
(i) said DNAs together contain all the phage structural protein genes necessary to produce a packaged phage particle containing a copy of said first DNA;
(ii) said first DNA comprises none, or at least one, but not all, of said genes and said one or more second DNAs comprise the remainder of said genes;
(iii) said first DNA comprises a phage packaging signal for producing said packaged phage particle;
(iv) said second DNA lacks a nucleotide sequence (e.g., packaging signal) necessary for packaging said second DNA into a phage particle;
operable to produce a packaged phage containing said first DNA when said DNA coexists in a host bacterium, said phage replicating said second DNA to produce additional phage particles; a kit that requires said second DNA to do so.
A method of producing a phage is also provided, the method comprising expressing a phage protein gene in a cell containing the DNA, wherein a packaged phage containing the first DNA is produced, the phage producing the second DNA. It requires a second DNA to replicate and produce additional phage particles.

In a second configuration,
A population of helper phages, wherein said helper phage is capable of packaging a first phage, said first phage being different from said helper phage, said helper phage being incapable of self-replicating. a group of

In a third configuration,
A composition comprising a first population of phages, wherein said first phages require helper phages according to said first configuration for replication, and less than 20% of all phages contained in said composition. A composition that is a helper phage.

In a fourth configuration,
A method of producing a first phage, said first phage requiring a helper phage to replicate, said method comprising:
(a) providing a DNA containing a packaging signal;
(b) introducing said DNA into a host bacterial cell;
(c) wherein said host bacterial cell contains a helper phage or helper phage is introduced into said bacterial cell simultaneously or sequentially with step (b);
(d) wherein said helper phage is according to the invention;
(e) causing or enabling said helper phage to produce a phage protein, wherein said packaging signal is recognized in said host cell such that said protein is used by the first phage; wherein said first phage packages said DNA;
(f) wherein replication of helper phage in said host cell is inhibited or reduced, thereby limiting helper phage availability;
said method comprising (g) optionally lysing said host cell to obtain said first phage;
(h) thereby producing a composition comprising a first phage that requires said helper phage for replication, wherein growth of the first phage is prevented or reduced by limiting helper phage availability; done, method.

In a fifth configuration,
A phage production system for producing a phage (NSI-phage) comprising a nucleotide sequence of interest (eg the first phage according to any of the preceding claims), said system comprising components (i) to ( iii), i.e.
(i) a first DNA;
(ii) a second DNA, and (iii) an NSI-phage production factor (NPF) or an expressible nucleotide sequence encoding the NPF,
a) said first DNA encodes a helper phage (e.g. said first helper phage recited in any of the preceding claims);
b) said second DNA comprises said nucleotide sequence of interest (NSI);
c) when said system is contained in a bacterial host cell, helper phage proteins are expressed from said first DNA to form phage which, in the presence of said NPF, package said second DNA, thereby forming an NSI- produce phages,
d) whether the system lacks a helper phage production factor (HPF) necessary to form phage that packages the first DNA, or lacks an expressible nucleotide sequence encoding a functional HPF; or said system comprises a nucleotide sequence comprising or encoding a functional HPF, said system eliminating or minimizing the production of helper phage comprising said first DNA of said HPF sequence in said host cell. further comprising a means for targeted inactivation, whereby said system is capable of producing a product comprising a population of NSI-phage, each NSI-phage requiring said helper phage for proliferation. and wherein said NSI-phage in said product is not mixed with helper phage, or said helper phage constitutes less than 20% of the total phage contained in said product.

The present invention also provides the following.
1. A composition for use in antibacterial treatment of bacteria, said composition comprising an engineered mobile genetic element (MGE) capable of translocating in a first bacterial host cell of a first species or strain. wherein said cell comprises a first phage genome, in said cell said MGE translocates using a protein encoded by said phage to inhibit first replication, said MGE encodes an antimicrobial agent or compositions encoding components of such agents.

A nucleic acid vector comprising said MGE integrated therein, wherein said vector is capable of transferring said MGE or a copy thereof into a host bacterial cell.

A non-self-replicating transduction particle comprising said MGE or vector of the invention.

A composition comprising a plurality of transducing particles, each particle comprising an MGE or a vector according to the invention, said transducing particles carrying said MGE or a nucleic acid encoding said agent or component, or a copy thereof. can be transfected into target bacterial cells,
(i) the target cell is killed by the antimicrobial agent;
(ii) the growth or proliferation of target cells is reduced, or (iii) the target cells are sensitized to an antibiotic whereby said antibiotic is toxic to said cells.

a composition comprising a plurality of non-self-replicating transducing particles, each particle comprising an MGE or a plasmid according to the invention, said transducing particles said MGE or a nucleic acid encoding said agent or component; or A targeting agent whose copy can be introduced into a target bacterial cell, the agent is a CRISPR/Cas system, and the component guides the Cas to a target nucleic acid sequence of the cell to modify the sequence. comprising a nucleic acid encoding a crRNA or guide RNA that is operable with Cas in a bacterial cell so that (i) the target cell is killed by the antimicrobial agent;
(ii) the growth or proliferation of target cells is reduced, or (iii) the target cells are sensitized to an antibiotic whereby said antibiotic is toxic to said cells.

A method of producing a plurality of transducing particles, comprising combining a composition of the invention with a host bacterial cell of said first species, wherein said cell is capable of introducing a plurality of said MGEs into said host cell. said host under conditions wherein a protein encoded by said first phage is expressed and copies of MGE are packaged by said first phage protein to produce a plurality of transducing particles; A method comprising culturing a cell and optionally separating the transduced particles from the cell to obtain a plurality of transduced particles separated from the cell.

A bacterial host cell comprising a first phage and an MGE, vector, or particle of the invention, wherein said agent is toxic to cells of the same species as said host cell, and said agent is toxic to said host cell A bacterial host cell, wherein said host cell has been engineered to be non-toxic.

A bacterial host cell comprising a first phage, said cell being included in a kit, said kit further comprising a composition of the invention, said agent being toxic to cells of the same species as said host cell. , a bacterial host cell, wherein said host cell is engineered such that said agent is not toxic to said host cell.

A bacterial host cell comprising a first phage and an MGE, vector, or particle of the invention, wherein said agent is not toxic to said host cell, but said agent is a species or strain different from said host cell species or strain. toxic to a second cell of the strain and mobile in a transducing particle that said MGE can be produced by said host cell capable of transferring said MGE or a copy thereof into said second cell and wherein said second cell is exposed to said antimicrobial agent.

A bacterial host cell comprising a first phage, said cell being included in a kit, said kit further comprising a composition of the invention, said agent not being toxic to said host cell, but said agent being said produced by said host cell that is toxic to a second cell of a species or strain different from the host cell species or strain, said MGE being capable of transfecting said MGE or a copy thereof into said second cell A bacterial host cell that is mobile within a transducing particle capable of exposing said second cell to said antimicrobial agent.

A bacterial host cell comprising an MGE, vector, or particle of the invention and a nucleic acid under the control of one or more inducible promoters, wherein said nucleic acid packages a copy of said MGE or plasmid to produce transduced particles. and wherein said agent is not toxic to said host cell, but said agent is toxic to a second cell of a different species or strain than said host cell species or strain. and said MGE is mobile in a transduction particle that can be produced by said host cell capable of transferring said MGE or a copy thereof into said second cell, thereby said second cell is exposed to said antimicrobial agent.

(a) a nucleotide sequence encoding an antimicrobial agent or component thereof for expression in a target bacterial cell;
(b) a constitutive promoter for controlling the expression of said agent or component;
(c) optionally a terS nucleotide sequence;
(d) an origin of replication (ori), and (e) a phage packaging sequence (optionally pac, cos, or homologues thereof)
A plasmid comprising
(f) all nucleotide sequences encoding the phage structural proteins necessary for the production of transducing particles (optionally phage), or a plasmid lacking at least one such sequence, and (g) optionally terL
lacking a plasmid.

a genome of a helper phage incapable of self-replication, optionally wherein said genome is present as a prophage, and a bacterial host cell comprising a plasmid according to the invention, wherein said helper phage comprises the plasmid in a transduction particle and said particles are capable of infecting bacterial target cells to which said antimicrobial agent is toxic.

A method of making a plurality of transducing particles, comprising culturing a plurality of host cells according to the invention, optionally inducing a lytic cycle of said helper phage, and packaged copies of said plasmid. A method comprising incubating said cells under conditions in which transduced particles are created, and optionally separating said particles from said cells to obtain a plurality of transduced particles.

A plurality of transducing particles obtainable by the method of the invention for use in medicine, for example to treat or prevent infection of a human or animal subject by target bacterial cells, said transducing particles infecting target cells and antibacterial A transducing particle administered to said subject to kill said cell using an agent.

A method of making a plurality of transducing particles, comprising:
(a) producing a host cell whose genome comprises nucleic acids encoding structural proteins necessary to produce a transducing particle capable of packaging the first DNA, said genome comprising devoid of phage packaging signals and expression of said protein is under the control of an inducible promoter;
(b) producing a first DNA encoding an antimicrobial agent or component thereof, said DNA comprising a phage packaging signal;
(c) introducing said DNA into said host cell;
(d) inducing the production of said structural protein in a host cell, whereby a transducing particle that packages said DNA is produced;
(e) optionally isolating a plurality of transduction particles; and (f) optionally formulating said particles into a pharmaceutical composition for administration to humans or animals for medical use. .

A plurality of transduced particles obtainable by the method.

a) a first DNA and b) one or more second DNAs
A host bacterial cell comprising
(i) said DNAs together contain all the genes necessary to produce a transduction particle comprising a copy of said first DNA packaged by phage structural proteins;
(ii) said first DNA lacks at least one functional essential gene (e.g., encoding a phage structural protein) required to produce said particle, and said one or more second DNAs lack said functional contains a sex-essential gene,
(iii) said first DNA comprises a phage packaging signal for producing said particle;
(iv) said second DNA lacks a nucleotide sequence necessary for packaging said second DNA into a transduction particle;
said second DNA is required to package said first DNA to produce particles, said DNA producing transducing particles comprising phage structural proteins that package copies of said first DNA A host bacterial cell that is capable of being manipulated in said cell to do so.

Genetic map of the P2 genome with non-essential genes boxed. Schematic representation of SaPIbov1. (A) Maps of P2 and P4, showing the genomic structure and regulatory networks of phage P2 and satellite phage P4. The gray boxed region in P2 has been deleted and replaced with the kanamycin marker sequence, and the dashed box indicates the region of P4 containing the cos site that was included in CGV. . (B) Genetic map of p94. Schematic representation of SA100 producing strains. Defective helper phage turn on phage production only after induction. Since the phage packaging DNA sequences are removed from the helpers and loaded onto the CGV™, only the CGV™ molecule is packaged into synthetic phage particles. FIG. 4 shows the efficiency of infection, the percentage of EMG-2 cells infected with increasing ratio of SA100 particles to EMG-2 (MOI). Total CFU after CGV™ delivery by SA100: (A) infection of MG1655_pks cells with SA100 containing p114 or p94, or (B) infection of X1-blue_pks cells with SA100 containing p114, and FIG. 3 shows a CFU containing a CGV™; Target cell killing by SA100-delivered CGV™, i.e., SA100-delivered CGV™ using wild-type and copy number optimized CGV™ compared to uninfected controls FIG. 13 shows the killing efficiency of two target strains (second, fourth, and sixth bars) by . FIG. 10 is a plot of amounts expressed in colony forming units (CFU) per 5 milliliters of stool sample (n=15). Two groups are shown: induced and non-induced CRISPR. No differences were observed between groups when CFU was determined on streptomycin-containing agar plates. FIG. 10 is a plot of amounts expressed in colony forming units (CFU) per 5 milliliters of stool sample (n=15). Two groups are shown: induced and non-induced CRISPR. Statistical differences were calculated using Student's t-test and statistically significant increases in amounts after 48 hours in the CRISPR non-induced group on agar plates supplemented with spectinomycin and streptomycin are shown. (p-value = 0.011). This indicates that CGV (containing the spectinomycin marker) can be delivered by non-self-replicating particles SA100. 1. The CRISPR system is inserted into the phage genome containing at least the packaging signal and the DNA replication module, thereby generating a vector such as a plasmid (which we call CRISPR Guided Vector™, CGV). A CRISPR system may comprise a nucleic acid encoding a Cas (eg, Cas3 or Cas9) and/or one or more crRNA or gRNA, optionally also a cascade protein when the Cas is Cas3. 2. An essential gene is removed or mutated in the phage and function is conferred by expression in trans from a plasmid (or function is instead transferred to the essential gene integrated into the chromosome of the host bacterial cell (aka producer strain)). derived from). 3. The CGV replicates and packages into phage-like transduction particles encoded by functions encoded on the CGV, but at least one essential function is expressed in trans from a plasmid or chromosome. 4. Production via growth cycles in production strains. Growth in other bacterial strains is advantageously prevented.

The present invention relates to the production of phages using DNA (eg, plasmids with helper phages), as well as phages, helper phages, compositions, and methods involving them. The present invention finds utility, for example, for containing phages in ex vivo and in vivo environments, reducing the risk of acquisition of antibiotic resistance or other genes by phages, and controlling phage dosing in the environment. Contamination of useful phage populations by helper phages is limited or eliminated in an example, thereby controlling phage growth and phage compositions such as pharmaceuticals, herbicides, and other agents that phages can be used to benefit. The percentage of desired phage in the can be enhanced. That is, the present invention provides the following embodiments.

a) a first DNA and b) one or more second DNAs
A kit comprising
(i) said DNAs together comprise all phage structural protein genes necessary to produce a packaged phage particle or transduced particle containing a copy of said first DNA;
(ii) said first DNA comprises none, or at least one, but not all, of said genes and said one or more second DNAs comprise the remainder of said genes;
(iii) said first DNA comprises a phage packaging signal for producing said packaged phage particle; and (iv) said second DNA packages said second DNA into a phage particle. lacks the nucleotide sequence required for
operable to produce a packaged phage containing said first DNA when said DNA coexists in a host bacterium, said phage replicating said second DNA to produce additional phage particles; a kit that requires said second DNA to do so.

For example, the second DNA lacks a packaging signal for packaging the second DNA. Additionally or alternatively, the second DNA lacks nucleotide sequences necessary for helper phage replication. Optionally, the nucleotide sequence encodes a sigma factor, or a sigma factor recognition site, a DNA polymerization recognition site, or a gene required for replication of helper phage DNA if the second DNA is contained in the helper prophage. Contains promoters.

In one example, the second DNA is contained in an M13 or M13-based helper phage. M13 encodes the following proteins required for phage packaging.
a) pIII: host recognition,
b) pV: coat protein;
c) pVII, pVIII, pIX: membrane proteins,
d) pI, pIV, pXI: Channels for translocation of phage into the extracellular space.

In this example, the second DNA lacks one or more of the genes encoding these proteins, e.g., the gene encoding pill, the gene encoding pV, the gene encoding pVII, the gene encoding pVIII. the gene encoding pIX, the gene encoding pI, the gene encoding pIV, and/or the gene encoding XI.

In one embodiment, the phage particle of (i) is capable of infecting a target bacterium, the phage comprising a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in the target bacterium, or the NSI is It contains regulatory elements that are operable in the target bacterium. In one example, the NSI can recombine with the target cell's chromosome or episome contained within the target cell to modify the chromosome or episome. Optionally, this means that the chromosome or episome is cleaved (e.g. at predetermined sites using guided nucleases such as Cas, TALENs, zinc fingers, or meganucleases, or restriction endonucleases) and simultaneously or sequentially A method in which a cell is infected with a phage particle containing a first DNA, the DNA is introduced into the cell and the NSI or its sequence is introduced into the chromosome or episome at or near the cleavage site. In one example, the first DNA comprises one or more CRISPR/Cas system components operable to effect cleavage (e.g., nucleotides encoding guide RNA or crRNA targeting the site to be cleaved). sequence), further including NSI.

In one embodiment, the presence of the NSI or its encoded protein or RNA in the target bacterium mediates target cell death or downregulation of target cell growth or proliferation, or is encoded by the target cell's genome. It mediates the switch-off or down-regulation of expression of one or more RNAs or proteins.

In one embodiment, the presence of the NSI or the protein or RNA encoded thereby in the target bacterium mediates upregulation of growth or proliferation of the target cell, or one or more of the proteins encoded by the genome of the target cell. mediates the switch-on of, or upregulation of, RNA or protein expression.

In one embodiment, the NSI encodes a component of the CRISPR/Cas system that is toxic to target bacteria.

In one embodiment, the DNA is the first DNA defined in any paragraph above.

In one embodiment, the first DNA is contained in a vector (eg, plasmid or shuttle vector).

In one embodiment, the second DNA is contained in a vector (eg, plasmid or shuttle vector), helper phage (eg, helper phagemid), or integrated into the genome of the host bacterial cell.

One embodiment provides a bacterial cell comprising first and second DNA. Optionally, the cell lacks a functional CRISPR/Cas system prior to transferring therein the first DNA, e.g., a first DNA comprising a component of the CRISPR/Cas system that is toxic to the target bacterium. there is One embodiment provides an antimicrobial composition comprising a plurality of cells, each cell optionally according to this paragraph for administration to a human or animal subject for medical use.

A method is provided for producing a phage, the method comprising expressing a phage protein gene in a host bacterial cell to produce a packaged phage comprising the first DNA, the phage replicating it. requires a second DNA to produce additional phage particles. Optionally, the method includes isolating the phage particles.

A composition comprising a population of phage particles obtainable by the present method is provided for administration to a human or animal subject to treat infection of target bacterial cells, wherein the phage infect and infect target cells. can be annihilated.

A method of treating an environment ex vivo is provided comprising exposing the environment to a population of phage particles obtainable by the method, the environment comprising target bacteria, the phage infecting the target bacteria. to kill it. In one example, the subject is further administered an agent concurrently or sequentially with administration of the phage. In one example, the agent is a herbicide, insecticide, insecticide, plant fertilizer, or cleaning agent.

Optionally, the method is to contain the treatment in the environment.

Optionally, the method is for controlling dosing of phage treatment in the environment.

Optionally, the method is to reduce the risk of acquisition of foreign gene sequences by phages in the environment.

A method of treating an infection of a target bacterium in a human or animal subject, the method comprising exposing the bacterium to a population of phage particles obtainable by the method of production, wherein the phage infect the target bacterium and thereby is provided.

Optionally, the method for treatment is to contain the treatment in a subject.

Optionally, the method for treatment is to contain the treatment within the environment in which the subject exists.

Optionally, the method for treatment is to control dosing of phage treatment in a subject.

Optionally, the method for treatment is to reduce the risk of acquisition of foreign gene sequences by phage in the subject.

Optionally, the method for treatment is to reduce the risk of acquisition of foreign gene sequences by phage in the environment in which the subject is present.

Optionally, the target bacterium herein is contained in the subject's microbiome, eg, the gut microbiome. Alternatively, the microbiome is the skin, scalp, hair, eye, ear, oral cavity, throat, lung, blood, rectal, anal, vaginal, scrotal, penis, nose, or tongue microbiome.

In one example, the subject is further administered a medicament concurrently or sequentially with administration of the phage. In one example, the medicament is an antibiotic, an antibody, an immune checkpoint inhibitor (eg, anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies), adoptive cell therapy (eg, CAR-T therapy), or a vaccine.

In one example, the invention employs helper phage to package phage nucleic acids of interest. That is, the present invention provides the following illustrative aspects.
1. A population of helper phages, wherein said helper phages are capable of packaging a first phage nucleic acid to produce a first phage particle, said first phage being distinct from said helper phages, said helper phages A population of helper phages that are incapable of producing helper phage particles by themselves.
2. A composition comprising a first population of phage, wherein said first phage require the helper phage of aspect 1 for replication of said first phage particle, and optionally all A composition wherein less than 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.2, or 0.1% of the phage particles are particles of such helper phage.

In one example, the composition comprises helper phage and less than 1% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.5% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.1% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage, and less than 0.01% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.001% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.0001% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.00001% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.000001% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.0000001% of the total phage particles contained in the composition are particles of such helper phage. In one example, the composition comprises helper phage and less than 0.00000001% of the total phage particles contained in the composition are particles of such helper phage.

In one example, the population comprises at least 10 ³ , 10 ⁴ , 10 ⁵ , or 10 ⁶ phage particles, eg, as indicated by a transduction assay. In one example, the population comprises at least 10 ³ phage particles and eg no more than 10 ¹⁴ particles. In one example, the composition or population comprises at least 10 ⁴ phage particles and, eg, no more than 10 ¹⁴ particles. In one example, the population comprises at least 10 ⁵ phage particles and eg no more than 10 ¹⁴ particles. In one example, the population comprises at least 10 ⁶ phage particles and eg no more than 10 ¹⁴ particles. A standard transduction assay can be performed to obtain a measure of the concentration of the first phage, eg, if the genome of the first phage contains an antibiotic marker. That is, in this case the first phage is capable of infecting the target bacterium and the population contains at least 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ transducing particles in a 1 ml sample, which is infected with susceptible bacteria at a multiplicity of infection of less than 0.1 and plated in vitro on selective agar plates corresponding to antibiotic markers at 20-37°C, e.g., 20°C or 37°C. It can be determined by determining the number.

Optionally, at least 99.9, 99.8, 99.7, 99.6, 99.5, 99.4, 99.3, 99.2, 99.1, 90 of the total phage particles contained in the composition , 85, 80, 70, 60, 50, or 40% are particles of the first phage.

In one example, the first phage genome comprises the f1 origin of replication.

In one example, the helper phage is E. coli phage. In one example, the first phage is an E coli, C difficile, Streptococcus, Klebsiella, Pseudomonas, Acitenobacter, Enterobacteracea, Firmicutes, or Bacteroidetes phage. In one example, the helper phage is an engineered M13 phage.

In one example, the genome of the first phage comprises a phagemid, the phagemid comprising packaging signals for packaging particles of the first phage in the presence of helper phage.

The first phage particle comprises a nucleotide sequence of interest (NSI), e.g., the configuration of a CRISPR/Cas system operable in a target bacterium capable of infecting the first phage particle, as defined herein It may contain an NSI, etc., which encodes the element. Once inside the target bacterium, the first phage DNA cannot be packaged to form first phage particles in the absence of the helper phage. This usefully includes the activity of the genome of the first phage and the products (proteins and/or nucleic acids) encoded by it, and the NSI and its activity in an environment comprising the target bacterium exposed to the first phage. to limit and control the dose of the product encoded by This is useful for controlling medical treatments of the environment, eg, contained in human or animal subjects, plants, or other environments (eg, soil or foodstuffs or food ingredients).

3. A helper phage or composition according to any preceding aspect, wherein the genome of each first phage lacks a gene encoding a first phage structural protein.
4. A composition according to aspects 2 or 3, wherein the composition comprises helper phage DNA.
5. 5. The composition of aspect 4, wherein said DNA comprises a helper DNA segment.
6. A helper phage or composition according to any preceding aspect, wherein said helper phage is in the form of a prophage.
That is, the prophage is integrated into the host cell's chromosome.

