Methods for growing propagator cells and phages, particularly for delivering CRISPR-CAS components via probiotic organisms

Abstract

To provide propagator cells, and to provide methods for propagating phage and transducing particles.SOLUTION: Provided is a method for producing a population of phage, the phage being of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of the species or strain, and the method comprising: (a) providing a population of second bacterial cells, wherein the second bacterial cells comprise the receptor on the surface thereof, are of a second species or strain different from the first species or strain, and can be infected with the phage of the first type; (b) infecting the second cells with the phage of the first type; and (c) propagating the phage in the second cells, thereby producing the population of phage.SELECTED DRAWING: None

Description

[Background technology]

[0001] Bacteriophages (phages) are a group of viruses that affect bacteria and Phages differ from bacterial and plant viruses in that they have a "lytic" life cycle, potentially They have either a "lysogenic" life cycle, which can become lytic, or a "non-lytic" life cycle. Phages that replicate by the lytic cycle may have the normal course of their life cycle. As an essential part of the lysogenic cycle, it causes the lysis of the host target bacterial cell. The phages that produce them are called temperate phages and replicate by a lytic life cycle. can cause lysis of the host bacterium or transfer their DNA to the host bacterium. They can either integrate into the bacterial DNA or become non-infectious prophage. Bacteriophages are those that only infect and grow within their specific bacterial hosts. Specifically, the host may be a strain level, a species level, or more rarely a bacterial virus. They are commonly found at the genus level. Specifically, this allows for the use of phages to identify dangerous Adsorption of bacteriophages to host cells can be achieved in some cases. In all cases except this, it is a prerequisite for the initiation of the infection process.

[0002] The specificity of phage-bacterial interactions, as well as the natural mechanisms by which phages infect and kill bacteria, The ability of phage therapy to inhibit lytic bacteria is a fundamental phenomenon on which the concept of phage therapy is based. Phages with a life cycle are suitable candidates for phage therapy. The use of phages in food production will increase the safety of fresh and ready-to-eat foods in particular. as a novel method for the biological control of unwanted pathogens, and as an option for the food industry. It is a limb.

[0003] International Patent Application WO00 / 69269 describes Enterococcus faecium for treating infections caused by vancomycin-susceptible as well as resistant bacteria. The use of certain phages is disclosed in International Patent Application WO 01 / 93904, which describes Clostridium alone or in combination with other agents to prevent or treat gastrointestinal diseases associated with Clostridium species The use of bacteriophages in combination with antimicrobial measures such as

[0004] U.S. Patent Application Publication No. 2001 / 0026795 describes a method for delaying inactivation by the host defense system. , thus modifying the phage to increase the period during which it is active in killing bacteria. Methods for producing bacteriophages have been described.

[0005] US Patent Application Publication No. 2002 / 0001590 describes the use of multidrug-resistant bacteria, particularly methicillin-resistant yellow bacillus. The use of phage therapy against Staphylococcus aureus was disclosed and an international patent was issued. WO 02 / 07742 discloses the development of bacteriophages with multiple host ranges. .

[0006] See, for example, U.S. Patent Application Publication Nos. 2002 / 0044922; 2002 / 0058027 and International Patent Application WO00 / 09994. No. 1 / 93904 discloses the use of phage therapy for the treatment of certain bacterial infectious diseases. .

[0007] However, commercial scale production of bacteriophage compositions for therapeutic use remains challenging. With current technology, the titers of phage compositions are low and usually exceed laboratory standards. 10 in model 9 ~10 11 pfu / ml, and 10 at commercial scale 7 ~10 9 range, while typical for therapeutic use The titer required for practical purposes remains limited. The titer of is low, typically 10 9 ~10 11 pfu / ml, and 10 at commercial scale 7 ~10 9 In the range of whereas the titer typically required for phage therapy is 10 12 pfu / ml. To reach the desired titer, very large volumes of liquid are required.

[0008] US20160333348 describes a method for delivering phages to host target bacterial cells using phages as vectors. The use of CRISPR / Cas systems to transduce phages has been described. In principle, phages can be used to transduce bacteria. Culture techniques and equipment can be used to grow cells in the same volume as the host cell. However, the propagation of such phages or lytic phages in target host cells is mediated by lysis. and / or by CRISPR / Cas targeting of host DNA or encoded by phage nucleic acid resident phages, by any other anti-host mechanisms or factors active in the host cell This can be hindered by host cell death due to the virus.