Examples of phage structural proteins include phage coat proteins, collar proteins, and phage tail fiber proteins.
7. Helper phage DNA comprising a sequence of 20 or more contiguous nucleotides (eg, 20-25, 30, 35, 40, 50, or 100 nucleotides), such as no helper phage DNA, embodiment 2 or 3. The composition according to 3.
This can be determined, for example, by conventional PCR using DNA probes (designed based on known helper phage genomic sequences). In one example, the composition may contain residual helper prophage DNA, but is otherwise essentially devoid of helper DNA.
8. 8. The composition of any one of aspects 2-5 and 7, wherein said helper phage is capable of infecting host bacteria and said composition is free of host bacteria.
9. Said composition is a lysate of a host bacterial cell, said lysate comprising helper prophage DNA, eg said DNA comprises 20 or more contiguous nucleotides (eg 20-25, 30, 35) of helper phage DNA. , 40, 50 or 100 nucleotides).
10. The composition is a lysate of host bacterial cells, and the lysate is treated (e.g., filtered) to remove all or some helper phage DNA, or the composition is host bacterial cells devoid of cellular material. 9. The composition of any one of aspects 2-8, which is a lysate of
11. 11. The composition of any one of aspects 2-10, which is free of helper phage particles.
12. 12. The composition of any one of aspects 2-11, wherein at least 95% (eg 100%) of the phage particles comprised in said composition are first phage particles.

In another embodiment, the composition comprises a second phage particle, wherein the second phage, unlike the first phage, is not a helper phage.
13. 13. The composition of any one of aspects 2-12, wherein said population comprises at least 10< ³ >, 10< ⁴ >, 10< ⁵ >, or 10< ⁶ > phage particles as indicated by a transduction assay.
14. an antibacterial agent such that said first phage is capable of replicating in a host bacterium and in the presence of said helper phage (e.g., helper prophage), said first phage killing a target bacterium of a first strain or species A helper phage or composition according to any preceding aspect, comprising means, wherein said target bacterium is of a different strain or species and said antimicrobial means is not operable to kill said target bacterium.
15. A composition comprising a population of phage, said population comprising
(a) a first subpopulation of first phage that requires a helper phage to package said first phage;
(b) a composition comprising a second subpopulation of phage comprising helper phage as recited in any of the preceding aspects.
16. A helper phage or composition according to any preceding aspect, wherein said helper phage is a phagemid.
17. (a) a population of helper phages as recited in any of the preceding aspects, and (b) a population of nucleic acid vectors comprising vector DNA comprising a first phage packaging signal;
A composition comprising
(c) a composition wherein said helper phage is capable of packaging said vector DNA to produce a first phage;
18. 18. The composition of aspect 17, wherein said vector is a phage.
19. 18. The composition of aspect 17, wherein said vector is a plasmid or phagemid.
20. Said vector is a shuttle vector (e.g. a pUC vector) capable of replicating in a first bacterium, said vector being further replicated in the presence of said helper phage, and said vector being further replicated in a second bacterium (host bacterium) 20. The composition of aspect 19, wherein said first bacterium is a different strain or species than said host bacterium strain or species.
21. 21. The composition of aspect 20, wherein said first phage is capable of infecting a third bacterium of a different strain or species than said second (and optionally said first) bacterium.
22. an antibacterial agent such that said first phage is capable of replicating in a host bacterium and in the presence of said helper phage (e.g., helper prophage), said first phage killing a target bacterium of a first strain or species 22. A composition according to any one of aspects 17-21, comprising means, wherein said host bacteria are of a different strain or species and said antimicrobial means are not operable to kill said host bacteria.
23. A helper phage or composition according to any preceding aspect, wherein said genome lacks a packaging signal (eg SEQ ID NO: 2 below) and said helper phage is incapable of self-replication.
24. A helper phage or composition according to aspect 23, wherein said signal is a pac or cos sequence.
25. A helper phage or composition according to any preceding aspect, wherein said helper phage genome is capable of replication in a host cell.

That is, the genome is capable of nucleic acid replication but is incapable of helper phage packaging.
26. 25. A helper phage or composition according to any one of aspects 1-24, wherein said genome lacks nucleotide sequences necessary for the production of helper phage particles.
27. A helper phage according to aspect 26, wherein said nucleotide sequence encodes a sigma factor (eg sigma-70) or comprises a sigma factor recognition site, a DNA polymerization recognition site, or the promoter of a gene required for helper phage DNA replication; or Composition.
28. A helper phage or composition according to any preceding aspect, wherein said helper phage is a mild phage.
29. 28. A helper phage or composition according to any one of aspects 1-27, wherein said helper phage is a lytic phage.
30. whether said first phage is capable of infecting a target bacterium and comprises a nucleotide sequence of interest (NSI) which enables said first phage to express a protein or RNA (e.g. gRNA or crRNA) in said target bacterium; or a helper phage or composition according to any preceding aspect, wherein said NSI comprises regulatory elements operable in the target bacterium.
31. The presence of the NSI or the protein or RNA encoded thereby in the target bacterium mediates target cell death or down-regulation of target cell growth or proliferation, or one or more encoded by the genome of said target cell. 31. A helper phage or composition according to aspect 30, which mediates switching off or down-regulating expression of multiple RNAs or proteins.
32. the presence of the NSI or the protein or RNA encoded thereby in the target bacterium mediates upregulation of the growth or proliferation of the target cell, or one or more RNAs or proteins encoded by the genome of said target cell; 31. A helper phage or composition according to aspect 30, which mediates switching on or upregulating the expression of
33. 33. The antimicrobial of any one of aspects 2-32, wherein said first phage is capable of infecting a target bacterium, each first phage comprising engineered antimicrobial means for killing the target bacterium. Composition.

Use of the term "engineered" makes it readily apparent to those skilled in the art that the relevant means have been introduced and are not naturally occurring in the phage. For example, the means are recombinant, artificial, or synthetic.
34. 34. The composition of aspects 14, 22, or 33, wherein said antimicrobial means comprises one or more components of the CRISPR/Cas system.
35. wherein said component is (i) a DNA sequence encoding a guide RNA (e.g., a single guide RNA) or comprising a CRISPR array for producing a guide RNA, said guide RNA comprising said genome of a target bacterium; 4. A DNA sequence according to claim 3 comprising a targetable DNA sequence, (ii) a DNA sequence encoding a Cas nuclease, and/or (iii) a DNA sequence encoding one or more components of Cascade. Composition.

In one example, Cas herein is Cas9. In one example, Cas herein is Cas3. The Cas may be identical to the Cas encoded by the target bacterium.
36. 36. The composition of any one of aspects 14, 22, or 33-35, wherein said antimicrobial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.
37. Any of the above, wherein the helper phage is for use in medicine practiced on human or animal subjects, or the composition is a pharmaceutical composition for use in medicine practiced on human or animal subjects. A helper phage or composition according to any aspect.

In one example, the animal is a livestock or companion pet animal (eg, cow, pig, goat, sheep, horse, dog, cat, or rabbit). In one example, the animal is an insect (an insect at any stage of its life cycle, eg, eggs, larvae, or pupae). In one example, the animal is a protozoan. In one example, the animal is a cephalopod.
38. The composition is a herbicide, pesticide, food or beverage processing agent, food or beverage additive, petrochemical or fuel treatment agent, water purification agent, cosmetic additive, detergent additive, or environmental (e.g., soil) additive. 37. The composition of any one of aspects 2-36, which is an additive or detergent.
39. 38. A helper phage or composition according to any one of aspects 1-37 for use in a contained method of treating a disease or condition in a human or animal subject, wherein said disease or condition is caused by said target bacterium. said target bacterium is contained in said subject, said method comprising administering said composition to said subject, whereby said target bacterium is killed by exposure to said antimicrobial means, and said first phage; helper phage or composition wherein the growth of the helper phage is contained.

The inability of the first phage to self-replicate and the need for a helper phage or second DNA to do this can be determined, for example, in the urine or urine of a subject that may otherwise contain the replicated first phage. It provides useful containment of the composition to the site of action (eg, intestine) and/or the environment of the subject when exposed to secretions such as faeces. The inability of the helper phage or the second DNA to self-package limits the availability of the factors required by the first phage to form the packaged particle and thus the first phage provide containment by limiting the growth of This would be useful to contain the antimicrobial activity provided by the primary phage, eg the CRISPR/Cas killing principle.
40. A bacterial cell or cells comprising a helper phage or composition according to any preceding aspect, wherein said first phage is capable of replicating in the presence of said helper phage in said cell. A cell or multiple bacterial cells.

The cell acts, for example, as a carrier for the genome of the first phage, and the first phage DNA is transferred once the carrier bacterium is to treat an environment, such as soil or human intestine or other environment as described herein. If administered at , horizontal transfer from the carrier to the target bacterium can occur. In one example, the environment is contained in a human or animal subject and the carrier is commensal or probiotic in the subject. For example, the carrier bacterium is a Lactobacillus (eg, L reuteri or L lactis), E coli, or Streptococcus (eg, S thermophilus) bacterium. Horizontal transfer can be transfer of a plasmid (eg, a conjugative plasmid) into the target bacterium, or infection of the target bacterium with a first phage, the first phage prepackaged in a carrier. The use of carriers is also useful for oral administration or other routes by which the carrier can protect the phage, helper, or composition from the acidic stomach or other hostile environment in the subject. Additionally, the carrier may be a beverage, such as a probiotic beverage, such as adapted Yakult™, Actimel™, Kevita™, Activia™, Jarrow™, or similar beverages for human consumption. can be blended into
41. 41. The cell of aspect 40 for administration to a human or animal subject for medical use comprising killing a target bacterium using the first phage, wherein said target bacterium is diseased or A cell that mediates a state.

In one example, the subject is not an embryo when the subject is human.
42. 42. The cell of aspect 41, comprising a helper phage and is synbiotic or probiotic in said subject.
43. 66. A method of killing target bacteria in the environment, optionally wherein said method is not performed on a human or animal body, said method comprising a cell according to aspect 42 or a cell according to any one of aspects 57-65. exposing said environment to a composition obtained or obtainable by the method, wherein said environment has been or has been exposed to said first phage or said vector and in the presence of said helper phage said first A method of producing a phage, said first phage being able to replicate in said environment and killing the target bacterium.
44. Said cells are E. 44. A cell or method according to any one of aspects 40-43, which is an E. coli, Lactobacillus (e.g. Lactis or retueri), or Streptococcus (e.g. thermophilus) cell.
45. 45. The cell or method of aspects 40-44, wherein said subject is or has been administered cells comprising the first phage.
46. 46. The composition of any one of aspects 2-45 in combination with a target bacterial cell, wherein said first phage is capable of infecting said target bacterial cell.
47. Any of aspects 1 to 42 and 44 to 46 in the manufacture of an antimicrobial agent that kills target bacteria for containment of said antimicrobial agent in an environment, e.g., ex vivo containment or containment in a human or animal subject containing said environment. Use of a helper phage, composition or cell according to any one of claims 57 to 65 or a composition obtained or obtainable by a method according to any one of aspects 57-65.
48. helper phage according to any one of aspects 1-42 and 44-46 in the manufacture of an antimicrobial agent that kills said target bacterium to reduce the risk of foreign gene acquisition by said first phage; composition; or use of a cell or composition obtained or obtainable by a method according to any one of aspects 57-65.

For example, this is useful for reducing the risk of antibiotic resistance genes by phages, eg, when the phages are in the presence of other phages or plasmids in the environment.
49. Aspects 1-42 and 44-46 of any one of aspects 1-42 and 44-46 in the manufacture of an antimicrobial agent that kills said target bacterium to reduce the risk of acquisition of one or more antibiotic resistance genes by said first phage or a composition obtained or obtainable by a method according to any one of aspects 57-65.
50. A method for reducing the risk of foreign gene acquisition by a first phage, comprising:
(a) providing a composition according to any one of aspects 2-42 and 44-46 or a composition obtained or obtainable by a method according to any one of aspects 57-65; (b) exposing a target bacterium to the composition, wherein the first phage infects the target bacterium;
(c) the helper phage is incapable of self-replicating, thereby limiting the growth of the first phage and preventing or reducing the growth of the first phage, thereby exogenous genes (e.g. antibiotic resistance genes) wherein the risk of acquisition of the first phage of is reduced.
51. A method of confining antimicrobial activity in the environment (e.g., ex vivo) comprising:
(a) providing an antimicrobial composition according to any one of aspects 2-42 and 44-46 or a composition obtained or obtainable by a method according to any one of aspects 57-65; and (b) exposing a target bacterium in said environment to said composition, wherein said bacterium is killed upon exposure to said first phage and antimicrobial means,
(c) the helper phage is incapable of self-replicating, thereby limiting the growth of the first phage and preventing or reducing the growth of the first phage, thereby locking in antibacterial activity;
52. A method of controlling dosing of a first phage in an environment (e.g., ex vivo) comprising:
(a) providing an antimicrobial composition according to any one of aspects 2-42 and 44-46 or a composition obtained or obtainable by a method according to any one of aspects 57-65; and (b) exposing a target bacterium in said environment to said composition, said bacterium being infected with a first phage,
(c) the helper phage is incapable of self-replicating, thereby limiting the growth of the first phage, preventing or reducing the growth of the first phage, thereby dosing the first phage in the environment; The method by which is controlled.
53. 48. The method according to any one of aspects 43 to 45, 51 and 52, or the use according to aspect 47, wherein said environment is a human or animal microbiome, such as a gut microbiome.
54. The environment is a soil microbiome, i.e. plants, plant parts (e.g. leaves, fruits, vegetables, or flowers) or plant products (e.g. pulp), water, watercourses, fluids, foodstuffs or components thereof, beverages or components thereof, medicines any one of aspects 43-45, 51, and 52, which is a chemical device, cosmetic, detergent, blood, bodily fluid, medical device, industrial device, oil rig, petrochemical processing, storage or transportation device, vehicle or container. 48. Use according to aspect 47.
55. The environment is an ex vivo bodily fluid (e.g., urine, blood, blood products, sweat, tears, sputum, or saliva), body solids (e.g., faeces), or tissue of a human or animal subject to which the composition has been administered. , a method according to any one of aspects 43 to 45, 51 and 52, or a use according to aspect 47.
56. The environment is an in vivo body fluid (e.g., urine, blood, blood products, sweat, tears, sputum, or saliva), body solids (e.g., faeces), or tissue of a human or animal subject to whom the composition has been administered. , a method according to any one of aspects 43 to 45, 51 and 52, or a use according to aspect 47.
57. A method of producing a first phage, said first phage requiring a helper phage to replicate, said method comprising:
(a) providing a DNA containing a packaging signal;
(b) introducing said DNA into a host bacterial cell;
(c) said host bacterial cell contains a helper phage or a helper phage is introduced into said bacterial cell simultaneously or sequentially with step (b);
(d) said helper phage is according to any of the above aspects;
The method comprises (e) causing or enabling the helper phage to produce a phage coat protein, wherein the packaging signal is recognized in the host cell such that the first phage can produce the protein. wherein said first phage packages said DNA;
(f) the production of helper phage particles in said host cell is inhibited or reduced, thereby limiting the availability of helper phage particles;
said method comprising (g) optionally lysing said host cell to obtain said first phage;
(h) thereby producing a composition comprising a first phage that requires said helper phage for replication, wherein further production of first phage particles limits the availability of helper phage in said composition; A method that is prevented or reduced by

In one embodiment, the DNA is contained in a phagemid or cloning vector (eg shuttle vector, eg pUC vector).

There may be a small amount of helper phage DNA replication, or replication of helper phage DNA may be eliminated entirely to allow efficient production of the first phage protein.
58. 58. The method of aspect 57, wherein in (c) said helper phage is a prophage integrated into said bacterial cell chromosome.
59. 60. A method according to aspect 59, wherein (e) comprises inducing helper phage DNA replication and/or expression of said protein using, for example, UV, mitomycin.
60. 60. The method of any one of aspects 57-59, wherein (g) comprises further separating said first phage from cellular material or helper phage DNA.
61. 61. The method of any one of aspects 57-60, wherein said composition comprises a first population of phage particles and said composition does not comprise helper phage DNA and/or particles.
62. 62. The method of any one of aspects 57-61, wherein the DNA of (a) comprises engineered antimicrobial means to kill the target bacteria.
63. 63. The method of aspect 62, wherein said antimicrobial means comprises one or more components of the CRISPR/Cas system.
64. wherein said component is (i) a DNA sequence encoding a guide RNA (e.g. a single guide RNA) or comprising a CRISPR array for producing a guide RNA, wherein said guide RNA comprises said genome of a target bacterium; (ii) a DNA sequence encoding a Cas (e.g., Cas9, Cas3, Cpf1, CasX, or CasY) nuclease, and/or (iii) one or more components of Cascade ( 64. A method according to aspect 63, comprising a DNA sequence encoding for example CasA).
65. 65. The method of any one of aspects 62-64, wherein said antimicrobial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.
66. A helper phage, composition, or according to any one of aspects 1-42 and 44-46, for antimicrobial treatment of a target bacterium in a human or animal subject, whereby said antimicrobial treatment is contained within said subject A cell, or composition obtained or obtainable by the method of any one of aspects 57-65.
67. According to any one of aspects 1-42 and 44-46, for antimicrobial treatment of target bacteria in the gut of a human or animal subject, whereby antimicrobial activity in one or more body exudates of said subject is reduced. A helper phage, composition or cell as described or a composition obtained or obtainable by a method according to any one of aspects 57-65.

This is useful as a safety measure to reduce or eliminate activity of the first phage outside the subject.
68. 68. A helper phage, composition, or cell according to aspect 67, wherein said antimicrobial activity in one or more bodily exudates of said subject is eliminated.
69. A helper phage, composition or cell according to any one of aspects 1-42 and 44-46 for controlling the dosage of an antibacterial treatment of a target bacterium in a human or animal subject, e.g. in the gut of said subject , or a composition obtained or obtainable by the method of any one of aspects 57-65.

Beneficially, proliferation of the first phage is limited or eliminated so that dosing in the subject can be controlled or even predetermined within expected narrow ranges. This is useful, for example, for pharmaceuticals containing the first phage or composition, and may aid approval of such pharmaceuticals by the FDA and similar authorities.

Alternatively, the dosing is the dosing of the environment, such as soil disclosed herein, and limiting the activity of the first phage or composition is also desirable to limit the spread of activity in nature and other fields.
70. A helper phage, composition or cell according to any one of aspects 1-42 and 44-46 for modifying the dosage of an antimicrobial treatment of a target bacterium in a human or animal subject, e.g. in the gut of said subject , or a composition obtained or obtainable by the method of any one of aspects 57-65.
71. A phage production system for producing a phage (NSI-phage) comprising a nucleotide sequence of interest (eg the first phage according to any of the above aspects), said system comprising components (i) to (iii) ), i.e.
(a) a first DNA;
(b) a second DNA, and (c) an NSI-phage producing factor (NPF) or an expressible nucleotide sequence encoding the NPF;
(d) said first DNA encodes a helper phage (e.g. said first helper phage recited in any of the above aspects);
(e) said second DNA comprises said nucleotide sequence of interest (NSI);
(f) when said system is contained in a bacterial host cell, helper phage proteins are expressed from said first DNA to form phage which, in the presence of said NPF, package said second DNA, thereby forming an NSI; - produce phage,
(g) the system lacks a helper phage production factor (HPF) required to form a helper phage particle that packages the first DNA, or contains an expressible nucleotide sequence encoding a functional HPF; in said host cell for eliminating or minimizing production of helper phage lacking or wherein said system comprises a nucleotide sequence comprising or encoding a functional HPF and said system comprises said first DNA; further comprising means for targeted inactivation of HPF sequences;
Said system is thereby capable of producing a product comprising a population of NSI-phages, each NSI-phage requiring said helper phage for propagation, optionally said NSI-phage in said product A phage production system which is not mixed with helper phage or wherein less than 20% of the total phage contained in said product is said helper phage.

It is within the concept of the present invention that, for example, the helper phage packaging signal or other HPF nucleotide sequence in the helper phage genome is mutated (e.g., by deletion, substitution, or addition of nucleotides therein) to produce a phage particle. The residual ability of the helper phage to replicate and produce particles when the ability to form is knocked down involves relatively low levels of helper phage particle production. Preferably, the production of helper phage particles is abolished, for example by deleting all or part of the sequences from the helper phage genome or by inactivating the sequences.
72. A method of producing a first phage, said first phage requiring a helper phage for replication,
(a) providing a system according to aspect 71 in a host cell;
(b) producing or allowing production of said helper phage protein, whereby said second DNA is packaged to produce a first phage; and (c) optionally lysing said host cell. obtaining a composition comprising the first phage.
73. 73. The method of aspect 72, wherein step (c) comprises separating said first phage from cellular material.
74. wherein said composition comprises a first population of phages, wherein less than 20, 10, 5, 4, 3, 2, 1, 0.5, or 0.1% of all phages contained in said composition are helper phages; 74. A method according to aspect 72 or 73.
75. 75. The method of any one of aspects 72-74, wherein said second DNA comprises engineered antimicrobial means to kill the target bacteria.
76. 76. The method of aspect 75, wherein said antimicrobial means comprises one or more components of the CRISPR/Cas system.
77. wherein said component is (i) a DNA sequence encoding a guide RNA (e.g. a single guide RNA) or comprising a CRISPR array for producing a guide RNA, wherein said guide RNA comprises said genome of a target bacterium; 77. A method according to aspect 76, comprising a targetable DNA sequence, (ii) a DNA sequence encoding a Cas nuclease, and/or (iii) a DNA sequence encoding one or more components of Cascade. .
78. 78. A method according to any one of aspects 75-77, wherein said antimicrobial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.
79. said first phage is capable of infecting a target bacterium, said NSI is capable of expressing protein or RNA in said target bacterium, or said NSI comprises a control element operable in said target bacterium; 79. A system or method according to any one of aspects 71-78.
80. the presence of said NSI or the protein or RNA encoded thereby in the target bacterium mediates target cell death or downregulation of target cell growth or proliferation, or one encoded by the genome of said target cell; or a system or method according to aspect 79, which mediates switching off or down-regulating expression of multiple RNAs or proteins.
81. the presence of said NSI or the protein or RNA encoded thereby in the target bacterium mediates upregulation of target cell growth or proliferation; 80. A system or method according to aspect 79, which mediates switching on of protein expression or upregulation thereof.
82. each of said NPF and HPF is a packaging signal, e.g., SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98, or 99% identical thereto, or is a homolog from a different species; A system or method according to any one of aspects 71-81.
83. 83. A system or method according to aspect 82, wherein each signal is a pac or cos sequence or is a homologue.
84. 82. The system or method of any one of aspects 71-81, wherein said HPF is a nucleotide sequence necessary for helper phage replication.
85. The HPF encodes a sigma factor (eg, sigma-70), or has a sigma factor recognition site, a DNA polymerization recognition site, or a promoter of a gene required for helper phage DNA replication, helper phage integrase, helper phage excitase. 82. A system or method according to any one of aspects 71 to 81, comprising a helper phage origin of replication.
86. 86. A composition comprising a population of first phage obtainable by the method of any one of aspects 72-85, wherein the genome of each said first phage lacks a gene encoding a phage protein.
87. 87. The composition of aspect 86, wherein said first phage comprises an antimicrobial means as recited in any one of aspects 75-78.
88. 88. The composition of aspect 87, comprising the same DNA or fragment thereof as said first DNA.
89. 89. The composition of aspect 88, wherein the DNA of said composition is identical to the first DNA and lacks the helper phage packaging signal.
90. 90. A composition according to any one of aspects 86-89 for antimicrobial treatment of a target bacterium in a human or animal subject, whereby said antimicrobial treatment is contained within said subject.
91. 90. A composition according to any one of aspects 86 to 89 for the antimicrobial treatment of target bacteria in the gut of a human or animal subject, whereby antimicrobial activity in one or more body exudates of said subject is reduced, the composition.
92. 92. The composition of aspect 91, wherein antimicrobial activity in one or more bodily exudates of said subject is eliminated.
93. 90. Composition according to any one of aspects 86-89, for controlling the dosing of antimicrobial treatment of target bacteria in a human or animal subject, eg in the gut of said subject.
94. 90. Composition according to any one of aspects 86-89, for modifying the dosing of antimicrobial treatment of target bacteria in a human or animal subject, eg in the gut of said subject.
95. An isolated DNA containing all the structural protein genes of the helper phage genome required to produce a phage particle, the helper phage production factors required to produce the helper phage in which said DNA is packaged ( HPF), optionally wherein said DNA comprises one or more promoters for expression of said gene when said DNA is integrated into said genome of a host bacterial cell .
96. 96. The DNA of aspect 95, which lacks any phage packaging signals.
97. The HPF is a nucleotide sequence encoding a sigma factor, or a sigma factor recognition site, a DNA polymerization recognition site, a promoter of a gene required for helper phage DNA replication, a nucleotide sequence encoding helper phage integrase, or a helper phage 97. DNA according to aspects 95 or 96, comprising a nucleotide sequence encoding an exhibitionase, or a helper phage origin of replication.
98. 98. The DNA of any one of aspects 95-97, comprising a nucleotide sequence encoding a CRISPR/Cas system repressor.
99. 99. The DNA of any one of aspects 95-98, wherein said DNA is integrated into the chromosome of a host bacterial cell and said gene is expressible in said host cell.
100. 100. The DNA of aspect 99, wherein said cell lacks an active CRISPR/Cas system.
101. 101. The DNA of any one of aspects 95-100 in combination with a second DNA, wherein said second DNA comprises said HPF.
102. 101. The DNA of any one of aspects 95-100 in combination with a second DNA, wherein said second DNA comprises a phage packaging signal and optionally said first DNA lacks a phage packaging signal. DNA.
103. 103. The DNA of aspects 101 or 102, wherein said second DNA is contained in a phagemid or plasmid (eg shuttle vector).

In one example, the kit, DNA, first phage, helper phage, or composition is contained in a medical container such as a syringe, vial, IV bag, inhaler, eyedropper, or nebulizer. In one example, the kit, DNA, first phage, helper phage, or composition is contained in a sterile container. In one example, the kit, DNA, first phage, helper phage, or composition are contained in a medically compatible container. In one example, the kit, DNA, first phage, helper phage, or composition is contained in a fermentation vessel, such as a metal, glass, or plastic vessel.

In one example, the kit, DNA, first phage, helper phage, or composition is administered to a human or animal subject by administration, e.g., orally, IV, subcutaneously, intranasally, intraocularly, vaginally, topically, rectally, or by inhalation. It is included in the medicament in combination with an instruction manual or packaging label containing instructions for administering the medicament. In one example, the kit, DNA, first phage, helper phage, or composition is included in an oral pharmaceutical formulation. In one example, the kit, DNA, first phage, helper phage, or composition is included in an intranasal or intraocular pharmaceutical formulation. In one example, the kit, DNA, first phage, helper phage, or composition is included in a personal hygiene composition (eg, shampoo, soap, or deodorant) or cosmetic formulation. In one example, the kit, DNA, first phage, helper phage, or composition is included in a cleaning formulation. In one example, the kit, DNA, first phage, helper phage, or composition is included in a cleaning formulation, eg, for cleaning medical or industrial devices or equipment. In one example, the kit, DNA, first phage, helper phage, or composition is included in a food product, food ingredient, or food processing agent. In one example, the particles, clusters, or compositions of the invention are included in a beverage, beverage ingredient, or beverage processor. In one example, the kit, DNA, first phage, helper phage, or composition is contained in a medical bandage, fabric, plaster, or swab. In one example, the kit, DNA, first phage, helper phage, or composition is included in a herbicide or pesticide. In one example, the kit, DNA, first phage, helper phage, or composition is included in an insecticide.

In one example, the first phage is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In one example, the helper phage is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In one example, the helper phage is filamentous M13, Noviridae, tailed phage (eg, Myoviridae, Siphoviridae, or Podoviridae), or non-tailed phage (eg, Tectiviridae).

In one example, both the first phage and helper phage are Corticoviridae. In one example, both the first phage and the helper phage are Cystoviridae. In one example, both the first phage and the helper phage are Inoviridae. In one example, both the first phage and helper phage are Leviviridae. In one example, both the first phage and helper phage are Microviridae. In one example, both the first phage and helper phage are Podoviridae. In one example, both the first phage and the helper phage are Siphoviridae. In one example, both the first phage and helper phage are Tectiviridae.

In one example, the CRISPR/Cas components are components of a Type I CRISPR/Cas system. In one example, the CRISPR/Cas components are components of a Type II CRISPR/Cas system. In one example, the CRISPR/Cas components are components of a Type III CRISPR/Cas system. In one example, the CRISPR/Cas component is a component of a Type IV CRISPR/Cas system. In one example, the CRISPR/Cas component is a component of a Type V CRISPR/Cas system. In one example, the CRISPR/Cas component comprises a nucleotide sequence encoding Cas9 (eg, S pyogenes Cas9, S aureus Cas9, or S thermophilus Cas9). In one example, the CRISPR/Cas component comprises a nucleotide sequence encoding Cas3 (eg, E coli Cas3, C difficile Cas3, or Salmonella Cas3). In one example, the CRISPR/Cas component 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.

In one example, the first DNA, first phage, or vector encodes a protein of interest from a nucleotide sequence comprising a CRISPR/Cas component or a promoter operable in the target bacterium.

In one example, the host bacterium and/or target bacterium is E coli. In one example, the host bacterium and/or target bacterium is C difficile (eg, the vector is a shuttle vector operable in E coli and the host bacterium is C difficile). In one example, the host bacterium and/or target bacterium is a Streptococcus, eg, S thermophilus (eg, the vector is a shuttle vector operable in E coli and the host bacterium is Streptococcus). In one example, the host bacterium and/or target bacterium is Pseudomonas, eg, Paeruginosa (eg, the vector is a shuttle vector operable in E coli and the host bacterium is Paeruginosa). In one example, the host bacterium and/or target bacterium is Klebsiella (eg, the vector is a shuttle vector operable in E coli and the host bacterium is Klebsiella). In one example, the host bacterium and/or target bacterium is Salmonella, eg, Styphimurium (eg, the vector is a shuttle vector operable in E coli and the host bacterium is Salmonella).

Optionally, the host and/or target bacterium is a Gram-negative bacterium (eg, Helix or Vibrio). Optionally, the host and/or target bacteria are Gram-positive bacteria. Optionally, the host and/or target bacterium is Mycoplasma, Chlamydia, Spirochete, or Mycobacterium. Optionally, the host and/or target bacterium is Streptococcus (eg pyogenes or thermophilus). Optionally, the host and/or target bacterium is Staphylococcus (eg aureus, eg MRSA). Optionally, the host and/or target bacterium is E. E. coli (eg O157:H7) host, eg where Cas is encoded by a vector or the nuclease activity of the endogenous host Cas is derepressed. Optionally, the host and/or target bacterium is Pseudomonas (eg aeruginosa). Optionally, the host and/or target bacterium is Vibro (eg cholerae (eg 0139) or vulnificus). Optionally, the host and/or target bacterium is Neisseria (eg gonnorrhoeae or meningitidis). Optionally, the host and/or target bacterium is Bordetella (eg pertussis). Optionally, the host and/or target bacterium is Haemophilus (eg influenzae). Optionally, the host and/or target bacterium is Shigella (eg dysenteriae). Optionally, the host and/or target bacterium is Brucella (eg abortus). Optionally, the host and/or target bacterium is a Francisella host. Optionally, the host and/or target bacterium is a Xanthomonas host. Optionally, the host and/or target bacterium is an Agrobacterium host. Optionally, the host and/or target bacterium is an Erwinia host. Optionally, the host and/or target bacterium is Legionella (eg pneumophila). Optionally, the host and/or target bacterium is Listeria (eg monocytogenes). Optionally, the host and/or target bacterium is a Campylobacter (eg jejuni). Optionally, the host and/or target bacterium is Yersinia (eg pestis). Optionally, the host and/or target bacterium is Borelia (eg burgdorferi). Optionally, the host and/or target bacterium is Helicobacter (eg pylori). Optionally, the host and/or target bacterium is Clostridium (eg difficile or botulinum). Optionally, the host and/or target bacterium is Erlichia (eg chaffeensis). Optionally, the host and/or target bacterium is Salmonella (eg typhi or enterica, eg serovar typhimurium, eg DT104). Optionally, the host and/or target bacterium is Chlamydia (eg pneumoniae). Optionally, the host and/or target bacterium is a Parachlamydia host. Optionally, the host and/or target bacterium is Corynebacterium (eg, amycolatum). Optionally, the host and/or target bacterium is Klebsiella (eg pneumoniae). Optionally, the host and/or target bacterium is Enterococcus (eg faecalis or faecim, eg linezolid resistant). Optionally, the host and/or target bacterium is Acinetobacter (eg baumannii, eg multidrug resistant).

Further examples of target cells using the present invention and targeting of antibiotic resistance in such cells are as follows.
1. Optionally, the target bacterium is a Staphylococcus aureus cell resistant to an antibiotic selected from, for example, methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin, and teicoplanin.
2. Optionally, the target bacterium is Pseudomonas aeruginosa resistant to antibiotics, e.g. selected from cephalosporins (e.g. ceftazidime), carbapenems (e.g. imipenem or meropenem), fluoroquinolones, aminoglycosides (e.g. gentamicin or tobramycin) and colistin. are cells.
3. Optionally, the target bacteria are, for example, carbapenem-resistant Klebsiella (eg, pneumoniae) cells.
4. Optionally, the target bacterium is a Streptococcus (eg, thermophilus, pneumoniae, or pyogenes) cell resistant to antibiotics, eg, selected from erythromycin, clindamycin, beta-lactams, macrolides, amoxicillin, azithromycin, and penicillins.
5. Optionally, the target bacteria are Salmonella (eg, serotype Typhi) cells resistant to antibiotics, eg, selected from ceftriaxone, azithromycin, and ciprofloxacin.
6. Optionally, the target bacteria are Shigella cells resistant to antibiotics, eg selected from ciprofloxacin and azithromycin.
7. Optionally, the target bacterium is a mycobacterium tuberculosis cell resistant to an antibiotic selected from, for example, resistance to isoniazid (INH), rifampicin (RMP), fluoroquinolones, amikacin, kanamycin, and capreomycin, and azithromycin.
8. Optionally, the target bacteria are Enterococcus cells that are resistant to, for example, vancomycin.
9. Optionally, the target bacterium is an Enterobacteriaceae cell resistant to antibiotics, eg selected from cephalosporins and carbapenems.
10. Optionally, the target bacterium is E. coli resistant to an antibiotic selected from, for example, trimethoprim, itrofurantoin, cephalexin, and amoxicillin. coli cells.
11. Optionally, the target bacteria are Clostridium (eg difficile) cells resistant to antibiotics, eg selected from fluoroquinolone antibiotics and carbapenems.
12. Optionally, the target bacterium is a Neisseria gonorrhoea cell resistant to an antibiotic selected from, for example, cefixime (eg, oral cephalosporins), ceftriaxone (injectable cephalosporins), azithromycin, and tetracyclines.
13. Optionally, the target bacterium is an Acinetobacter baumannii cell resistant to an antibiotic selected from, for example, beta-lactams, meropenems, and carbapenems.
14. Optionally, the target bacteria are Campylobacter cells resistant to antibiotics, eg selected from ciprofloxacin and azithromycin.
15. Optionally, the target cell produces beta (β)-lactamase.
16. Optionally, the target cell is a bacterial cell resistant to the antibiotics cited in any one of Examples 1-14.

Mobile genetic elements, genomic islands, pathogenicity islands, etc. Genetic variation in bacteria and archaea can be achieved by mutation, rearrangement, and horizontal introgression and recombination. In addition to the core genes encoding basic metabolic activities, information processing, and housekeeping functions such as bacterial structural and regulatory components, the growing number of genomic sequence data show that bacteria adapt under certain environmental conditions. It has been demonstrated that a large number of accessory genes encoding antimicrobial resistance, toxins, and enzymes that contribute to and survival are acquired by horizontal introgression of mobile genetic elements (MGEs). Mobile genetic elements are heterogeneous groups of molecules including plasmids, bacteriophages, genomic islands, chromosomal cassettes, virulence islands, and integral and complex elements. Genomic islands are relatively large segments of DNA ranging from 10-200 kb that are often integrated into tRNA gene clusters flanked by direct repeats of 16-20 bp. They are recognized as discrete DNA segments acquired by horizontal introgression, as they may differ from the rest of the chromosome in terms of GC content (%G+C) and codon usage.

Pathogenicity islands (PTIs) are a subset of horizontally transferred genetic elements known as genomic islands. There is a specific family of highly mobile PTIs in Staphylococcus aureus that are induced to excise and replicate by certain resident prophages. These PTIs are packaged in microcephalic phage-like particles and are transfected at a frequency commensurate with the plaque-forming titer of the phage. This process is termed the SaPI excision replication-packaging (ERP) cycle, and the high frequency of SaPI transfer is characterized by SaPI-specific transfer (SPST) to distinguish it from classical generalized transduction (CGT). ). SaPI possesses a highly conserved genetic organization that is comparable to that of bacteriophages and distinct from all other horizontally acquired genomic islands. The integrases encoded by SaPI1 and SaPIbov2 are used for both excision and integration of the corresponding elements, and the same is hypothesized to be true for other SaPIs. Phage 80α can induce several different SaPIs, including SapI1, SapI2, and SapIbov1, while φ11 can induce SapIbov1 but neither of the other two SapIs.

See "Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases", Quiles-Puchalt et al, PNAS April 2601-616. Staphylococcal virulence islands (SaPIs) are highly mobile, carrying and disseminating superantigens and other virulence genes. SaPI has been reported to hijack the packaging machinery of the phage it sacrifices using two unrelated and complementary mechanisms. Phage packaging begins with the recognition in the phage DNA of specific sequences called "pac" or "cos" depending on the type of phage. SaPI's strategy relies on the helper phage machinery and involves the delivery of helper phage pac- or cos-like sequences in the SaPI genome that ensure packaging of SaPI in full-sized phage particles. These strategies interfere with phage reproduction, which ultimately is of key benefit to the bacterial population by reducing the number of phage particles.

Staphylococcal virulence islands (SaPIs) are archetypal, widely distributed families of chromosomally-located mobile genetic elements that contribute substantially to intra- and interspecific introgression, host adaptation, and virulence. is a member. A key feature of its mobility is induction of SaPI excision and replication by certain helper phages and efficient encapsidation into phage-like infectious particles. Most SaPIs use abundant packaging mechanisms and encode small terminase subunit (TerS) homologs that recognize SaPI-specific pac sites and determine SapI packaging specificity. Some of the known SaPIs do not encode recognizable TerS homologues, yet are efficiently packaged and frequently transfected by helper phage. Quiles-Puchalt et al. reported on one non-terS-coding SaPI, SaPIbov5, and found that it used two different, undescribed packaging strategies. SaPIbov5 is packaged into full-sized phage-like particles by typical pac-type helper phages or by cos-type phages. That is, it is both pac and cos in nature and uses two different TerS encoded by the phage. This is an example of SaPI packaging by the cos phage, in which it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is even more unique in that it requires a HNH nuclease that is carried only by the cos phage in addition to a large terminase subunit for cleavage and melting of the cos-site.

Characterization of phage-inducible SaPI and some of its helper phages established that the pac (or abundance) mechanism is used for encapsidation. In keeping with this notion, some SaPIs encode homologs of TerS, which complex with the large phage-encoded terminase subunit TerL to bind SaPI DNA in infectious particles composed of phage proteins. enable packaging. They also contain a morphogenetic (cpm) module that initiates the formation of small capsids to match the small SaPI genome. Among the SaPI sequences initially characterized, there were several sequences containing neither TerS nor cpm homologues, several SaPIs subsequently identified from bovine sources and many phages from other species. The same is true for inducible chromosomal islands. For some of these islands, it was hypothesized that they were either defective derivatives of the elements that originally harbored these genes, or that the terS and cpm genes were present but not recognized by homology.

Quiles-Puchalt et al. observed that key features of φSLT/SapIbov5 packaging are required for the HNH nuclease, which in turn is encoded in the φSLT terminase module. Proteins that carry HNH domains are widely distributed in nature and are present in organisms in all living kingdoms. HNH motifs are small degenerate nucleic acid binding and cleavage modules of about 30-40 amino acid residues that bind a single divalent metal ion. HNH motifs have been found to play important roles in many different cellular processes, including bacterial killing, DNA repair, replication, and recombination, and RNA-related processes, among other enzymes. ing. HNH endonucleases are present in many cos-site bacteriophages of Gram-positive and negative bacteria and are always flanked by genes encoding terminase and other morphogenic proteins. Quiles-Puchalt et al. demonstrated that the HNH nucleases encoded by φ12 and the closely related φSLT have non-specific nuclease activity and are required for packaging of these phages and SaPIbov5. Quiles-Puchalt et al. showed that HNH and TerL are jointly required for cleavage of the cos site. Quiles-Puchalt et al. also observed that only cos phages from Gram-negative as well as Gram-positive bacteria encode HNH nucleases, consistent with the specific requirement for cos-site cleavage as opposed to pac-site cleavage, which produces blunt-ended products. did. The demonstration that HNH nuclease activity is required for some cos phages but not others suggests that there are differences between the TerL proteins of the two types of phages. That is, one can cleave both strands and the other requires a second protein to enable the generation of a double-stranded break.

The invention also includes the use of mobile genetic elements (MGEs) in certain configurations. That is, the following sections are provided. Any of the other configurations, aspects, embodiments, or descriptions of the invention above or elsewhere in this specification, mutatis mutandis, may be combined with any of these sections.
1. 1. A composition for use in antibacterial treatment of bacteria, said composition comprising an engineered mobile genetic element (MGE) capable of translocating in a first bacterial host cell of a first species or strain. wherein said cell comprises a first phage genome, in said cell said MGE translocates using a protein encoded by said phage to inhibit first replication, said MGE encodes an antimicrobial agent or compositions encoding components of such agents.

Alternatively, instead of bacteria, the host cells are archaea, and instead of phages, there are viruses capable of infecting archaea.

In one example, the MGE can integrate into the genome of the host cell, including the genome of the first phage, eg, into the chromosome of the host cell and/or its episome.

Optionally, MGE inhibits replication of the first phage.

In one example, replication of the first phage is completely inhibited. In one example, replication of the first phage is reduced by at least 50, 60, 70, 80, or 90% compared to replication in the absence of MGE in the host cell. This can be assessed by a standard in vitro plaque assay that determines the relative amount of primary phage plaque formation.

optionally in the presence of a drug
(i) the host cell is killed by the antimicrobial agent;
(ii) the growth or proliferation of the host cell is reduced and/or (iii) the host cell is sensitized to the antibiotic so that the antibiotic is toxic to the cell.
2. 2. The composition of clause 1, wherein said agent is toxic to cells of the same species or strain as said host cells.
3. 3. The composition of clause 1 or 2, wherein said agent is toxic to cells of a different species or strain than said host cell strain or species.
4. 2. The composition of clause 1, wherein said agent is toxic to cells of the same species as said host cell and said host cell is engineered such that said agent is not toxic to said host cell.
5. said agent is a guided nuclease system (optionally a CRISPR/Cas system), wherein a cell of the same species as said host cell comprises a target sequence to be cleaved by said nuclease, said target sequence being removed or 5. The composition of clause 4, which has been modified such that said nuclease is unable to cleave said target sequence.

Viruses undergo a lysogenic and lytic cycle in the host cell. If a lysogenic cycle is adopted, the phage chromosome can integrate into the bacterial chromosome or establish itself as a stable plasmid in the host, where it can remain dormant for long periods of time. . Once the lysogen is induced, the phage genome is excised from the bacterial chromosome and initiates a lytic cycle that leads to cell lysis and release of phage particles. The lytic cycle leads to the production of new phage particles, which are released by host lysis.
6. The composition of any preceding paragraph, wherein said first phage is a mild phage.
7. The composition of any preceding clause, wherein said first cell comprises said first phage as a prophage.
8. 6. The composition of any one of clauses 1-5, wherein said first phage is a lytic phage.
9. The composition of any preceding paragraph, wherein translocation of said MGE in the presence of the first phage causes host cell lysis.
10. The composition of any preceding paragraph, wherein said MGE can be packaged into a transducing particle comprising some, but not all, structural proteins of said first phage.

A "transduction particle" is a phage or particle smaller than a phage that is capable of transducing a nucleic acid or other DNA encoding an antibiotic or component thereof into a target bacterial cell.

In some instances, instead of a nucleic acid encoding an antibiotic or component thereof, the purpose is to encode a protein or RNA of interest for expression in a cell in which transduction from the transducing particle can occur. is used (eg, any NSI disclosed herein).

Examples of structural proteins include phage proteins selected from one, more, or all of major head-to-tail proteins, portal proteins, tail fiber proteins, and minor tail proteins.

The MGE packages the MGE (or at least its nucleic acid encoding an agent or one or more components thereof) into a transducing particle capable of infecting host cells of the same species or strain as the first host cell. It includes a packaging signal sequence operable with the protein encoded by the first phage to do so.
11. Transfer of said MGE packages a copy of said MGE or nucleic acid encoding said agent or component into a transducing particle capable of transferring said copy into target bacterial cells for antimicrobial treatment of said target cell. A composition according to any preceding clause, comprising:
12. 12. The composition of paragraphs 10 or 11, wherein said transducing particles are particles of a second phage capable of infecting cells of said first species or strain.
13. 13. The composition of any one of clauses 10-12, wherein said transducing particles are non-self-replicating particles.

A "non-self-replicating transducing particle" is capable of delivering a nucleic acid molecule encoding an antimicrobial agent or component (or any protein or RNA) to a bacterial cell, but transducing its own replicated genome. Refers to particles that do not package into particles (eg, phage or phage-like particles, or particles produced from genomic islands (eg, SaPI) or modified versions thereof). Alternatively herein, viruses that infect animal, human, plant, or yeast cells are used or packaged instead of phages. For example, adenovirus when the cells are human cells.
14. The composition of any preceding paragraph, wherein said MGE lacks genes encoding phage structural proteins.

Optionally, the MGE lacks one or more phage genes, rinA, terS and terL.

In one example, in a host cell, a protein complex comprising a small terminase protein (encoded by terS) and a large terminase protein (encoded by terL) is formed at or near the pac site (cos site contained in MGE). or other packaging signal sequences) can recognize and cleave MGE double-stranded DNA molecules, thereby allowing the MGE or plasmid DNA molecules to be packaged into phage capsids. When the first phage is induced as a prophage in the host cell, the phage lytic cycle produces the phage structural proteins and the phage large terminase protein. The MGE or plasmid is replicated and the small terminase protein encoded by the MGE or plasmid is expressed. Replicated MGE or plasmid DNA containing terS (and nucleotide sequences encoding antimicrobial agents or components) are packaged into phage capsids, resulting in non-self-replicating transduction particles carrying only MGE or plasmid DNA.
15. 14. The composition of any one of clauses 1-13, wherein said MGE comprises a phage structural gene and a packaging signal sequence, and said first phage lacks a packaging signal sequence.
16. The composition of any preceding clause, wherein said MGE is a modified version of MGE naturally found in bacterial cells of said first species or strain.
17. The composition of any preceding paragraph, wherein the MGE comprises modified genomic islands.

Optionally, the genomic island is an island naturally found in bacterial cells of the first species or strain. In one example, the genomic island is selected from the group consisting of SaPI, SaPI1, SaPI2, SaPIbov1, and SaPibov2 genomic islands.
18. The composition of any preceding paragraph, wherein said MGE comprises an altered virulence island.

Optionally, the pathogenicity island is an island naturally found in bacterial cells of the first species or strain, such as Staphylococcus SaPI or Vibro PLE or P. aeruginosa virulence island (eg, PAPI or PAGI, such as PAPI-1, PAGI-5, PAGI-6, PAGI-7, PAGI-8, PAGI-9, PAGI-10, or PAGI-).
19. 19. The composition of clause 18, wherein said pathogenicity island is SaPI (S aureus pathogenicity island).
20. 20. The composition of clause 19, wherein said first phage is φ11, 80α, φ12, or φSLT.

Staphylococcus phage 80α appears to recruit all known SaPIs. Thus, in one example, the MGE contains a modified SaPI and the first phage is 80α.
21. Said pathogenicity island is V . 19. The composition of paragraph 18, which is a B. cholerae PLE (phage-inducible chromosomal island-like element) and optionally said first phage is ICP1.
22. Said pathogenicity island is E. 19. The composition of clause 18, which is E. coli PLE.
23. 17. The composition of any one of clauses 1-16, wherein said MGE comprises P4 DNA, such as the P4 packaging signal sequence.
24. 24. The composition of paragraph 23, wherein said first phage is a P2 phage or a modified P2 phage deficient in self-replication, optionally present as a prophage.
25. The composition of any preceding paragraph, wherein said MGE comprises the pacA gene of Enterobacteriaceae bacteriophage P1.
26. The composition of any preceding clause, wherein said MGE comprises a packaging initiation site sequence, optionally a packaging initiation site sequence for P1.
27. any of the preceding paragraphs, wherein said MGE comprises a nucleotide sequence that is beneficial to cells of said first species or strain, optionally encoding a protein that is beneficial to cells of said first species or strain. The described composition.

Not only does the presence of MGE reduce replication of the first phage in the host cell, but the MGE is taken up to provide a survival, growth, or other advantage to the host cell, and the uptake of MGE by the host cell and/or Useful for promoting retention. In one example, the expression of the antimicrobial agent in the host cell is an inducible promoter or weak promoter that allows a period of favorable uptake of MGE into the host cell due to the presence of nucleotide sequences beneficial to cells of the first species or strain. Under the control of a promoter.
28. The composition of any preceding paragraph, wherein said MGE lacks rinA.
29. The composition of any preceding paragraph, wherein said MGE lacks terL.
30. The composition of any preceding clause, wherein said MGE comprises terS or a homologue thereof and optionally lacks any other terminase genes.

A terS homolog is a sequence that, like terS, recognizes a SaPI-specific pac site (or other packaging sequence) contained in an MGE or plasmid and determines packaging specificity for packaging MGE.

Examples of terminase genes are pacA, pacB, terA, terB, and terL.
31. The composition of any preceding paragraph, wherein said first phage is a pac-type phage (eg, φ11 or 80α) that is operational with pac contained in said MGE.
32. 31. The composition of any one of clauses 1-30, wherein said first phage is a cos-type phage (eg, φ12 or φSLT) operable with cos contained in said MGE.

Optionally, the phage is P2. Optionally, the first phage is a T7 or T7-like phage that recognizes direct repeat sequences contained in the MGE for packaging.
33. A composition according to any preceding clause, wherein said plasmid or MGE comprises pac and/or cos sequences, or homologues thereof.
34. The composition of any preceding clause, wherein said plasmid or MGE comprises terS or a homologue thereof and optionally lacks terL.

A terS homolog is a sequence that, like terS, recognizes a SaPI-specific pac site (or other packaging sequence) contained in an MGE or plasmid and determines packaging specificity for packaging MGE.

In one example, terS comprises the sequence of SEQ ID NO:1.
35. 35. The composition of paragraph 34, wherein said terS is S aureus bacteriophage φ80α terS or bacteriophage φ11 terS.
36. The composition of any preceding paragraph, wherein said MGE is a modified SaPIbov1 or SaPIbov5 and lacks terS.
37. to produce a transduction particle in which said first phage lacks a functional packaging signal sequence and said MGE packages a copy of said MGE or a copy of said agent or component-encoding nucleic acid; A composition according to any preceding paragraph, comprising a packaging signal sequence operable with a protein encoded by the phage.
38. The composition of any preceding clause, wherein said MGE or plasmid comprises a Ppi or homologue capable of complexing with the first phage TerS, thereby blocking the function of said TerS.
39. The composition of any preceding paragraph, wherein said MGE comprises morphogenic (cpm) modules.
40. The composition of any preceding paragraph, wherein said MGE comprises cpmA and/or cpmB.

Optionally cpmA and B are from any SaPI disclosed herein. In one example, the optional SaPI is a SaPI disclosed in Table 2 and optionally the host or target cell is any corresponding Staphylococcus disclosed in the Table.
41. The composition of any preceding paragraph, wherein said MGE or first phage comprises one, more than one or all genes cp1, cp2 and cp3.

In one example, the MGE contains a modified SaPI and contains one or more or all of the genes cp1, cp2, and cp3.
42. The composition of any preceding paragraph, wherein said MGE or first phage encodes an HNH nuclease.
43. The composition of any preceding clause, wherein said MGE or first phage comprises an integrase gene encoding an integrase for excising said MGE and integrating said MGE into the bacterial cell genome.
44. The composition of any preceding clause, wherein said MGE lacks a functional integrase gene and the first phage or host cell genome (eg, bacterial chromosome or bacterial episome) comprises a functional integrase gene.
45. The composition of any preceding clause, wherein transcription of said MGE nucleic acid is under the control of a constitutive promoter for transcription of copies of the agent or component in the host cell.

Optionally, constitutive transcription and production of agents in target cells can be used when killing target cells, eg, in a medical setting.

Optionally, transcription of the MGE nucleic acid is under the control of an inducible promoter for transcription of copies of the agent or component in the host cell. This would be useful, for example, to control the switch-on of antimicrobial activity against target bacterial cells in an environment (eg, soil or water) containing target cells, or in industrial culture or fermentation vessels, and the like. For example, target cells are useful in industrial processes (e.g. brewing or fermentation in the dairy industry) and induction allows the process to be controlled (e.g. stopped or reduced) by using antimicrobial agents against the target bacteria. can do.
46. 46. The composition of clause 45, wherein said promoter is exogenous to said host cell.
47. 47. The composition of paragraphs 45 or 46, wherein said promoter comprises a nucleotide sequence that is at least 80% identical to an endogenous promoter sequence of said host cell.
48. A composition according to any preceding paragraph comprising a nucleic acid separate from said MGE, wherein said nucleic acid comprises all genes necessary to produce a first phage particle.
49. 48. The composition of any one of clauses 1-47 comprising a nucleic acid separate from said MGE, wherein said nucleic acid comprises less than all genes required to produce a first phage particle, but said antimicrobial A composition comprising genes encoding structural proteins for the production of transduction particles that package an MGE nucleic acid encoding an agent or one or more components thereof.

If the agent comprises multiple components, for example, the agent is a CRISPR/Cas system, or encodes a CRISPR array or guide RNA (e.g., a single guide RNA) encoding crRNAs operable with Cas in the host cell If a nucleic acid, the crRNA or gRNA guides Cas to a target sequence in the host cell and modifies the target (eg, cleaves it or suppresses transcription from it).
50. 50. The composition of clauses 48 or 49, wherein said gene is contained in said host cell chromosome and/or one or more host cell episomes.
51. 51. The composition of clause 50, wherein said gene is contained in a prophage integrated into the chromosome of said first phage.
52. The composition of any preceding paragraph, wherein the agent is a guided nuclease system or a component thereof, and wherein the agent is capable of recognizing and cleaving host cell DNA (eg, chromosomal DNA).

In examples, such cleavage causes one or more of the following.
(i) host cells are killed by the antimicrobial agent;
(ii) host cell growth or amplification is reduced; and/or (iii) the host cell is sensitized to the antibiotic so that the antibiotic is toxic to the cell.
53. 53. The composition of clause 52, wherein said guided nuclease system is selected from a CRISPR/Cas system, a TALEN system, a meganuclease system, or a zinc finger system.
54. Said system is a CRISPR/Cas system, each MGE encoding (a) a CRISPR array encoding a crRNA or (b) a nucleic acid encoding a guide RNA (gRNA, e.g., a single guide RNA), wherein said crRNA or gRNA is operable with Cas in a target bacterial cell, and said crRNA or gRNA guides said Cas to a target nucleic acid sequence in said host cell and modifies said target sequence (e.g., cleaves it or prevents transcription from it). inhibit), a composition according to clause 52.

Optionally, the Cas is Cas encoded by a functional endogenous nucleic acid of the host cell. For example, the target is contained in the host cell's DNA or RNA.
55. 53. According to clause 52, wherein said system is a CRISPR/Cas system and each MGE encodes a Cas (e.g., a Cas nuclease) operable in a target bacterial cell to modify a target nucleic acid sequence contained in said target cell composition.
56. 56. The composition of paragraphs 53, 54, or 55, wherein said Cas is Cas3, Cas9, Casl3, CasX, CasY, or Cpf1.
57. 57. Any one of clauses 52-56, wherein said system is a CRISPR/Cas system and each MGE encodes one or more Cascade Cas (e.g., Cas, A, B, C, D, and E) composition.
58. 58. The composition of any one of clauses 52-57, wherein each MGE further encodes a Cas3 operable with said Cascade Cas in target bacterial cells.
59. The composition of any preceding clause, wherein said first species or strain is a Gram-positive species or strain.
60. 59. The composition of any one of clauses 1-58, wherein said first species or strain is a Gram-negative species or strain.
61. The composition of any preceding clause, wherein said first species or strain is selected from Table 1.

In one example, the first species or strain is a Staphylococcus (eg, Saureus) species or strain, optionally the MGE is a modified SaPI, optionally the first phage is φ80α or φ11. In one example, the first species or strain is a Vibrio (eg, V cholerae) species or strain and optionally the MGE is a Vibrio (eg, V cholerae) PLE.
62. The composition of any preceding paragraph, wherein said first species or strain is selected from Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas, and Enterobacter.

These are species that the P2 phage can infect. Thus, in one embodiment, the MGE comprises one or more P4 sequences (eg, the P4 packaging sequence) and the first phage is P2. That is, MGE is packaged by the P2 structural protein, and the resulting transduced particles infect two or more of a broad spectrum of species: Shigella, E. coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas, and Enterobacter. can be done.
63. A nucleic acid vector comprising an MGE integrated therein, wherein said MGE is from any of the clauses above, and wherein said vector is capable of transferring said MGE or a copy thereof into a host bacterial cell. .

Suitable vectors are plasmids (eg conjugative plasmids) or viruses (eg phage or packaged phagemids).
64. 64. The vector of clause 63, wherein said vector is a shuttle vector.

A shuttle vector is a vector (usually a plasmid) constructed so that it can be propagated in two different host species. Therefore, DNA inserted into the shuttle vector can be tested or manipulated in two different cell types.
65. 64. The vector of clause 63, wherein said vector is a plasmid and said plasmid is capable of transforming into a host bacterial cell containing the first phage.
66. A non-self-replicating transducing particle comprising said MGE or vector according to any preceding paragraph.

"Non-replicating" means that the MGE is incapable of self-replicating itself. For example, MGE lacks one or more nucleotide sequences that encode proteins (eg, structural proteins) necessary to produce transduction particles containing copies of MGE.
67. A composition comprising a plurality of transducing particles, each particle comprising an MGE or vector according to any one of clauses 1-65, wherein said transducing particle encodes said MGE or said agent or component the nucleic acid or a copy thereof can be transferred into the target bacterial cell;
a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
Composition.

In one example, the reduction in host cell growth or proliferation is at least 50, 60, 70, 80, 90, or 95%. The antibiotic agent can be any antibiotic agent disclosed herein.
68. wherein said agent is a guided nuclease system or a component thereof, said agent being capable of recognizing and cleaving host cell DNA (eg, chromosomal DNA), thereby a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
A composition according to clause 67.
69. A composition comprising a plurality of non-self-replicating transducing particles, each particle comprising an MGE or plasmid according to any one of clauses 1-65, wherein said transducing particle comprises said MGE or said agent or construct. Nucleic acids encoding elements or copies thereof can be introduced into target bacterial cells, wherein said agent is a CRISPR/Cas system, said elements guide said Cas to target nucleic acid sequences in said cells, and said sequences a nucleic acid encoding a crRNA or guide RNA operable with Cas in a target bacterial cell to modify a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
Composition.

In one example, the reduction in host cell growth or proliferation is at least 50, 60, 70, 80, 90, or 95%. The antibiotic agent can be any antibiotic agent disclosed herein.
70. A kit comprising the composition of clause 69 and said antibiotic.
71. 70. The composition of clause 69, wherein said composition comprises said antibiotic agent.
72. 70. The composition of any one of clauses 67-69, wherein less than 10% of the transducing particles contained in said composition are first phage particles.
73. 69. The composition of any one of clauses 67-69, wherein no first phage particle is present in said composition.
74. An MGE, vector, particle, composition or kit according to any preceding paragraph for medical use in a human or animal patient.
75. The MGE, vector, particle, composition, or kit of any preceding paragraph for treating or preventing infection by a target bacterial cell in a human or animal patient, wherein said antimicrobial agent is directed against said target cell MGE, vector, particle, composition or kit that is toxic to.
76. The MGE, vector, particle, composition, or kit of any preceding paragraph for treating or preventing infection by a target bacterial cell in a human or animal patient, wherein in the presence of said antimicrobial agent,
a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, and/or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
MGE, vector, particle, composition or kit.
77. Sections 1 to 62 of a method of producing a plurality of transducing particles, said first species host bacterial cell comprising a first phage, which allows for the introduction of a plurality of said MGEs into the host cell; , 67-69, and 71-76, wherein the protein encoded by the first phage is expressed and copies of MGE are packaged by the first phage protein to form a plurality of and optionally separating said transduced particles from said cells to obtain a plurality of transduced particles separated from said cells.
78. 78. The method of clause 77, comprising separating said transducing particles from any first phage, optionally by filtration or centrifugation, thereby obtaining a plurality of transducing particles in the absence of first phage. Method.
79. 79. The method of paragraphs 77 or 78, wherein said particle encodes a guided nuclease system (optionally a CRISPR/Cas system) or a component thereof for cleaving a target nucleic acid sequence contained in a target bacterial cell.
80. 80. The method of clause 79, wherein said sequence is included in an antibiotic resistance gene and said method comprises combining said plurality of particles with said antibiotic in a kit or mixture.
81. 81. The method of any one of clauses 77-80, wherein said conditions comprise induction of a lytic cycle of said first phage.
82. A bacterial host cell comprising a first phage and an MGE, vector, or particle as recited in any one of paragraphs 1-66, wherein said agent is toxic to cells of the same species as said host cell, said A bacterial host cell, wherein the host cell is engineered such that said agent is not toxic to said host cell.
83. a bacterial host cell comprising a first phage, said cell being included in a kit, said kit further comprising a composition recited in any one of paragraphs 1-62, 67-69, and 71-76; A bacterial host cell wherein said agent is toxic to cells of the same species as said host cell and said host cell is engineered such that said agent is not toxic to said host cell.
84. said agent is a guided nuclease system (optionally a CRISPR/Cas system), wherein a cell of the same species as said host cell comprises a target sequence to be cleaved by said nuclease, said target sequence being removed or 84. The cell of clause 83, which has been modified such that said nuclease is unable to cleave said target sequence.
85. A bacterial host cell comprising a first phage and an MGE, vector, or particle as recited in any one of paragraphs 1-66, wherein said agent is not toxic to said host cell, but said agent is said host cell is toxic to a second cell of a species or strain different from the species or strain of said MGE is capable of being produced by said host cell capable of transfecting said MGE or a copy thereof into said second cell A bacterial host cell that is mobile within the transduction particle, thereby exposing said second cell to said antimicrobial agent.
86. a bacterial host cell comprising a first phage, said cell being included in a kit, said kit further comprising a composition recited in any one of paragraphs 1-62, 67-69, and 71-76; wherein said agent is not toxic to said host cell, but said agent is toxic to a second cell of a different species or strain than said host cell species or strain, and said MGE kills said MGE or a copy thereof; A bacterial host cell that is mobile in a transducing particle that can be produced by said host cell that can be transfected into a second cell, thereby exposing said second cell to said antimicrobial agent.
87. 87. The cell of clause 86, wherein said first phage is a prophage.
88. A bacterial host cell comprising an MGE, vector, or particle as recited in any one of paragraphs 1 to 66, under the control of one or more inducible promoters, and a nucleic acid, wherein said nucleic acid is of said MGE or plasmid. a species or strain that encodes all structural proteins necessary to produce a transducing particle that packages a copy, wherein said agent is not toxic to said host cell, but said agent is different from said host cell species or strain toxic to a second cell of the strain and mobile in a transducing particle that said MGE can be produced by said host cell capable of transferring said MGE or a copy thereof into said second cell and wherein said second cell is exposed to said antimicrobial agent.
89. 89. The cell of clause 88, wherein said structural protein is a lytic phage structural protein.
90. 89. The cell of clauses 88 or 89, wherein said nucleic acid comprises terS and/or terL.
91. 91. any one of clauses 88-90, wherein said host cell and said second cell are cells of the same species and said host cell is engineered such that said antibiotic is not toxic to said host cell Cells as indicated.
92. 92. The cell of any one of clauses 88-91, wherein said nucleic acid is contained in a plasmid.
93. said agent is a guided nuclease system (optionally a CRISPR/Cas system), said second cell comprises a target sequence to be cleaved by said nuclease, said target sequence being present in said host cell's genome; 93. The cell of any one of clauses 88-92, wherein said nuclease is unable to cleave said host cell's genome.
94. The composition, vector, particle, kit of any preceding clause, wherein said cells, host cells or target cells are selected from Staphylococcal, Vibrio, Pseudomonas, Clostridium, E coli, Helicobacter, Klebsiella and Salmonella cells. , or how.
95. The plasmid
a. a nucleotide sequence encoding an antimicrobial agent or component thereof for expression in a target bacterial cell;
b. a constitutive promoter to control the expression of an agent or component;
c. an optional terS nucleotide sequence;
d. an origin of replication (ori), and e. a phage packaging sequence (optionally pac, cos, or homologues thereof), and f. a plasmid lacking
g. all nucleotide sequences encoding the phage structural proteins necessary for the production of transduction particles (optionally phage), or a plasmid lacking at least one such sequence, and h. optionally, terL
including.
96. The antibacterial agent is a CRISPR/Cas system, and the plasmid is a crRNA or guide RNA (a crRNA or guide RNA (such as a single gRNA), thereby
a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
A plasmid according to clause 95.
97. crRNA or guide RNA (e.g. a single gRNA) in the target cell such that the antimicrobial agent is the CRISPR/Cas system and the plasmid guides the Cas to a target nucleotide sequence to modify (e.g. cleave) the sequence code a Cas that can operate with
a. target cells are killed by the antimicrobial agent;
b. target cell growth or proliferation is reduced, or c. target cells are sensitized by an antibiotic whereby said antibiotic is toxic to said cells;
A plasmid according to paragraphs 95 or 96.
98. 98. The plasmid of clause 97, further encoding said crRNA or gRNA.
99. 99. A host cell comprising the plasmid of any one of clauses 95-98, wherein said host cell does not contain said target nucleotide sequence.
100. 100. The host cell of clause 99, wherein said cell is capable of replicating said plasmid and packaging said replicated plasmid into a transducing particle capable of infecting a target bacterial cell.
101. said host cell integrated into said cell chromosome and/or one or more episomes of said cell;
a. terL,
b. optionally terS, and c. comprising an expressible nucleotide sequence encoding all structural proteins necessary for the production of transduced particles that package a copy of said plasmid;
d. the chromosome (other than the plasmid) and episomes of said cell lack phage packaging sequences, and said phage packaging sequences contained in said plasmid are operable with said terS and terL products in the production of a packaged plasmid; is
A host cell according to clauses 99 or 100.
102. 102. The cell of clause 101, wherein nucleotide sequences encoding said terL, optionally terS, and said structural proteins are contained in a phage (optionally prophage) genome in said host cell.
103. A bacterial host cell comprising the genome of a helper phage incapable of self-replication, optionally present as a prophage, and a plasmid according to any one of paragraphs 95 to 98, wherein said helper phage is A bacterial host cell operable to package a copy of said plasmid, said particle being capable of infecting a bacterial target cell to which said antimicrobial agent is toxic.
104. said host cell is of a first species or strain, said target cell is of the same species or strain, and optionally said host cell is an engineered cell to which said antimicrobial agent is not toxic. , paragraph 103.
105. 104. The cell of clause 103, wherein said host cells are cells of a first species or strain, said target cells are cells of a different species or strain, and said antimicrobial agent is not toxic to said host cells.
106. A method of making a plurality of transduction particles, comprising culturing a plurality of host cells according to any one of paragraphs 103-105, optionally inducing a lytic cycle of said helper phage, and said plasmid and optionally separating said particles from said cells to obtain a plurality of transduced particles.
107. A plurality of transducing particles obtainable by the method of paragraph 106 are provided for use in medicine, e.g., for treating or preventing infection of a human or animal subject by target bacterial cells, wherein the transducing particles are and administered to a subject to kill the cells using an antibacterial agent.
108. A method of making a plurality of transducing particles, comprising:
i. Producing a host cell whose genome comprises nucleic acids encoding the structural proteins necessary to produce a transducing particle capable of packaging the first DNA, said genome carrying a phage packaging signal. lacking and expression of said protein is under the control of an inducible promoter;
ii. producing a first DNA encoding an antimicrobial agent or component thereof (e.g., as defined in any section above), said DNA comprising a phage packaging signal;
iii. introducing said DNA into said host cell;
iv. inducing the production of a structural protein in a host cell, whereby a transducing particle that packages said DNA is produced;
v. optionally isolating a plurality of said transducing particles; and vi. optionally comprising formulating said particles into a pharmaceutical composition for administration to humans or animals for medical use.
109. 109. The method of paragraph 108, wherein said DNA comprises an MGE as defined in any preceding paragraph.
110. 110. The method of paragraphs 108 or 109, wherein said structural protein is a P2 phage protein and optionally said packaging signal is a P4 phage packaging signal.
111. 110. The method of paragraphs 108 or 109, wherein said DNA comprises modified SaPI or genomic island DNA.
112. said cell in step (iv) comprises a gene encoding a helper phage activator, optionally said activator is P4 phage delta or ogr protein when said structural protein is P2 protein, or said MGE comprises a modified SaPI, said activator is SaPI rinA, ptiA, ptiB, or ptiM, optionally expression of said activator is controlled by an inducible promoter, such as the T7 promoter, paragraphs 108 to 111 The method according to any one of
113. 113. The method of any one of paragraphs 108-112, wherein said packaging signal is P4 phage Sid and/or psu, or said signal is SaPI cpmA and/or cpmB.

This is useful for packaging DNA into smaller capsids.
114. 114. The method of any one of clauses 108-113, wherein said cellular genome comprises prophages, each prophage comprising said nucleic acid encoding a structural protein.
115. said prophage is cos and optionally one or more selected from int, cox, orf78, B, orf80, orf81, orf82, orf83, A, orf91, tin, old, orf30, and fun(Z); or 115. The method of paragraph 114, wherein the P2 prophage lacks all genes and optionally said packaging signal of (ii) is a cos or P4 packaging signal.
116. 116. The method of paragraph 114 or 115, wherein said prophage is a P2 prophage that lacks cos and contains genes from Q to S, V to G, and F1 to _ogr .
117. Clause 114, wherein said prophage lacks a packaging signal and is a phi11 prophage comprising gene 29 (terS) to gene 53 (lysin), optionally in (ii) said packaging signal is a phi11 packaging signal The method described in .
118. A plurality of transduction particles obtainable by a method according to any one of paragraphs 108-117.
119. 119. The particle according to clause 118 for administration to humans or animals for medical use.

A further concept of the invention is as follows.

The invention is optionally for industrial or domestic use or is used in a method for such use. For example, this includes agriculture, oil or petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, Medical Industry, Healthcare Industry, Dental Industry, Energy Industry, Consumer Products Industry, Pharmaceutical Industry, Mining Industry, Cleaning Industry, Forestry Industry, Fisheries Industry, Leisure Industry, Recycling Industry, Cosmetics Industry, Plastics Industry, Pulp or Paper Industry, Textiles for or used in industry, the clothing industry, the leather or suede or animal leather industry, the tobacco industry, or the steel industry.

The invention is optionally for use in industry, or the environment is an industrial environment, and the industries are medical and healthcare, pharmaceuticals, human food, feed, plant fertilizers, beverages, dairy, meat processing, agriculture. , Animal Husbandry, Poultry Farming, Seafood Industry, Veterinary Medicine, Oil, Gas, Petrochemicals, Water Treatment, Sewage Treatment, Packaging, Electronics and Computers, Personal Health Care and Toiletries, Cosmetics, Dentistry, Non-Medical Dentistry, Ophthalmology, Non- Medical Ophthalmology, Mining and Mineral Processing, Metal Mining and Metalworking, Quarrying, Aviation, Automotive, Rail, Transportation, Space, Environment, Soil Treatment, Pulp and Paper, Clothing Manufacturing, Dyes, Printing, Adhesives, Air Conditioning, Solvents , biodefense, vitamin supplementation, cryopreservation, textile soaking and production, biotechnology, chemicals, industrial cleaning products, household cleaning products, soaps and detergents, consumer products, forestry, fisheries, leisure, recycling, plastics , Leather, Leather and Suede, Waste Disposal, Funeral and Contracting, Fuels, Construction, Energy, Steel, and the Tobacco Industry.

In one example, the first DNA, first phage, or vector comprises a CRISPR array targeting the target bacterium, wherein the array comprises one, two or more spacers to target the genome of the target bacterium. (eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers).

In one example, target bacteria are included in the environment as follows. In one example, the environment is the human microbiome, such as the oral or gut microbiome, or the bloodstream. In one example, the environment is not the environment in or on humans. In one example, the environment is not an environment in or on a non-human animal. In one embodiment, the environment is an air environment. In one embodiment, the environment is an agricultural environment. In one embodiment, the environment is an oil or petroleum recovery environment, such as an oil or petroleum field or well. In one example, the environment is the environment in or on food or beverages for consumption by humans or non-human animals.

In one example, the environment is a human or animal microbiome (eg, gut, vaginal, scalp, axillary, skin, or oral microbiomes). In one example, the target bacteria are contained in the human or animal microbiome (eg, the gut, vaginal, scalp, axillary, skin, or oral microbiome).

In one example, a DNA, phage, or composition of the invention is or is for administration intranasally, topically, or orally to a human or non-human animal. Those skilled in the art wishing to treat human or animal microbiomes can determine the best route of administration depending on the microbiome of interest. For example, if the microbiome is the gut microbiome, administration may be nasal or oral. Where the microbiome is the scalp or axillary microbiome, administration may be topical. If the microbiome is mouth or pharynx, administration may be oral.

For any use or method herein, in one embodiment, the first phage 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, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 , 200, 300, 400, 500, 600, or 700 multiplicity of infection (MOI). For example, the MOI is 20-200, 20-100, 50-200, 50-100, 75-150, 100 or about 100, or 200 or about 200. In one example, this involves taking a sample of the microbiome containing the target bacteria (e.g., a sample of a subject's waterway or gut microbiome), determining the number of CFU/ml or mg in the sample, and using this to It can be determined by titrating the dose of phage at the desired MOI to be exposed to the biome or administered to the environment or subject to be treated.

In one example, the environment includes drinking or water (eg, waterways or drinking water for human consumption) or soil. The water is optionally in heating, cooling, or industrial systems, or in potable water storage containers.

一例では、宿主および／または標的細菌はＡｎａｅｒｏｔｒｕｎｃｕｓ、Ａｃｅｔａｎａｅｒｏｂａｃｔｅｒｉｕｍ、Ａｃｅｔｉｔｏｍａｃｕｌｕｍ、Ａｃｅｔｉｖｉｂｒｉｏ、Ａｎａｅｒｏｃｏｃｃｕｓ、Ａｎａｅｒｏｆｉｌｕｍ、Ａｎａｅｒｏｓｉｎｕｓ、Ａｎａｅｒｏｓｔｉｐｅｓ、Ａｎａｅｒｏｖｏｒａｘ、Ｂｕｔｙｒｉｖｉｂｒｉｏ、Ｃｌｏｓｔｒｉｄｉｕｍ、Ｃａｐｒａｃｏｃｃｕｓ、Ｄｅｈａｌｏｂａｃｔｅｒ、Ｄｉａｌｉｓｔｅｒ、Ｄｏｒｅａ、Ｅｎｔｅｒｏｃｏｃｃｕｓ、Ｅｔｈａｎｏｌｉｇｅｎｅｎｓ、Ｆａｅｃａｌｉｂａｃｔｅｒｉｕｍ、Ｆｕｓｏｂａｃｔｅｒｉｕｍ、Ｇｒａｃｉｌｉｂａｃｔｅｒ、 Ｇｕｇｇｅｎｈｅｉｍｅｌｌａ、Ｈｅｓｐｅｌｌｉａ、Ｌａｃｈｎｏｂａｃｔｅｒｉｕｍ、Ｌａｃｈｎｏｓｐｉｒａ、Ｌａｃｔｏｂａｃｉｌｌｕｓ、Ｌｅｕｃｏｎｏｓｔｏｃ、Ｍｅｇａｍｏｎａｓ、Ｍｏｒｙｅｌｌａ、Ｍｉｔｓｕｏｋｅｌｌａ、Ｏｒｉｂａｃｔｅｒｉｕｍ、Ｏｘｏｂａｃｔｅｒ、Ｐａｐｉｌｌｉｂａｃｔｅｒ、Ｐｒｏｐｒｉｏｎｉｓｐｉｒａ、Ｐｓｅｕｄｏｂｕｔｙｒｉｖｉｂｒｉｏ、Ｐｓｅｕｄｏｒａｍｉｂａｃｔｅｒ、Ｒｏｓｅｂｕｒｉａ、Ｒｕｍｉｎｏｃｏｃｃｕｓ、Ｓａｒｃｉｎａ、Ｓｅｉｎｏｎｅｌｌａ、Ｓｈｕｔｔｌｅｗｏｒｔｈｉａ、Ｓｐｏｒｏｂａｃｔｅｒ、Ｓｐｏｒｏｂａｃｔｅｒｉｕｍ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ、Ｓｕｂｄｏｌｉｇｒａｎｕｌｕｍ、Ｓｙｎｔｒｏｐｈｏｃｏｃｃｕｓ、 Firmicutes selected from Thermobacillus, Turibacter, and Weisella.

In one example, the kit, DNA, first phage, helper phage, composition, use, or method is used to reduce pathogenic infection or to rebalance gut or oral microbial communities, e.g., humans or animals. to treat or prevent obesity or disease in For example, the first phage, helper phage, composition, use, or method is for knocking down Clostridium difficile or E coli bacteria in a human or animal gut microbiome.

In one example, the packaging signal, NPF, and/or HPF consists of or includes SEQ ID NO:2 or a structural or functional homologue thereof.

In one example, the packaging signal, NPF, and/or HPF are SEQ ID NO: 2 or at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 Consisting of or comprising nucleotide sequences that are % identical.

In one example, the disease or condition is a cancer, inflammatory or autoimmune disease or condition such as obesity, diabetes, IBD, GI tract conditions or oral cavity conditions.

Optionally, the environment or the target bacterium is a gut, skin, oral, throat, hair, axillary, vaginal, rectal, anal, or ocular flora. , nasal microorganisms, tongue microorganisms, lung microorganisms, liver microorganisms, kidney microorganisms, genital microorganisms, penis microorganisms, scrotal microorganisms, mammary gland microorganisms, ear microorganisms, urethral microorganisms, labial microorganisms, Included in the organ microbial group, or the dental microbial group. Optionally, the environment, or the target bacterium, is in a plant (e.g., tobacco, crop, fruit tree, vegetable, or tobacco, e.g., on or contained in a plant) or environment (e.g., soil or water or waterways or aqueous fluids). included.

Optionally, the human or animal subject disease or condition is
(a) a neurodegenerative disease or condition;
(b) a disease or condition of the brain;
(c) a CNS disease or condition;
(d) memory loss or damage;
(e) a heart or cardiovascular disease or condition, such as a heart attack, stroke, or atrial fibrillation;
(f) a disease or condition of the liver;
(g) kidney diseases or conditions, such as chronic kidney disease (CKD);
(h) a disease or condition of the pancreas;
(i) pulmonary diseases or conditions, such as cystic fibrosis or COPD;
(j) gastrointestinal disease or condition;
(k) diseases or conditions of the throat or oral cavity;
(l) diseases or conditions of the eye;
(m) diseases or conditions of the reproductive organs, such as diseases or conditions of the vagina, labia, penis, or scrotum;
(n) sexually transmitted diseases or conditions, such as gonorrhea, HIV infection, syphilis, or chlamydia infection;
(o) diseases or conditions of the ear;
(p) skin diseases or conditions;
(q) cardiac disease or condition;
(r) nasal diseases or conditions;
(s) blood diseases or conditions, such as anemia, such as anemia of chronic disease or cancer;
(t) viral infection;
(u) pathogenic bacterial infections;
(v) cancer,
(w) an autoimmune disease or condition, such as SLE;
(x) an inflammatory disease or condition such as rheumatoid arthritis, psoriasis, dermatitis, asthma, ulcerative 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. tumor or ocular),
(jj) common cold,
(kk) epilepsy;
(ll) fibrosis, such as liver or lung fibrosis;
(mm) fungal diseases or conditions such as thrush,
(nn) metabolic diseases or conditions such as obesity, anorexia, diabetes, type I or type II diabetes,
(oo) ulcers, such as gastric or skin ulcers,
(pp) dry skin;
(qq) Sjögren's syndrome;
(rr) cytokine storm,
(ss) deafness, loss or impairment of hearing;
(tt) slowed or accelerated metabolism (i.e. slowed or accelerated above average for the subject's weight, sex, and age)
(uu) fertility disorders such as infertility or subfertility;
(vv) jaundice,
(ww) skin rashes,
(xx) Kawasaki disease,
(yy) Lyme disease,
(zz) allergies, such as allergies to nuts, grasses, pollen, dust mites, cats, or dog fur or 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's disease, or hemophilia;
(ooo) shingles or herpes,
(ppp) menstruation,
(qqq) selected from decreased sperm count;

In one example, the neurodegenerative or CNS disease or condition for treatment or prevention according to the present invention is Alzheimer's disease, geriatric psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetes is selected from the group consisting of neuropathy, Parkinson's syndrome, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinson's syndrome.

In examples where the methods of the invention are practiced on a human or animal subject to treat a CNS or neurodegenerative disease or condition, the methods cause downregulation of Treg cells in the subject, thereby causing systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus and into the brain of a subject, thereby treating, preventing, or slowing the progression of a disease or condition (e.g., Alzheimer's disease). Reduce. In one embodiment, the method elicits an increase in IFN-gamma in the subject's CNS system (eg, in the brain and/or CSF). In one example, the method restores nerve fibers and/or reduces progression of nerve fiber damage. In one example, the method restores nerve myelin and/or reduces progression of nerve myelin damage. In one example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method can be used in conjunction with any method disclosed in WO2015136541 (disclosure of this document, e.g. Such methods, diseases, conditions, and potential therapeutic agents that can be administered to a subject to effect treatment and/or prevention of CNS and neurodegenerative diseases and conditions, such as immune checkpoint inhibitors, such as anti-PD. -1, anti-PD-L1, anti-TIM3, or other antibodies disclosed herein, which are hereby incorporated by reference in their entirety).

Cancers for Treatment or Prevention by the Present Methods Cancers that may be treated include non-vascularized or substantially non-vascularized tumors as well as vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors. Cancer types to be treated by the present invention include carcinoma, blastoma, and sarcoma, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant diseases such as sarcoma, carcinoma, and melanoma. including but not limited to: Also included are adult tumors/cancers and pediatric tumors/cancers.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematopoietic) cancers include acute leukemia (acute lymphocytic leukemia, acute myelogenous leukemia, acute myelogenous leukemia, and myeloblasts, promyelocytic, myelomonocytic, monocyte, and erythroleukemia), chronic leukemia (including chronic myelogenous (granulocytic) leukemia, chronic myelogenic leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma ( inactive and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

A solid tumor is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Various types of solid tumors derive their names from the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma tumor, colon cancer, lymphatic malignancy, 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, thyroid Papillary carcinoma, pheochromocytic adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminiferous tumor, Bladder cancer, melanoma, and CNS tumors such as glioma (including brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma , CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngiogioma, ependymoma, pinealoma, hemangioblastoma, auditory neuroma, oligodendroglioma, meninges menangioma, neuroblastoma, retinoblastoma, and brain metastases).

AUTOIMMUNE DISEASE FOR TREATMENT OR PREVENTION BY THE METHODS 1. Acute disseminated encephalomyelitis (ADEM)
2. Acute necrotizing hemorrhagic leukoencephalitis3. Addison's disease4. Agammaglobulinemia5. alopecia areata6. amyloidosis7. Ankylosing myelitis8. Anti-GBM/anti-TBM nephritis9. antiphospholipid syndrome (APS)
10. Autoimmune angioedema11. Autoimmune aplastic anemia 12. Autoimmune autonomic neuropathy 13. autoimmune hepatitis 14. Autoimmune hyperlipidemia15. Autoimmune immunodeficiency 16. Autoimmune inner ear disease (AIED)
17. Autoimmune myocarditis 18. Autoimmune oophoritis19. Autoimmune pancreatitis20. Autoimmune retinitis 21. Autoimmune thrombocytopenic purpura (ATP)
22. Autoimmune thyroid disease23. Autoimmune urticaria24. Axonal and neuronal neuropathy25. Barrow's disease26. Behcet's disease27. Bullous pemphigoid28. Myocardial myopathy29. Castleman's disease30. celiac disease31. Chagas disease32. Chronic fatigue syndrome33. Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)
34. Chronic Relapsing Osteomyelitis (CRMO)
35. Churg-Strauss Syndrome36. Cicatricial pemphigoid/benign mucosal pemphigoid37. Crohn's disease 38. Cogans Syndrome39. Cold agglutinin disease 40. Congenital heart block 41. Coxsackie myocarditis 42. CREST disease 43. Mixed essential cryoglobulinemia 44 . Demyelinating neuropathy 45. Dermatitis herpetiformis 46. Dermatomyositis 47. Devitch's disease (neuromyelitis optica)
48. Discoid Loops 49. Dressler syndrome50. Endometriosis51. Eosinophilic esophagitis52. Eosinophilic fasciitis53. Erythema nodosum 54 . Experimental allergic encephalomyelitis55. Evans Syndrome56. fibromyalgia57. fibrosing alveolitis58. Giant cell arteritis (temporal arteritis)
59. Giant cell myocarditis60. Glomerulonephritis61. Goodpasture syndrome62. Endoblastomatosis with polyangiitis (GPA) (previously called Wegener's endoblastomatosis)
63. Graves' disease64. Guillain-Barré Syndrome65. Hashimoto's encephalitis66. Hashimoto's thyroiditis67. Hemolytic anemia68. Henoch-Schoenlein purpura69. gestational herpes70. Hypogammaglobulinemia71. Idiopathic thrombocytopenic purpura (ITP)
72. IgA nephropathy73. IgG4-associated sclerosing disease74. immunomodulatory lipoproteins 75. Inclusion body myositis 76 . Interstitial cystitis77. Juvenile arthritis78. juvenile diabetes (type 1 diabetes)
79. Juvenile myositis 80. Kawasaki syndrome81. Lambert-Eaton syndrome82. Leukocytoclastic vasculitis 83 . Lichen planus84. lichen sclerosus85. Woody conjunctivitis 86. Linear IgA disease (LAD)
87. Lupus (SLE)
88. Lyme disease, chronic 89. Meniere's disease90. Microscopic polyangiitis 91. mixed connective tissue disease (MCTD)
92. Mollen's ulcer 93. Mukka Habermann disease94. multiple sclerosis 95. Myasthenia gravis 96. Myositis 97. Narcolepsy 98. Neuromyelitis optica (Debitch's)
99. Neutropenia 100. Ocular cicatricial pemphigoid 101. Optic neuritis 102. Relapsing rheumatoid arthritis 103. PANDAS (Streptococcus Associated Pediatric Autoimmune Neuropsychiatric Disorder)
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. Types I, II, and III autoimmune polyglandular syndrome 116. Polymyalgia rheumatoid arthritis 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. Plasmodium aplasia 128. Raynaud's phenomenon 129. Reactive arthritis 130. Reflex sympathetic dystrophy 131. Reiter Syndrome 132. Relapsing Polychondritis 133. Restless leg syndrome 134. Retroperitoneal fibrosis 135. Rheumatic fever 136. Rheumatoid arthritis 137. sarcoidosis138. 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. Susak 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. Type 1 diabetes 153. Ulcerative colitis 154. Undifferentiated Connective Tissue Disease (UCTD)
155. Uveitis 156. Vasculitis 157. Vesicular bullous dermatosis 158. Vitiligo 159. Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA))

Inflammatory diseases for treatment or prevention by this method 1. Alzheimer's disease2. Ankylosing spondylitis3. Arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis)
4. Asthma5. Atherosclerosis6. Crohn's disease7. colitis8. 9. Dermatitis Diverticulitis10. fibromyalgia11. Hepatitis 12. irritable bowel syndrome (IBS)
13. Systemic lupus erythematosus (SLE)
14. Nephritis 15. Parkinson's disease16. Ulcerative colitis

Paragraphs By way of example, the invention provides the following paragraphs (which may optionally be combined with any of the above disclosures).
1. An antimicrobial composition comprising a population of first phages, wherein said first phages require helper phages for replication of first phage particles, said helper phages packaging first phage nucleic acids to produce a first phage particle, wherein said first phage differs from said helper phage, said helper phage is incapable of producing helper phage particles by itself, and said first phage is An antimicrobial composition capable of infecting a target bacterium, wherein each first phage comprises antimicrobial means for killing the bacterium.
2. The composition of paragraph 1, wherein at least 95% of the phage particles contained in said composition are first phage particles.

Optionally, the composition includes helper phage. Optionally, the composition comprises no more than 1 helper phage particle per 1×10 ⁶ phage particles or more. Optionally, the composition comprises no more than 1 helper phage particle per 1×10 ⁸ phage particles or more. Optionally, the composition comprises no more than 1 helper phage particle per 1×10 ⁹ phage particles or more. Optionally, the composition comprises no more than 1 helper phage particle per 1×10 ¹⁰ phage particles or more.
3. each first phage comprises one or more components of the CRISPR/Cas system, said components encoding or producing a guide RNA (optionally a single guide RNA) 3. The composition of paragraph 1 or 2, wherein said guide RNA is capable of targeting said genome of a target bacterium.
4. A composition according to any preceding paragraph, wherein each first phage genome lacks a genome encoding a phage protein.
5. A composition according to any preceding paragraph for use in antibacterial treatment of bacteria, wherein said composition is engineered capable of translocating in a first bacterial host cell of a first species or strain. a mobile genetic element (MGE), wherein the cell contains a helper phage genome, and in the cell the MGE is translocated using proteins encoded by the helper phage to inhibit helper phage replication; Said MGE encodes an antibacterial agent or encodes a component of such agent, said MGE includes modified genomic island, modified virulence island, SaPI (Saureus virulence island), V cholerae PLE ( phage-like inducible chromosomal island-like elements), or E coli PLE.
6. (a) a first DNA, and (b) one or more second DNAs
A kit comprising
(i) said DNAs together contain all the phage structural protein genes necessary to produce a packaged phage particle containing a copy of said first DNA;
(ii) said first DNA comprises none, or at least one, but not all of said genes, and said one or more second DNAs comprise the remainder of said genes;
(iii) said first DNA comprises a phage packaging signal for producing said packaged phage particle;
(iv) said second DNA lacks a nucleotide sequence necessary for packaging said second DNA into a phage particle;
operable to produce a packaged phage (first phage) comprising said first DNA when said DNA coexists in a host bacterium, said first phage containing said second DNA to produce additional first phage particles.
7. said phage particle of (i) is capable of infecting a target bacterium, said phage comprises a nucleotide sequence of interest (NSI) that enables said phage to express a protein or RNA in said target bacterium; 7. The kit of paragraph 6, wherein the presence of the NSI-encoded protein or RNA mediates target cell death or downregulation of target cell growth or proliferation.
8. Said phage particle of (i) is capable of infecting a target bacterium and said phage comprises a nucleotide sequence of interest (NSI) that enables said protein or RNA to be expressed in said target bacterium, or said NSI comprises said 7. A kit according to paragraph 6, comprising control elements operable in the target bacterium.
9. the presence of said NSI or the protein or RNA encoded thereby in said target bacterium mediates target cell death or down-regulation of target cell growth or proliferation, or is encoded by said target cell's genome. 9. A kit according to paragraph 8, which mediates switching off or down-regulating expression of one or more RNAs or proteins.
10. The presence of said NSI or protein or RNA encoded thereby in said target bacterium mediates upregulation of growth or proliferation of said target cell or one or more 9. A kit according to paragraph 8, which mediates the switch-on of RNA or protein expression or upregulation thereof.
11. 10. The kit of paragraphs 7, 8, or 9, wherein said NSI encodes a component of the CRISPR/Cas system that is toxic to said target bacterium.
12. The kit of paragraphs 7, 8, or 9, wherein said NSI comprises engineered antimicrobial means for killing target bacteria.
13. 13. The kit of any one of paragraphs 6-12, wherein the packaged phage particle genome lacks genes encoding phage proteins.
14. 14. The kit of any one of paragraphs 6-13, wherein said first DNA does not contain any of said structural protein genes.
15. 15. The kit of any one of paragraphs 6-14, wherein said second DNA lacks a phage packaging signal.
16. 16. A kit according to any one of paragraphs 6 to 15, wherein each signal is a pac or cos sequence or a homologue thereof.
17. any of paragraphs 6 to 16, wherein each signal comprises SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98, or 99% identical thereto, or is a homolog from a different species or the kit described in 1.
18. An isolated DNA that is the first DNA as defined in any one of paragraphs 6-17.
19. 19. The kit or DNA of any one of paragraphs 6-18, wherein said first DNA is contained in a vector (optionally a plasmid, phagemid or shuttle vector).
20. Paragraph 6, wherein said second DNA is contained in a vector (optionally a plasmid, phagemid, or shuttle vector), helper phage (optionally helper phagemid), or integrated into said genome of a host bacterial cell; 20. The kit or DNA of any one of 19 to 19.
21. A host bacterial cell comprising said first and second DNAs as defined in any one of paragraphs 6 to 17, 19 and 20, or comprising the DNA according to paragraphs 18, 19 or 20.
22. 22. A method of producing a phage composition, said method comprising expressing said phage protein in the cell of paragraph 21, wherein packaged first phage particles comprising said first DNA are produced; said first phage requires said second DNA to replicate said second DNA to produce further first phage, said method optionally producing an amount of first phage from cellular material; A method comprising separating, wherein a quantity of purified phage is obtained.

In one example, purified phage are mixed with a pharmaceutically acceptable excipient, carrier, or diluent (eg, an aqueous liquid or water) to produce a pharmaceutical composition.

In one example, any composition or kit of the invention is combined with a label or instructions for use in treating and/or preventing a disease or condition in humans, optionally the label or instructions include a marketing authorization number (e.g. FDA or EMA clearance number) and optionally the kit includes an injection pen or IV container containing the first DNA or first phage.
23. 23. The method of paragraph 22, comprising isolating said first phage particle.
24. A composition comprising a first population of phage particles obtainable by a method according to paragraphs 22 or 23 (eg an antimicrobial composition, eg for medical use).

In one example, the first phage particle is obtained by the above method.

In one example, any composition of the invention comprises at least 1×10 ³ first phage per ml or mg, eg, when the composition is contained in a fluid (eg, liquid) or solid. In one example, any composition of the invention contains at least 1×10 ⁴ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ⁵ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ⁶ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ⁷ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ⁸ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ⁹ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ¹⁰ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ¹¹ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ¹² first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ¹³ first phage per ml or mg. In one example, any composition of the invention contains at least 1×10 ¹⁴ first phage per ml or mg.

In one example, any composition of the invention contains up to 1×10 ¹⁴ first phage per ml or mg, eg, when the composition is contained in a fluid (eg, liquid) or solid. In one example, any composition of the invention contains up to 1×10 ¹³ first phage per ml or mg. In one example, any composition of the invention contains up to 1×10 ¹² primary phage per ml or mg. In one example, any composition of the invention contains up to 1×10 ¹¹ primary phage per ml or mg. In one example, any composition of the invention contains up to 1×10 ¹⁰ first phage per ml or mg. In one example, any composition of the invention contains up to 1×10 ⁹ first phage per ml or mg.

In one example, any composition of the invention contains at least 1×10 ³ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 11 , per ml or mg, for example when the composition is contained in a fluid (eg, liquid) or solid. Contains 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first phage. In one example, any composition of the invention has at least 1×10 ⁴ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first of phages. In one example, any composition of the invention has at least 1×10 ⁵ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first of phages. In one example, any composition of the invention has at least 1×10 ⁶ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first of phages. In one example, any composition of the invention has at least 1×10 ⁷ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first of phages. In one example, any composition of the invention has at least 1×10 ⁸ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ¹⁰ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ¹¹ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ¹² to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ¹³ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages. In one example, any composition of the invention has at least 1×10 ¹⁴ to 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ , or 1×10 ¹⁴ first per ml or mg. of phages.

In one example, a composition comprises one or more doses of a first phage for administration to a subject for medical use, eg, to treat or prevent a disease or condition in a subject. In one example, the composition contains a single dose. In one example, the compositions are , 24, 25, 26, 27, 28, 29, or 30 doses. In one example, each dose comprises (or at least) 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200, or a dose of 250 mg or ml (ie, doses are the amounts described above, including phage and, for example, excipients, diluents, or carriers).

In one example, the composition includes one or more doses of a first phage for administration to a subject for non-medical use, eg, for agricultural use. In one example, the composition contains a single dose. In one example, the compositions are , 24, 25, 26, 27, 28, 29, or 30 doses. In one example, each dose comprises (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, 50000, 100000 mg or ml, i. including). The dose may be dissolved or diluted in a solvent (eg, aqueous solvent or water) prior to use to contact the target bacteria. In one example, one imperial gallon contains one dose of the first phage, for example for agricultural use, such as spraying crops, or for animal or livestock use, such as for use as a beverage.
25. 24. The method of paragraph 22 or 23 or the composition of paragraph 24, wherein said second DNA is comprised in helper phage DNA and wherein less than 5% of all phage particles comprised in said composition are helper phage particles.
26. 25. The method of any one of paragraphs 22-24, wherein said second DNA is comprised of helper phage DNA and said composition comprises no more than 1 helper phage particle per 1 x ¹⁰⁶ phage particles or more. Or composition.

This was demonstrated in Example 3 and shown in Example 4 to be effective in killing target bacteria. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁷ or more phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁸ or more phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁹ or more phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ¹⁰ or more phage particles.

In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁶ phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁷ phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁸ phage particles. In one embodiment, the composition comprises no more than 1 helper phage particle per 1×10 ⁹ phage particles.

In one embodiment, the composition comprises only 1 helper phage particle per 1×10 ⁶ to 1×10 ⁹ phage particles. In one embodiment, the composition comprises only 1 helper phage particle per 1×10 ⁶ to 1×10 ⁸ phage particles. In one embodiment, the composition comprises only 1 helper phage particle per 1×10 ⁶ to 1×10 ⁷ phage particles. In one embodiment, the composition comprises only 1 helper phage particle per 1×10 ⁷ to 1×10 ⁹ phage particles. In one embodiment, the composition comprises only 1 helper phage particle per 1×10 ⁷ to 1×10 ⁸ phage particles.

In one example, the percentage of helper phage is determined as plaque-forming units (PFU), eg, PFU/ml of phage composition comprising said number of phage particles. In an example, the percentage of non-helper phage (ie, first phage) is determined as the number of transduced units (TFU) of the target bacteria, eg, TFU/ml of phage composition. PFU is determined in a lawn of susceptible indicator bacteria, while TFU is sensitive in phage compositions that ensure an excess of indicator cells (i.e. at low multiplicity of infection (MOI < 0.01)). is determined in a transduction assay in which a culture of certain indicator bacteria is infected, and the number of transduced cells is determined by plating onto selective plates.

That is, the composition may contain one or more helper phage particles. But the level of helper particles is very low. This is beneficial (and thus useful as a pharmaceutical, for example) because the composition is relatively pure. This is also useful because the first phage has a very low chance of replicating, and offers the advantage of controlling phage dosing, phage containment (e.g., in humans or animals or in the environment), and The lack of replication reduces the chances of acquisition of unwanted genes (eg, antibiotic resistance genes) by the phage.
27. Each first phage particle contains a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in a target bacterium, and the presence of the protein or RNA encoded by said NSI in said target bacterium determines the presence of said target cell. or down-regulation of target cell growth or proliferation. Or the composition according to 26.
28. 28. The method or composition of paragraph 27, wherein said NSI encodes a component of the CRISPR/Cas system that is toxic to target cells.
29. wherein said component is (i) a DNA sequence encoding a guide RNA (e.g. a single guide RNA) or comprising a CRISPR array for producing a guide RNA, wherein said guide RNA comprises said genome of a target bacterium; 29. A method according to paragraph 28, comprising the targetable DNA sequence, (ii) a DNA sequence encoding a Cas nuclease, and/or (iii) a DNA sequence encoding one or more components of Cascade. Or composition.
30. The NSI encodes the Cas nuclease and guide RNA of the system (or the NSI encodes the Cas nuclease and a CRISPR array for producing the guide RNA), the guide RNA targeting the genome of the target bacterium. 30. The method or composition of paragraph 29, wherein said guide RNA is capable of guiding said Cas in target cells to mediate target cell death or downregulation of target cell growth or proliferation.

Optionally, the NSI encodes Cas9 and tracrRNA and CRISPR arrays for producing guide RNA.
31. The method or composition of any one of paragraphs 1-5 and 22-30, wherein said first phage particle does not contain a phage structural protein gene.
32. The kit, DNA, method, or composition of any preceding paragraph, wherein said first DNA or first phage DNA is contained in a high copy number plasmid.

Optionally, the first DNA or first phage DNA is contained in a medium copy number plasmid.

The meanings of low, medium, and high copy number ori and plasmid are known to those skilled in the art and are technical terms. As known to those skilled in the art, copy number refers to the average number of plasmid copies per cell. For example, a low copy number plasmid is a plasmid that is present at 1-10 copies per bacterial cell in which the plasmid is contained. Medium copy number plasmids are present at 11-50 (eg 11-40 or 20-30 or 40) copies per cell and high copy numbers are >50 (eg 100, 200, 250, 300, 400, 500) copies per cell. , 600, or 700) copies. In one example, the plasmid or vector containing the first DNA is a medium copy number plasmid or vector. In one example, the plasmid or vector containing the first DNA is a high copy number plasmid or vector. Examples of common ori and plasmids are shown in Table 7.
33. 33. The method or composition of any one of paragraphs 28-32 when dependent on paragraph 27 or paragraph 27, wherein said NSI comprises an engineered antimicrobial means for killing the target bacteria.
34. 34. A method or composition according to paragraph 33, wherein said antimicrobial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.
35. A composition according to any one of paragraphs 1-5 and 24-34 for administration to a human or animal subject for medical use.
36. 35. The composition of any one of paragraphs 1-5 and 24-34 for administration to a human or animal subject to treat infection of a target bacterial cell, wherein said first phage is A composition capable of infecting and killing cells, optionally wherein said infection is an infection of the intestinal microbiome.

Optionally, the target cell is an E coli cell and the first DNA is contained in a high copy number plasmid.

In one example, the gut microbiome is the upper GI tract microbiome. In one example, the target cells are contained in the subject's upper GI tract. In one example, the first phage is delivered to the subject's upper GI tract.

In one example, the gut microbiome is the stomach or small intestine microbiome. In one example, target cells are contained in the subject's stomach or small intestine. In one example, the first phage is delivered to the subject's stomach or small intestine.
37. 35. The composition of paragraphs 33 or 34 for use in a contained method of treating a disease or condition in a human or animal subject, wherein said disease or condition is mediated by a target bacterium, said target bacterium contained in a subject (optionally contained in the gut microbiome), said method comprising administering said composition to said subject, whereby said target bacteria are exposed to said antibacterial means and killed; phage growth is contained.
38. A method of treating an environment ex vivo comprising exposing said environment to a composition comprising a first population of phage particles, said composition comprising any one of paragraphs 22, 23 and 25-34. or said composition is according to any one of paragraphs 1-5 and 24-34, wherein said environment comprises a target bacterium and said first phage comprises said A method of infecting and killing a target bacterium.
39. 39. The method of paragraph 38, wherein said treatment is for containment in said environment.
40. A composition according to paragraphs 1-5 and 24-34 for controlling dosage of treatment with said first phage in said subject, or for controlling dosage of treatment with said first phage in said environment , paragraph 38 or 39.
41. 35. The composition of any one of paragraphs 1-5 and 24-34 for reducing the risk of acquisition of foreign gene sequences by said first phage in said subject, or said first phage in said environment. 40. A method according to paragraph 38 or 39 for reducing the risk of acquisition of foreign gene sequences by phage of

Concepts The present invention provides the following concepts exemplified in Example 6.
1. c) a first DNA, and d) one or more second DNAs
A host bacterial cell comprising
(v) said DNAs together comprise all the genes necessary to produce a transduction particle comprising a copy of said first DNA packaged by phage structural proteins;
(vi) said first DNA lacks at least one functional essential gene (e.g., encoding a phage structural protein) required to produce said particle, and said one or more second DNAs lack said functional contains a sex-essential gene,
(vii) said first DNA comprises a phage packaging signal for producing said particle;
(viii) said second DNA lacks a nucleotide sequence necessary for packaging said second DNA into a transducing particle;
said second DNA is required to package said first DNA to produce particles, said DNA producing transducing particles comprising phage structural proteins that package copies of said first DNA A host bacterial cell that is capable of being manipulated in said cell to do so.
2. said host bacterial cell comprising:
a) said first DNA and b) said one or more second DNAs
including
(i) together said DNA encodes all phage structural proteins necessary to produce a packaged transduction particle containing a copy of said first DNA;
(ii) said first DNA encodes none of said structural proteins, or encodes at least one, but not all, of said structural proteins, and said one or more second DNAs encode the remainder of said structural proteins; ,
(iii) said first DNA comprises a phage packaging signal for producing said particle;
(iv) said second DNA lacks a nucleotide sequence necessary for packaging said second DNA into a transduction particle;
said second DNA is required to package said first DNA to produce particles, said DNA producing transducing particles comprising phage structural proteins that package copies of said first DNA. The cell of concept 1, wherein the cell is operable in said cell for
3. (i) said first DNA is contained in an episome (e.g. plasmid) lacking said essential or structural protein gene and/or said second DNA is contained in an episome (e.g. plasmid) or chromosome of said cell. 1. The cell according to 1.
4. The cell of concepts 1, 2, or 3, wherein all of said essential genes or phage structural protein genes are contained in said second DNA and said first DNA lacks said genes.
5. A cell according to any preceding concept, wherein said first DNA encodes a guided nuclease or a component of the CRISPR/Cas system (optionally a crRNA or guide RNA).
6. said first DNA comprises a phage origin of replication and/or said first DNA comprises a phage replication gene and/or a phage lysis gene and/or (ii) said first DNA comprises a phage origin of replication does not contain a bacterial origin of replication.
7. A cell according to any preceding concept, wherein each transducing particle is a non-self-replicating transducing particle.
8. A cell according to any preceding concept, wherein each particle comprises a phage tail filament.
9. Any of the preceding concepts, wherein the essential gene or structural protein gene and packaging signal are those of a tailed phage, such as P2, T4, T7, Phi92, lambda, K1-5, or 933w phage. cells as described in .
10. The first DNA is contained in a phage genome, the phage genome is integrated into a plasmid, and optionally each particle is capable of infecting a target bacterium, the first DNA being a protein or protein in the target bacterium. A cell according to any of the preceding concepts, comprising a nucleotide sequence of interest (NSI) capable of expressing RNA, said NSI replacing said essential gene or structural protein gene of said phage.
11. each particle is capable of infecting a target bacterium, said first DNA comprising a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in said target bacterium, wherein said NSI in said target bacterium is the presence of the encoded protein or RNA mediates target cell death or down-regulation of target cell growth or proliferation, or (ii) each particle is capable of infecting a target bacterium and said first DNA is any of the preceding concepts, comprising a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in said target bacterium, or wherein said NSI comprises a regulatory element operable in said target bacterium. cell.
12. the presence of said NSI or the protein or RNA encoded thereby in said target bacterium mediates target cell death or down-regulation of target cell growth or proliferation, or is encoded by said target cell's genome. 12. A cell according to Concept 11, which mediates switching off or down-regulating expression of one or more RNAs or proteins.
13. presence of said NSI or a protein or RNA encoded thereby in said target bacterium mediates upregulation of target cell growth or proliferation, or one or more RNAs encoded by said target cell's genome; or a kit according to Concept 11, which mediates switching on or upregulating expression of a protein.
14. A cell according to any preceding concept, wherein said packaging signal is a pac or cos sequence, or a homologue thereof, or a direct terminal repeat (DTR).
15. Any of the preceding concepts, wherein the packaging signal comprises SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98, or 99% identical thereto, or is a homolog from a different phage. cells as described in .
16. An isolated DNA comprising a first DNA as defined in any preceding concept or comprising a second DNA as defined in any preceding concept.
17. 16. The first DNA or second DNA according to any one of Concepts 1 to 15, contained in a plasmid.
18. 18. The second DNA of concept 16 or 17, contained in cells that do not contain the first DNA.
19. 19. A kit comprising a cell as recited in Concept 18, wherein said kit comprises a vector (optionally a plasmid) comprising said first DNA, said vector not contained in said cell.
20. 20. The kit of concepts 18 or 19, wherein said cells are bacterial or archaeal cells.
21. 16. A method of producing a transducing particle composition, said method comprising expressing a phage structural protein in the cell of any one of Concepts 1-15 and replicating said first DNA. wherein transducing particles comprising the packaged first DNA are produced, said method optionally comprising separating an amount of transducing particles from cellular material, obtaining an amount of purified transducing particles; can be done, how.
22. 22. The method of Concept 21, comprising isolating the transducing particles.
23. 23. A composition comprising a population of transducing particles obtainable by a method according to concept 21 or 22.
24. The method of Concept 21 or 22 or the composition of Concept 23, wherein said second DNA is comprised of plasmid DNA, and wherein less than 5% of the total DNA comprised in said composition is DNA of said plasmid.
25. Each transduction particle contains a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in the target bacterium, and the presence of the protein or RNA encoded by said NSI in said target bacterium is the target cell's 25. The method or composition of any one of Concepts 21-24, which mediates killing or downregulation of target cell growth or proliferation.
26. 26. The method or composition of Concept 25, wherein said NSI encodes a component of a guided nuclease or CRISPR/Cas system that is toxic to target cells.
27. wherein said component is (i) a DNA sequence encoding a guide RNA (e.g. a single guide RNA) or comprising a CRISPR array for producing a guide RNA, wherein said guide RNA comprises said genome of a target bacterium; 27. The method of Concept 26, comprising a DNA sequence that can be targeted, (ii) a DNA sequence encoding a Cas nuclease, and/or (iii) a DNA sequence encoding one or more components of Cascade. Or composition.
28. The NSI encodes the Cas nuclease and guide RNA of the system (or the NSI encodes the Cas nuclease and a CRISPR array for producing the guide RNA), the guide RNA targeting the genome of the target bacterium. 27. The method or composition of concept 26, wherein said guide RNA can guide said Cas in target cells to mediate target cell death or downregulation of target cell growth or proliferation.
29. 26. The method or composition of Concept 25, wherein said NSI comprises an engineered antimicrobial means for killing target bacteria.
30. 30. The method or composition of concept 29, wherein said antimicrobial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.
31. A cell according to any one of concepts 1-15, a DNA according to any one of concepts 16-18, or any one of concepts 23-30 for administration to a human or animal subject for medical use. 1. The composition according to 1.
32. A cell according to any one of Concepts 1-15, a DNA according to any one of Concepts 16-18, or Concept 23, for administration to a human or animal subject to treat an infection of the target bacterial cell. 31. The composition of any one of Claims 30 to 30, wherein said particles are capable of infecting and killing said target cells, and optionally said infection is in the intestinal, blood, lung, or uterine tube microbiome. A cell, DNA, or composition that is an infection.
33. 33. The composition of concepts 31 or 32 for use in a contained method of treating a disease or condition in a human or animal subject, wherein said disease or condition is mediated by a target bacterium, and wherein said target bacterium is contained in said subject (optionally contained in the intestinal, blood, lung, or uterine tube microbiome), said method comprising administering said composition to said subject, whereby said target bacteria are killed upon exposure to an antibacterial means encoded by the DNA of said transduction particles and the growth of said transducing particles is contained.
34. A method of treating an environment ex vivo, comprising exposing said environment to a composition comprising a population of particles, said particles transducing a first DNA into said target cells contained in said environment. wherein said first DNA encodes an antimicrobial means that is toxic to target cells, thereby killing said target cells, said composition according to any one of concepts 21, 22, and 24-30 or said composition is according to any one of concepts 23 to 30.
35. 35. The method of Concept 34 for containing said treatment in said environment.
36. 31. The composition of any one of concepts 23-30 for controlling dosing of a transducing particle treatment of a target bacterial cell infection in a human or animal subject, wherein the particle comprises the first DNA into said target cell, wherein said first DNA encodes an antimicrobial means that is toxic to said target cell, thereby killing said target cell, or said particle treatment in said environment 36. The method of concept 34 or 35 for controlling dosing of
37. 31. A composition according to any one of concepts 23 to 30 for reducing the risk of acquiring foreign gene sequences in a human or animal subject by said particles in said subject or foreign by said particles in said environment 36. The method of concept 34 or 35 for reducing the risk of obtaining a gene sequence of

Optionally, each particle is capable of infecting a target bacterium and the first DNA contains a nucleotide sequence of interest (NSI) capable of expressing the protein or RNA in the target bacterium. For example, NSI codes for antimicrobial agents. For example, NSIs encode guided nucleases (eg, Cas, TALENs, zinc finger nucleases, or meganucleases). For example, NSI encodes the components of the CRISPR/Cas system. For example, a nuclease or system can be operated in a target cell to cleave a target nucleic acid (DNA or RNA) sequence contained in the target cell (eg contained in its chromosome or episome).

In one embodiment, essential genes are omitted from the first DNA, such that the first DNA lacks essential genes. In another embodiment, the first DNA may contain a variant of an essential gene that does not provide an essential gene function (ie it is not a functional essential gene). In another embodiment, the first DNA comprises a non-expressible form of an essential gene, e.g. the regulatory elements of the gene have been deleted or mutated so that the gene does not function, e.g. not expressed.

Optionally, the essential gene is not the phage terminase gene. Optionally, the first DNA lacks all phage terminase genes. Optionally, the first DNA does not lack phage structural protein genes. Optionally, the essential gene is not a phage structural protein gene. Optionally, the essential gene is neither a phage terminase gene nor a phage structural protein gene. Optionally, the first DNA does not contain an origin of replication (ori) operable in a bacterial host cell for replication of the first DNA (and optionally the first DNA further comprises a phage origin of replication).

Optionally, each particle comprises a tail fiber (eg, a tail fiber comprising one or more tail fiber domains of wild-type phage).

Optionally, each particle comprises a phage capsid protein, a packaging signal (contained in the first DNA), and optionally a phage replication gene, all of which are proteins of the same phage (e.g., wild-type phage) , packaging signals, and genes.

Optionally, each particle comprises a phage capsid protein, a packaging signal (contained in the first DNA), and optionally a phage replication gene, wherein the packaging signal and the gene are components of the same phage (e.g. wild-type phage) and the capsid protein is a protein of a different phage (eg wild-type phage).

Optionally, the first DNA lacks all phage structural protein genes and the second DNA contains all phage structural protein genes necessary to package the first DNA and produce particles, e.g. The packaging signal is a phage packaging signal selected from Caudovirales, Myovirideae, Podovirideae, or Siphovirideae phages. Optionally, the first DNA lacks all phage structural protein genes and the second DNA contains all phage structural protein genes necessary to package the first DNA and produce particles, e.g. The packaging signal is a phage packaging signal selected from P2, Phi92, T7, lambda, 933w, K1-5, and T4 phages.

Optionally, the first DNA lacks all phage structural protein genes and the second DNA contains all phage structural protein genes necessary to package the first DNA to produce particles, e.g. The phage structural protein gene of 2 DNA is a phage gene selected from Caudovirales, Myovirideae, Podovirideae, or Siphovirideae phage. Optionally, the first DNA lacks all phage structural protein genes and the second DNA contains all phage structural protein genes necessary to package the first DNA to produce particles, e.g. The phage structural protein genes of 2 DNA are phage genes selected from P2, Phi92, T7, lambda, 933w, K1-5, and T4 phages.

The essential or structural protein genes and packaging signals may be tailed phage structural protein genes and packaging signals. Alternatively, the essential or structural protein gene and packaging signal may be a mild phage essential or structural protein gene and packaging signal. Alternatively, the essential or structural protein gene and packaging signal may be a lytic phage essential or structural protein gene and packaging signal.

Optionally, the essential or structural protein genes and packaging signals may be P2, T4, T7, Phi92, lambda, K1-5, or 933w phage essential or structural protein genes and packaging signals. Optionally, the essential or structural protein gene and packaging signal may be a tailed phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a Caudvirales phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein genes and packaging signals may be Myovirideae, Podovirideae, or Siphovirideae phage essential or structural protein genes and packaging signals. Optionally, the essential or structural protein gene and packaging signal may be a P2 phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a T7 phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a Phi92 phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a lambda phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a 933w phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a K1-5 phage essential or structural protein gene and packaging signal. Optionally, the essential or structural protein gene and packaging signal may be a T4 phage essential or structural protein gene and packaging signal.

Optionally, any phage herein may be a tailed phage. Optionally, any phage herein may be a Caudovirales phage. Optionally, any phage herein can be a Myovirideae, Podovirideae, or Siphovirideae phage. Optionally, any phage herein can be a P2, T4, T7, Phi92, lambda, K1-5, or 933w phage. Optionally, any phage herein may be a P2 phage. Optionally, any phage herein may be a T7 phage. Optionally, any phage herein may be a Phi92 phage. Optionally, any phage herein may be a lambda phage. Optionally, any phage herein may be a 933w phage. Optionally, any phage herein may be a K1-5 phage. Optionally, any phage herein may be a T4 phage.

An essential gene according to the invention may be any nucleic acid sequence (not necessarily protein-encoding) required to produce a particle or phage. In one example, an essential gene encodes a protein. In one example, the essential gene is not a packaging signal or phage origin of replication.

For example, each essential gene is
- a phage gene encoding a phage structural protein;
a phage gene encoding a gene expression activator (eg RNA polymerase, eg RNA polymerase of Coliphage T4, T3, or K1-5), gene Q (eg gene Q of coliphage lambda), gene Rin (eg of Staphylococcal phage NMI) Rin), gene ogr (eg ogr of coliphage P2), or gene delta (eg delta of colisatellite phage P4),
- phage RNA metabolism genes (i.e., encode proteins involved in the phage RNA metabolism system),
phage DNA metabolism genes, i.e., encoding proteins involved in the phage DNA metabolism system;
- phage DNA packaging genes (i.e., encode proteins involved in the phage DNA packaging system);
• is selected from phage genes that encode proteins required for lysis of bacterial cells;

For example, each essential gene is selected from the following P2 genes (functions are shown in parentheses).
- gene B (DNA replication),
・Gene Q (portal protein),
- genes P and M (terminase),
- genes O, L, and N (capsid),
- gene X, R, S, v, W, J, I, H, G, FI, FII, E, E', T, U, D (tail and tail fiber),
• Genes X, Y, lysA, lysB, lysC (lysis cassette), and • Ogr (activator of late gene expression).

For example, each essential gene is selected from the following T7 genes (functions are shown in parentheses).
- gene 1 (RNA polymerase),
-Genes 1.3, 2.5, 3, 3.5, 4a, 5, 6 (DNA and RNA metabolism),
- genes 6.7, 7.3, 8, 9, 10A, 10b, 14, 15, 16 (capsid and internal core),
・Genes 11, 12, 17 (tail),
• Genes 17.5, 18.5, 18.6, 18.6 (lysis), and • Genes 18 and 19 (terminase).

A phage is a virus capable of infecting a target bacterial cell, or a transducing particle is capable of transducing a target bacterial cell, i.e. introducing a first DNA or portion thereof into the target cell. .

In one embodiment, the kit includes cells (eg, bacterial cells) containing the first and second DNAs. For example, the cell is a bacterial cell, the first DNA is contained in an episome (eg plasmid) lacking the gene for said essential or structural protein and the second DNA is contained in the chromosome of the cell. Also optionally all essential or phage structural protein genes are included in the second DNA and the first DNA and episome lack said genes. Optionally, the first DNA (or portion thereof) encodes a guided nuclease or CRISPR/Cas system component (eg, Cas, Cascade protein, crRNA, guide RNA, or tracrRNA). Optionally, the first DNA (or portion thereof) encodes crRNA or guide RNA.

Optionally, the first DNA contains a phage origin of replication.

Optionally, the transducing particles are non-self-replicating transducing particles.

Optionally, the first DNA contains phage replication genes and/or phage lysis genes.

Optionally, the phage or particle comprises a tail fiber, eg, a wild-type phage tail fiber (or domain thereof) comprising structural proteins.

In alternatives to bacterial cells, the cells are archaeal cells, the phages are viruses capable of infecting archaea, or the transducing particles are capable of transducing archaea.

Optionally, the packaging signal herein is a pac or cos sequence, or a homologue thereof, or a direct terminal repeat (DTR).

It will be appreciated that the particular embodiments described herein are presented by way of illustration and not as limitations of the invention. The principal features of the invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to ascertain numerous equivalents to the specific procedures described herein without using more than routine experimentation. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. U.S. patent application Ser. Incorporated herein by reference to the same extent as specifically and individually indicated. The use of the word "one ([a] or [an])" may mean "one" when used in conjunction with the term "comprising" in the claims and/or specification but this is also consistent with the meanings of "one or more," "at least one," and "one or more." Although this disclosure supports definitions that mean only alternatives and "and/or," use of the term "or" in the claims is expressly indicated to mean alternatives only, or alternatives are mutually exclusive. is used to mean "and/or", except where it is exclusive to Throughout this application, the term "about" is used to indicate that a value includes inherent error variation for a device and the method employed to determine the variation that exists between that value or consideration. be done.

As used herein and in the claims, the words "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and having ], e.g., [have] and [has]), "including" (and any form of [including], e.g., [includes] and [include]), or "containing" ( Any form of and [containing], eg, [contains] and [contain], is inclusive or open-ended and does not exclude additional elements or method steps not recited.

The term "or combinations thereof" or like terms as used herein means all permutations or combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" means at least one of A, B, C, AB, AC, BC, or ABC, and where order is important in the particular context, Also intended to include at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, eg, BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any other part of this disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, no departure from the concept, spirit, and scope of the present invention may be made to the compositions and/or methods, or to any of the methods described herein. It will be apparent to those skilled in the art that variations can be applied to the steps or the sequence of steps. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined in the appended claims.

[Example 1]
PRODUCTION OF EFFICIENT PHAGE CRISPR DELIVERY VEHICLES BACKGROUND ART We designed a strategy to efficiently produce phage particles containing components of the CRISPR/Cas system to kill target E. coli Nissle strain bacteria. did. Thus, our phage composition consisted of a lysate containing primarily CRISPR/Cas system components packaged into phage particles, which lacked sequences encoding phage proteins. and have no or very low percentage of helper phages. This strategy also alternatively works in phage/bacterial strain combinations. Demonstrations made are shown in Examples 3 and 4.

Overview of strategies for packaging of CRISPR/Cas components into hitherto unknown phage: (a) High or medium copy number cloning/cloning, including E. coli ori for replication in E. coli cloning production strains; identifying a shuttle vector that can be cloned and propagated in a first E coli strain (cloning production strain) and then transferred to a second bacterial host strain of interest (target host strain);
(b) isolating mild phage against the target host (second) bacterium;
(c) have an inactive CRISPR/Cas system (e.g., suppressed Cas3 or other nuclease) and/or lack the target protospacer found in the second strain to produce a cloned production strain; and identifying or engineering a phage-producing strain of a host target bacterium (or other bacterium) that can be infected with and lysogenized with mild phage, or suppressing or inactivating said system in said producing strain.
(d) using a mild phage (helper phage) to generate a lysogen in the production strain and test that it is inducible;
(e) Using Phage Term (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557969/) on whole-genome-sequenced phage, packaging sequences (pac or cos) identify,
(f) deleting the pac/cos packaging signal sequence in the helper phage within the producing strain bacterium;
(g) The packaging signal is linked to a CRISPR-array or a single gRNA coding sequence (and optionally other components of the CRISPR/Cas system, such as a nucleotide sequence encoding Cas9 and optionally a tracrRNA coding sequence, or Cas3) and/or a sequence encoding a cascade) into a shuttle vector;
(h) transforming the vector into a production host strain;
(i) Phage containing the CRISPR/Cas components are induced (eg, by UV or mitomycin C) and harvested. Alternatively, a system with inducible RecA is used in trans to simulate SOS (required to become active RecA).

The above example, specifically for E coli Nissle, using phage P2:
Nissle is useful because of its GRAS (Generally Regarded as Safe) status, and P2 has a relatively broad host range (DNA delivery to most E. coli, Shigella, Klebsiella, Salmonella e.g. Pseudomonas; Kahn et al. al 1991, "Bacteriophage P2 and P4", Methods in Enzymology, vol 204, pp264-280).

We use pUC19 or other high or medium copy number cloning vectors. Mild phage P2 is able to lysogenize Nissle. Most E. coli K strains have an inactive CRISPR/Cas system and can be infected with P2, thus all common cloning hosts can be used (herein by E. coli TOP10 exemplified).

P2 is introduced into TOP10 to produce lysogens. Although P2 cannot be induced by mitomycin C or UV, we use an epsilon antirepressor derived from paracytophage P4 that derepresses P2 and directs P2 to the lytic phase. We express this gene from an inducible promoter in the production host strain.

The 325 bp packaging signal sequence of SEQ ID NO:2 is used.

The packaging sequence is deleted in the P2 prophage of the lysogenic TOP10 strain.

A pUC19 shuttle vector is constructed that encodes a guide RNA (or alternatively contains a CRISPR array for producing such guide RNA) that targets the genome of the target Nissle strain, and the packaging signal SEQ ID NO:2 is added. If the target Nissle contains its own endogenous CRISPR/Cas system, we use an activation strategy that activates endogenous Cas3 by including the Cas activation gene in the vector. Otherwise, we place the exogenous Cas3-encoding nucleotide sequence (and optionally one or more nucleotide sequences encoding one or more of the required cascade components) into the vector. Include and express at the target Nissle. We transform this vector into a TOP10 producer, induce the P4 anti-repressor, and pick phage containing the CRISPR/Cas components.

Since the induced (helper) phage DNA does not contain the packaging signal, we are able to isolate particles with only the vector DNA packaged. We thus obtain compositions comprising such phages which can be used to infect target Nissle E coli bacteria and to introduce CRISPR/Cas components into said bacteria to kill the target bacteria. .

[Example 2]
MGE, genomics islands, etc. A review of various possible MGE packaging strategies follows.

Applicable to different types of phage:
- Identifying the packaging signal and structural gene in the helper phage - Deleting the packaging signal in the helper phage and placing it on a plasmid containing MGE - Placing both helper and plasmid in the production strain - Transcription of the helper's structural gene to produce MGE packaged in helper phage

Induction of helper phage activators from one, more or all of the parasite element Delta within P4 or ptiA/B/M within SaPI for use with a parasite migratory element (such as the P4 phage or SaPI) Activation of helper phage structural genes is achieved by

If smaller size particles are desired, packaging into parasite-sized capsids (typically 10-20 kb) by including Sid of P4 and cpmA/B of psu or SaPI in MGE or vector. You can choose.

Defective helper phage can be used if at least the packaging signal has been removed and the structural gene is either on a plasmid or integrated into the production host as a cryptic prophage. If for some reason it is not possible to use this approach and it is necessary to use a functional helper phage, the genes on parasite that hijack the phage packaging machinery can be included in the MGE or vector to generate parasite DNA ( In our case CGV™) is preferentially packaged over phage DNA.

List of minimal genes that can be included in a P4-derived plasmid vector.
P4 sequence: https://www. ncbi. nlm. nih. See gov/nuccore/x51522 Cos packaging site: SEQ ID NO:3
Homologous sequence between P2 and P4; this can be used as an alternative packaging signal in MGE or vectors: SEQ ID NO:4

For smaller capsid sizes (packaging 11.4 kb instead of 33.5 kb), Sid and/or Psu can be included in the MGE or vector:
Sid: SEQ ID NO:5
Psu: SEQ ID NO:6

Delta of P4 can be included in the host cell genome (provided separately in the host cell and not on the packaged MGE or vector) in order to activate the helper phage P2.
Delta: SEQ ID NO:7

Minimal gene of P2 for inclusion in host chromosome/episomes.
P2 sequence (accession number: NC_001895)

FIG. 1 shows the genetic map of the P2 genome, with non-essential genes boxed. One, more or all of these can be left out (but Cos is always missing). Cos is deleted, preferably the entire region from int to Cos is deleted. This region can be exchanged for example with a resistance marker, while the orf30 and fun(Z) genes can be left intact or deleted.

Thus, in one embodiment of any aspect herein of the invention, int to Cos (i.e., inclusive of int and Cos) are omitted from the helper phage DNA, or a different phage is helper phage DNA or When used as a basis for a second DNA, the second DNA or homologous region is omitted.

The sequences of the P2 genome of various lengths are as follows:
"Q" to "S": SEQ ID NO:8
"V" to "G": SEQ ID NO:9
"FI" to "ogr": SEQ ID NO: 10

Minimal gene for inclusion in vector or MGE from SaPI.
There are several different SaPI systems. Figure 2 shows one well-characterized SaPI (SaPIbov1), which utilizes the phage phi11 or phi80alpha as helper phage. Sequence of SaPIbov1 (Accession number: AF217235.1)

Packaging Signal If a defective helper phage lacking a packaging signal is used, the packaging signal derived from the helper phage can be used and included in the DNA for packaging. S. An example of aureus phi11 (accession number: AF424781) is SEQ ID NO:11.

For smaller capsid sizes (packaging 15.8 kb instead of 43.6 kb), the MGE or vector can include cpmA and/or cpmB (SEQ ID NOS: 12 and 13).

One, more, or all of ptiA, B, and M can be included to activate the helper phage phi11 (provided separately in the production host cell and provided on the packaged MGE or vector). not) (SEQ ID NOS: 14-16).

Minimal gene of phi11 for inclusion in host chromosome/episomes.
Phi11 sequence (Accession number: AF424781)
Gene number 29 (terS) to gene number 53 (lysin): SEQ ID NO: 17

List of phages that work with SaPI Different SaPIs are associated with different helper phages (see Table 2 below).

A helper phage can be mutated to contain only the structural gene, causing it to package into a smaller capsid. Looking only at the genes involved in small capsid packaging (cpmA and cpmB), these genes are highly conserved among staphylococci, indicating that they are responsible for packaging in the following list (Table 1). 2) functions to redirect a wider variety of phages.

[Example 3]
Design, Construction, and In Vitro Efficacy of a Synthetic Transduced Particle Delivery Platform A synthetic DNA delivery vehicle comprising replicating transduction particles, which are capable of infecting and introducing DNA into target E coli host cells. The introduced DNA can then be used in a host cell to produce said component. We refer to such DNA as CRISPR-Guided Vectors (CGV™). As shown in this example, we generated such particles using defective helper phage. When synthetic particles are produced, we obtain a pure synthetic lysate devoid of all phage nucleic acids encoding phage proteins. The inventors have found that this lysate is in vitro infected with E. It shows that highly efficient CGV™ delivery to E. coli is possible. This particle is non-self-replicating and requires helper phage for replication. Beneficially, therefore, the lysate is pure, eliminating the possibility of replication of the CGV™-containing particles. This is useful, for example, to include the action of antimicrobial agents to allow for more controlled (e.g., predetermined) dosing for administration to human or animal subjects or environments, and , elimination of replication in a subject or environment reduces the chances of phages acquiring unwanted genes or traits (e.g., unwanted antibiotic resistance genes) from surrounding phages or bacteria, and furthermore, the particle replication of the present invention Restriction of provides a way to reduce or eliminate the spread of such genes or traits. This may be of interest to authorities such as the US FDA or USDA, which consider such aspects when approving drugs or antimicrobials for use in the environment.

Research Objectives Objective 1: Design and engineer a fully synthetic phage-based CGV delivery platform SA100.
Purpose 2: Using SA100 to detect E. CGV delivery to E. coli is demonstrated.

Materials and Methods Bacterial Strains and Growth Conditions We generated bacterial production strains for the production of synthetic SAE100 transduction particles . E. coli strain C-2323, which contains phage P2, was used as the starting material for engineering our SA100/CGV production strain (gift from Gail E. Christie, Commonwealth University of Virginia). Other strains used for SA100 and infectivity testing were EMG-2, C-1a, and C-1792 (purchased from the Coli Genetic Stock Center http://cgsc2.biology.yale.edu/). .

All bacterial cultures were grown in LB medium supplemented with ₂ mM MgCl2. When appropriate, cultures were supplemented with 50 μg/mL kanamycin or 50 μg/mL spectinomycin for selection of defective prophages and CGV, respectively. In SA100 infection studies, the medium was supplemented with 2.5 mM CaCl ₂ to promote adsorption of SA100 particles to bacterial cells. To induce the CRISPR/Cas system, cells were exposed to 0.5 mM IPTG and 2 mM theophylline.

Construction of defective helper prophage The pTK-red recombinant plasmid (p72) was transformed into strain C-2323. The KamR kanamycin marker gene was amplified using oligos with 50 bp homology to regions of the P2 prophage immediately upstream and downstream of the approximately 10 kbp region of P2 that we wanted to delete ( See Figure 3). After successful replacement of the desired region with the KanR marker gene, the thermosensitive pTK-red recombinant plasmid was cured.

Engineering CGV(p94) for packaging into SA100 particles CGV p77 was constructed using plasmid p53 as a template. Plasmid p53 is a plasmid that encodes the components of the CRISPR/Cas system, ie, a plasmid that contains nucleic acid sequences encoding tracrRNA and the Cas9 protein from Streptococcus pyogenes (SpCas). Nucleic acid sequences for producing crRNA were obtained and expression was driven by the plac promoter. The two fragments were assembled using Gibson assembly (NEB E5510S) and generated by E. E. coli competent cells were transformed. The assembled p77 plasmid was verified by total sequencing.

The p77 containing inducible CRISPR/Cas system was used as a scaffold to insert a fragment containing an arabinose-inducible promoter that transcribes the sid to cos region of satellite phage P4 (Fig. 3).

Both fragments were PCR amplified and cloned by restriction enzyme cloning. The sequence of the resulting SA100-packaging CGV (p94) was verified.

Production of CGV packaged in SA100 CGV p94 was transformed into the previously constructed strain containing the defective prophage, thereby generating production strain number 189.

Producer strains were grown exponentially in the presence of kanamycin and spectinomycin for approximately 10 generations to ensure even population growth. At an optical density (OD ₆₀₀ ) of 1, cultures were induced with 1% arabinose and incubation continued until optical density readings fell and stabilized. After removing cell debris by spinning, the lysate was filtered (0.45 μm) and stored at +4° C. until later use.

Transduction protocol for determining the titer of SA100.
Titer determinations were performed by mixing the SA100 lysate with the appropriate bacterial strain to ensure at least a 100-fold excess of bacterial cells in the presence of _2.5 mM CaCl2. After 30 minutes of incubation, bacteria were diluted and plated in LB containing spectinomycin to count bacteria that received p94 CGV by transduction. To verify the purity of the SA100 lysate, the undiluted lysate was spotted onto a lawn (C-1a) known to support P2 growth using a standard soft agar overlay. did. Absence of plaque formation was used to verify purity.

DNA Delivery Assay The transduction protocol involves diluting the SA100 lysate and then infecting the bacterial culture to achieve a multiplicity of infection ranging from 0.01 to 100, with the log ratio of SA100 to bacterial cells effectively ranging to 4. (MOI) was used.

After 30 minutes of infection in the presence of _2.5 mM CaCl2, bacteria were counted for delivery of p94 CGV on LB plates with and without spectinomycin selection. The numbers obtained were used to calculate the percentage of the population infected at a given MOI.

Results Design of SA100 Vehicle SA100 E. coli containing CGV and defective prophage. The overall design of the E. coli production strain is outlined schematically in FIG. We have found that native E. coli integrated as a prophage into the chromosome of the production strain. E. coli phage P2 was used as our starting material. In the prophage, we deleted genes essential for the phage to initiate and carry out its own propagation, thus generating defective helper phage.

We then placed the expression of the structural genes required to produce SA100 transduced particles under the control of an inducible promoter, which allowed us to turn on particle production. It has become possible. Finally, we removed the packaging sequence that phages normally use to package their DNA into phage particles derived from phage DNA and placed this in our CGV (Fig. 3). (see cos sites denoted by ). Upon induction of the phage structural genes, we packaged CGV DNA into particles, which were subsequently released by lysis of the producing cells.

Manipulation of Defective Helper Phage E. coli containing phage P2 on its chromosome. E. coli strain C-2323 was used as starting material to engineer a production strain with a defective helper phage. The region on the P2 phage containing the integrase, the promoter for initiating phage propagation, the origin of replication, the DNA replication gene, and the cos site (the DNA sequence when packaging the DNA into phage particles) is shown in FIG. See boxed P2 DNA shown), replaced by the kanamycin marker using recombination. Site-specific integration of markers and deletion of approximately 10 kbp of phage DNA was verified by sequencing.

Engineering CGVs to Enable SA100 Packaging To engineer our SA100 packaging-capable CGVs, we used a single A vector (p77) was used.

To enable activation of the defective P2 helper phage and packaging of CGV into synthetic transducing particles, we used the sid-delta-psu-cos (cos packaging site) from satellite phage P4. was cloned into the p77 plasmid. The cloned P4 region (SEQ ID NO: 18), found to be able to activate the P2 phage, had already been cloned into another vector, p93, in which it was expressed from an arabinose-inducible promoter. rice field. The resulting CGV (p94) containing all elements required for P2 activation and SA100 packaging was verified by total sequencing. The genomic structure of P4 and the regions cloned into p94 are shown in the lower boxes in FIG. 3A, and the genetic map of p94 is shown in FIG. 3B.

Production of CGV packaged in SA100 A strain containing a defective prophage was electroporated with p94 CGV to give producer strain number 189.

CGV packaged in SA100 was produced as described above. The titer of SA100-packaged CGV showed that SA100-packaged p94 was isolated from three strains of E. coli. approximately 10 ¹⁰ TFU/mL as determined by transducing E. coli. As expected, no native P2 phage contaminants could be observed (Table 3 below). Therefore, we can assume natural phage contaminants to be less than 1 in 1e9 per synthetic particle.

E.C. using SA100. CGV delivery to E. coli. To determine the number of phage required to infect 100% of the E. coli population, we varied the ratio of SA100 particles to bacterial cells, i.e. multiplicity of infection (MOI) from 0.01 to 100 carried out an infection experiment.

The inventors have found that E. We observed that an MOI of 1, meaning one SA100 particle per E. coli cell, was sufficient to infect 100% of the bacterial population (Fig. 4).

Discussion and Conclusion SA100, a fully synthetic phage-based CGV delivery vehicle, was designed and constructed. High-titer lysates of SA100-packaged CGV, devoid of natural phage contaminants, were produced when expressed in production strains that also contained matched CGV. Finally, the resulting CGV packaged in SA100 was isolated from E. Efficiently delivered to E. coli.

[Example 4]
E. coli in vitro by CGV after delivery by the SA100 synthetic transduction particle delivery platform. E. coli Killing Protocol We used the SA100 synthetic DNA delivery vehicle to deliver the CRISPR-guided vector (CGV™) to two E. coli. E. coli target strain. When the system was induced, we were able to demonstrate up to 4 logs of target population killing.

Introduction We have transformed CGV into E. A phage-based synthetic DNA delivery vehicle SA100 has been engineered for delivery to E. coli target cells (Example 3). Here, we optimized CGV for delivery by SA100 vehicle and then E. was tested for efficacy in killing E. coli target cells.

Study Objectives Objective 1: Optimize CGV for delivery by SA100.
Aim 2: E. coli by SA100-delivered CGV kill the E. coli.

Materials and Methods Bacterial strains and culture conditions All bacterial cultures were grown in LB medium supplemented with ₂ mM MgCl2. When appropriate, cultures were supplemented with 50 μg/mL kanamycin or 50 μg/mL spectinomycin for selection of defective prophages and CGV, respectively. In SA100 infection studies, the medium was supplemented with 2.5 mM CaCl ₂ to promote adsorption of SA100 particles to bacterial cells. To induce the CRISPR/Cas system, cells were exposed to 0.5 mM IPTG and 2 mM theophylline. By recombining a 20-nucleotide sequence complementary to the guide RNA spacer sequence in our CGV into the lacZ gene of MG1655 and XL1-blue, thereby generating target strains MG1655_pks and XL1-blue_pks, respectively. Strains MG1655_pks and XL1-blue_pks were constructed.

Production of CGV packaged in SA100 CGV (p94 and p114) were separately transformed into their respective strains containing defective prophages, thereby generating production strains #189 and #226, respectively.

Producer strains were grown exponentially in the presence of kanamycin and spectinomycin for approximately 10 generations to ensure even population growth. Cultures were concentrated 10-fold at an optical density (OD ₆₀₀ ) of 1, induced with 1% arabinose, and incubation continued until optical density readings fell and stabilized. After spinning out cell debris, the lysate was filtered (0.45 μm), further concentrated approximately 4-fold using Amicon Ultra-15 centrifugal filters, and stored at +4° C. until later use.

CGV delivery and killing Exponentially growing E. coli. E. coli target strain cultures were infected with SA100-packaged CGV p94 or CGV p114 at an MOI of 20 in the presence of 2.5 mM CaCl ₂ .

After incubation for 30 minutes at 37 degrees Celsius, serial dilutions of bacterial cultures were plated on LB plates containing IPTG and theophylline (to induce expression of CRISPR/Cas components) to prevent CGV-mediated killing. or seeded on LB + spectinomycin to examine CGV delivery to target populations.

Results Optimizing CGV DNA by increasing copy number CGV p94 is isolated from E. It has previously been shown to be efficiently delivered to E. coli (Example 3). This CGV is described by Stuitje et al. 1981, "Identification of mutations affecting replication control of plasmid Clo DF13", Nature, vol 290, pp264-267, including the CloDF13 origin of replication, where a single SNP increases copy number from 7 to 80. By incorporating the SNP into the oligos used for PCR amplification of whole CGV, we generated p114 containing the mutated CloDF13 ori (SEQ ID NO:23). Performed sequence validation.

Delivery of CGV by SA100 We have previously demonstrated efficient CGV delivery using SA100-packaged p94 (Example 3). Here, we compared the delivery of CGV p94 and CGV p114 to MG1655_pks (Fig. 6A) and also p114 to XL1-blue_pks (Fig. 6B). Using a multiplicity of infection (MOI) of 20, we demonstrate 100% delivery to the target population.

Killing Efficacy of SA100-delivered CGV Using the same experimental set-up as above (target strain, SA100-packaged CGV, and MOI of 20), we determined that expression of the CRISPR/Cas system was induced. The ability of the delivered CGV to kill target cells was examined.

p94 CGV targeting the pks DNA sequence in MG1655_pks was able to reduce the target strain by approximately 2 logs, while p114 targeting the same sequence reduced the target population by approximately 4 logs (Fig. 7). A similar degree of killing was observed when p114 was targeting pks in the XL1-blue_pks target population (FIG. 7).

Discussion and Conclusion After CGV delivery of SA100 to target cells, we were able to increase the killing of the target population from approximately 2 logs using p94 to 4 logs using p114, which is likely , due to the high copy number of CGV.

[Example 5]
Protocol for In Vivo Antimicrobial Delivery to the Gut Microbiome Using Non-Self-Replicating Particles and Killing of Target Cells in the Microbiome SettingE. E. coli AMG1655-pks was used to colonize the digestive tract of mice. We demonstrate statistically significant delivery of CGV™ pSNP114 to MG1655-PKS by the SA100 delivery vehicle (synthetic non-self-replicating phage particles, see Examples 3 and 4). The inventors have found that E. We observed that activation of CGV™ targeting E. coli MG1655-pks reduced survival.

INTRODUCTION We have found that E. E. coli ATCC43888 colonizes the mouse gut of the NMRI mouse model (data not shown) and E. coli ATCC43888 colonizes the mouse gut in the mouse model. We have previously shown that activation of the CRISPR array in E. coli ATCC43888 reduces survival and that guide RNAs produced using this array target the E. coli genome for Cas cleavage (data not shown). there is In this example, we sought to assess the survival of a synthetic Nanoboiotic™, a CRISPR/Cas-based antimicrobial (CGV™) delivered to MG1655-pks by SA100.

Purpose of research:
E. coli in the mouse gut microbiome by delivery of CGV™ pSNP114 with synthetic Nanoboiotic™ SA100. Establish a reduction in survival of E. coli MG1655-pks.

Materials and Methods The E. coli was E. coli MG1655, ie clade A, with a pks fragment inserted with a streptomycin resistance marker.

Cells were grown overnight in LB medium supplemented with 50 μg/mL spectinomycin. The next day, OD was measured and culture diluted to 10 ⁸ CFU/mL. Vehicle, induced and non-induced groups are shown in Table 4.
- 15 female NMRI mice, 26-30 grams (Taconic)
- an inducer solution containing theophylline and L-arabinose

Laboratory animal facilities and mouse housing Temperature was 21°C +/- 2°C, adjustable by heating and cooling, and light/dark periods were 6am-6pm/6pm-am. It was a 12 hour interval at 6:00. Mice had free access to food and domestic quality drinking water up to 3 days before the study. Mice were housed 6-7 mice per cage in standard facilities with Tapvei aspen-derived bedding and Sizzle-nest-derived paper strands as nesting material until 1 day prior to study. and then transferred to the GMO facility with one mouse per cage. Beginning on day 0, the wooden bedding was replaced with blank paper bedding to facilitate fecal collection from the cage.

Preparation of Streptomycin Drinking Water Six liters of 5 g/L streptomycin was prepared by dissolving 6.94 g of streptomycin sulfate per liter of sterile water. Mice were given streptomycin water starting on day -3 and throughout the study.

Inoculation of Mice Prior to inoculation, the inoculum was formulated as a ready-to-use suspension. At 8 am on day 0, mice were inoculated by oral gavage with 0.25 ml. A titer of approximately 10 ¹¹ TFU/ml was used.

Treatment of Mice At 7:00 am and 1:00 pm on day 1 and again at 7:00 am on day 2, mice were orally gavaged with inducer and 10 ¹¹ TFU/mL of SA100. Inducing agents were mixed just prior to each treatment time point, after which treated mice were gavaged with antacids.

Collection of Fecal Samples from Cages Immediately prior to inoculation, 0.5 ml fecal samples were collected from cages into three 15 ml Nunc tubes. On days 1 and 2 post-inoculation, 0.5 ml fecal samples were collected prior to treatment at 7:00 am and mice were transferred to new cages. Also on day 2 post-inoculation, fecal samples were collected 4 hours after treatment.

Determination of CFU 0.5 ml of stool was dissolved in 5 ml of sterile saline by vortexing multiple times for 1-4 hours. Colony numbers were determined by 10-fold serial dilution of fecal samples into sterile 0.9% NaCl and 20 μL spots were applied to agar plates. The lower limit of detection for fecal samples was 50 CFU/ml. All agar plates were incubated at 35° C. for 18-22 hours in ambient air.

CGV delivery was measured by plating on LB+streptomycin+spectinomycin plates. Target strain abundance was measured by inoculating LBs with streptomycin alone.

Clinical Monitoring of Mice Mouse weights were monitored throughout the study and mice were scored from 0 to 6 based on their behavior and clinical signs (Table 5). Mice were euthanized when a score of 3 was reached or there was 20% weight loss.

Results Microbiome Analysis of Pre-Inoculation Mice No bacteria were detected in 2 of 3 pooled pre-inoculation collected samples. One pooled sample had small white colonies at 6.54 log10 CFU/ml. MALDI analysis showed Paenibacillus odorifer with a score value of 1.71 and the second most likely Staphylococcus xylosus with a score value of 1.43, both of low ranked quality. Small white colonies with 3-4 log10 CFU/ml also appeared after 48 hours of incubation in two other pooled samples.

Twenty-four hours after inoculation, the range of CFU levels in fecal samples was 6.1-8.6 log10 CFU/sample as determined on LB+streptomycin agar plates. Similar levels were observed two days after inoculation. No significant difference was found in each of the two inoculation groups.

No colonies were observed on day 1 in either group on LB+streptomycin+spectinomycin agar plates. Day 2 CFU counts ranging from 1-5 log10 CFU/sample were observed in the inducer-treated group, but not in the vehicle-treated group.

Animal Study Design Twenty-four mice were used in each mouse intervention study.

Clinical Mouse Scores Mice did not show any clinical signs of infection or discomfort during the entire study period.

Induction of Killing of MG1655-pks Fecal extracts were plated on plates containing streptomycin to induce E. CFU of E. coli MG1655-PKS strain was determined (Fig. 8). CFU counts with streptomycin show no difference between the various treatment groups.

When seeding fecal samples on spectinomycin and streptomycin to determine delivery of CGV pSNP114 (because this CGV carries the spectinomycin resistance marker), we determined that after 48 hours: A significant increase in CFU and thus in CGV delivery is observed (Fig. 9). When the CRISPR system was induced, we observed a significant decrease in mean CFU numbers.

DISCUSSION AND CONCLUSIONS We used the Synthetic Nanobiotic™ platform, SA100, to study E. coli in a mouse gut model. successfully demonstrated the delivery of guided nuclease antimicrobial agents (CGV™) to E. coli MG1655-pks. Thus, the delivery of antimicrobial agents to the microbiome in vivo using non-self-replicating particles has been established. A statistically significant reduction in CFU count was observed using the activated CRISPR system on the CGV™.

[Example 6]
Production of transduction particles with essential functions in trans Engineering native phages to convert them into DNA carrier vehicles (transduction particles) for our CRISPR-Guided Vectors™ (CGV) do. The particles can be transduced into target bacteria to introduce vectors from which the CRISPR/Cas system or one or more components thereof are expressed. We use lytic growth in which essential helper functions are provided only by the host cells (SA800 and SA900).

SA800 and SA900
Production of SA800 and SA900 particles is based on lytic growth in the production host bacterial strain. We have developed native (wild-type) phages by inactivating essential phage functions (e.g., by deleting structural genes) and by adding nucleotide sequences encoding the CRISPR-Cas system. to operate.

By providing the essential genes on separate helper plasmids, we are able to perform CGV-phage hybrid propagation in production strains. However, propagation of CGV is not possible in any other bacterial strain due to the lack of helper plasmids.

We refer to FIG. To construct non-self-replicating particles containing CGVs containing nucleotide sequences encoding components of the CRISPR-Cas system, we constructed a to manipulate natural phages. A CGV may contain multiple phage genes, but it may lack one or more essential genes so that CGV-phage hybrids are non-self-replicating in strains that do not express essential functions. or mutated. In production strains, this basic function is provided expressed in trans from a plasmid or bacterial cell chromosome.

In one example, the producer cell contains a plasmid having a gene encoding a structural phage protein that is expressed during CGV propagation and packaging. A vector containing the genome of a native phage was constructed, in which this gene was mutated and a nucleotide sequence encoding a component of the CRISPR-Cas system was inserted to form a CGV-phage hybrid. Thus, CGV-phage hybrids are non-functional to package CGV DNA into transducing particles in the absence of helper functions provided in trans.

In the minimal case, all basic functions (i.e., essential for packaging), except the phage packaging signal and the phage DNA replication machinery (including the phage origin of replication) are instead carried on the CGV. It is carried by the production host cell (eg, on a chromosome or on one or more plasmids). Importantly, the DNA replication machinery need not be derived from the same phage as the helper plasmid or essential gene on the chromosome. For example, the DNA replication machinery of phage lambda can be contained in a CGV carrying the phage P2 packaging signal (and used to replicate it) and the P2 phage capsid (e.g., the capsid protein can be a helper plasmid or production (where encoded by the chromosome of the cell line).

CGV can replicate as a lytic phage (ie, propagate by infection-lytic-reinfection cycles) on suitable production host strains that possess helper functions. However, in non-producing cells such as clinical target bacteria, phages cannot propagate through the lytic cycle, but serve only as DNA delivery vehicles, thereby allowing CRISPR/Cas components (or alternatively, another antibacterial agent or A sequence encoding one or more components encoding another protein or RNA of interest is delivered.

Claims (24)

a) a first DNA, and b) one or more second DNAs
A host bacterial cell comprising
(i) said DNAs together contain all the genes necessary to produce a transduction particle comprising a copy of said first DNA packaged by phage structural proteins;
(ii) said first DNA lacks at least one functional essential gene required to produce said particle and said one or more second DNAs comprise said functional essential gene;
(iii) said first DNA comprises a phage packaging signal for producing said particle;
(iv) said second DNA lacks a nucleotide sequence necessary for packaging said second DNA into a transduction particle;
said second DNA is required to package said first DNA to produce particles, said DNA producing transducing particles comprising phage structural proteins that package copies of said first DNA A host bacterial cell that is capable of being manipulated in said cell to do so. said DNAs together encode all phage structural proteins necessary to produce a packaged transduction particle containing a copy of said first DNA, said first DNA encoding any of said structural proteins none, or encode at least one, but not all, of said one or more second DNAs encoding the remainder of said structural protein. 3. The cell of claim 1 or 2, wherein said first DNA is contained in an episome lacking said essential or structural protein gene and/or said second DNA is contained in an episome or chromosome of said cell. 3. The cell of any preceding claim, wherein all of said essential genes or phage structural protein genes are contained in said second DNA and said first DNA lacks said genes. 4. The cell of any preceding claim, wherein the first DNA encodes a guided nuclease or a component of the CRISPR/Cas system (optionally crRNA or guide RNA). 4. A cell according to any preceding claim, wherein said first DNA comprises a phage origin of replication and/or said first DNA comprises a phage replication gene and a phage lysis gene. 4. A cell according to any preceding claim, wherein each transducing particle is a non-self-replicating transducing particle. Any of the preceding wherein the essential genes, structural protein genes, and packaging signals are those of tailed phage, optionally P2, T4, T7, Phi92, lambda, K1-5, or 933w phage. A cell according to claim. The first DNA is contained in a phage genome, the phage genome is integrated into a plasmid, and optionally each particle is capable of infecting a target bacterium, the first DNA being a protein or protein in the target bacterium. 4. The cell of any preceding claim, comprising a nucleotide sequence of interest (NSI) capable of expressing RNA, said NSI replacing said essential gene or structural protein gene of said phage. each particle is capable of infecting a target bacterial cell, said first DNA comprising a nucleotide sequence of interest (NSI) capable of expressing a protein or RNA in said target bacterium, and said The presence of a protein or RNA encoded by an NSI mediates target cell killing or down-regulation of target cell growth or proliferation, and optionally a guided nuclease or CRISPR/NSI wherein said NSI is toxic to said target cell. 4. A cell according to any preceding claim, which encodes a component of the Cas system. 3. A cell according to any preceding claim, wherein said packaging signal is a pac or cos sequence, or a homologue thereof, or said packaging signal is a direct terminal repeat (DTR). 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 2, or is a homologue thereof from a different phage. A cell according to claim. comprising the first DNA as defined in any preceding claim, or comprising the second DNA as defined in any preceding claim, and optionally the isolated DNA contained in a plasmid. isolated DNA. (a) the cell comprises the second DNA as defined in any one of claims 1-12, provided that the cell contains the first DNA as defined in any one of claims 1-12; and (b) a vector (optionally a plasmid) comprising said first DNA and not contained in said cell.
A kit comprising
A kit, optionally wherein said cells are bacterial or archaeal cells. 1. A method of producing a transducing particle composition, said method comprising expressing a phage structural protein in a cell and replicating a first DNA in said cell, wherein the packaged first Transduced particles comprising DNA are produced, said method optionally comprising separating an amount of transduced particles from cellular material, resulting in an amount of purified transduced particles, said cells comprising: A cell according to any one of claims 1 to 12 when dependent on 2. 16. A composition comprising a population of transducing particles obtainable by the method of claim 15. 17. The method of claim 15 or the composition of claim 16, wherein said second DNA is comprised of plasmid DNA and less than 5% of the total DNA comprised in said composition is DNA of said plasmid. 18. The cell, DNA or composition of any one of claims 1-13, 16 and 17 for administration to a human or animal subject for medical use. 18. A cell, DNA or composition according to any one of claims 1 to 13, 16 and 17 for administration to a human or animal subject to treat infection of target bacterial cells, wherein A cell, DNA or composition, wherein said particle is capable of infecting and killing said target cell, optionally said infection is an intestinal, blood, lung or uterine tube microbiome infection. 18. A cell, DNA or composition according to any one of claims 1 to 13, 16 and 17 for use in a contained method of treating a disease or condition in a human or animal subject, wherein said disease or condition is mediated by a target bacterium, said target bacterium is contained in said subject (optionally contained in the intestinal, blood, lung, or uterine tube microbiome), and said method comprises administering said composition to said subject; so that the target bacteria are exposed to and killed by the antimicrobial means encoded by the first DNA, and the growth of the transduction particles is contained. 18. The composition of claim 16 or 17 for controlling dosing in a human or animal subject of transducing particle treatment of a targeted bacterial cell infection in said subject, wherein said particles target said first DNA. A composition capable of transducing cells, wherein said first DNA encodes an antimicrobial means that is toxic to target cells, thereby killing the target cells. 22. The composition of claim 18, 19, 20 or 21 for reducing the risk of acquisition of foreign gene sequences by said particles in said subject. A method of treating an environment ex vivo, comprising exposing said environment to a composition comprising a population of particles, wherein said particles transduce a first DNA into target cells contained in said environment. wherein said first DNA encodes an antimicrobial means that is toxic to target cells, thereby killing target cells, said composition being the composition of claim 16 or 17. the method is
(a) containment of the treatment in the environment;
24. The method of claim 23, for (b) reducing the risk of acquisition of foreign gene sequences by said particles in said environment and/or (c) controlling dosage of said particle treatment in said environment. .

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