[0009] The increasing use of bacteriophages in industrial applications has led to the identification of bacteriophages. There is a demand for commercial quantities of phage, thus providing good yield titers, and There is a need for methods for the production of phages that reduce and / or production volumes. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Patent Application WO00 / 69269 [Patent Document 2] International Patent Application WO01 / 93904 [Patent Document 3] U.S. Patent Application Publication No. 2001 / 0026795 [Patent Document 4] U.S. Patent Application Publication No. 2002 / 0001590 [Patent Document 5] International Patent Application WO02 / 07742 [Patent Document 6] U.S. Patent Application Publication No. 2002 / 0044922 [Patent Document 7] U.S. Patent Application Publication No. 2002 / 0058027 [Patent Document 8] US20160333348 [Patent Document 9] WO2016205276 [Non-patent literature]

[0011] [Non-Patent Document 1] Hill et al. J Bacteriol. 173(14):4363-70 (1991) [Non-patent document 2] O'Loughlin et al. PLoS One. 2015:e0118533 [Non-patent document 3] Roberts, RJ et al. Nucleic Acids Res. 31, pp. 1805-1812 (2003) [Non-patent document 4] Lobocka et al. J. Bacteriol. 186, 7032-7068 (2004) [Non-patent document 5] Iida et al. Virology. 1 57(1):156-66(1987) [Non-patent document 6] McGrath et al. Applied Environmental Microbiology. 65:1891~1899 (1999) [Non-Patent Document 7] Roberts et al. Nucleic Acids Res 43:D298-D299. http: / / dx.doi.org / 10.1093 / nar / gkul046 [Non-patent document 8] K. Terpe Appl. Microbiol, Biotechnol. 72:211~222 (2006) [Non-Patent Document 9] Hannig et al. Trends in Biotechnology 16:54-60 (1998) [Non-Patent Document 10] Srivastava Protein Expr Purif 40:221~229 (2005) [Non-Patent Document 11] Levskaya et al. 2005. Nature 438:441-442 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides a solution by providing propagator cells for propagating phages. To this end, the present invention provides: [Means for solving the problem]

[0013] In the first configuration A method for producing a population of phages, the method comprising: host cells) by binding to cell surface receptors contained in bacteria of said species or strains. , is of a first type capable of infecting, and the method comprises: (a) a second species or strain that contains a receptor on its surface and is different from the first species or strain; providing a second population of bacterial cells comprising: (b) infecting a second cell with the first type of phage; (c) propagating the phage in a second cell, thereby producing a population of phage. The process and (d) optionally isolating phages from said population and

[0014] In the second configuration A cell for propagating a phage (propagator cell), the phage being a first In cells (host cells) of a bacterial species or strain, a cell surface receptor contained in the bacteria of said species or strain is The first type is capable of infecting by binding to the the target cells contain a receptor on their surface and are of a second species or strain, and the second species or strain The first species or strain is different from the first species or strain, thereby allowing the propagator cells to a protease capable of being infected by said first type of phage for propagation of said protease; Paget cells.

[0015] In the third configuration Optionally, culturing propagator cells to propagate said first type of phage. 1. A population of propagator cells according to the invention contained in a fermentation vessel. [Brief explanation of the drawings]

[0016] [Figure 1]A bacterial production strain (1) was genetically engineered to express a receptor recognized by helper phage (2) (series X), while receptor-free cells (series Y) served as a control. Both lines were then transformed with CGV and infected with helper phage to generate CGC-PLP (3). Only in line X, which possesses the helper phage receptor, did CGV-PLP (4) be produced, which can be used to deliver DNA to a target cell population expressing the phage receptor. [Figure 2] Figure 1 is a graph showing delivery of CGV to target cells ATCC43888 (obtained from ATCC) or EMG-2 (obtained from Coli Genetic Stock Center, CGSC), both of which express a receptor recognized by CGV-PLP. Lysates used for infection were generated for producer strains bearing a receptor for helper phage (solid bars) or control strains lacking the receptor (open bars). Only producer strains bearing the receptor, which helper phage can infect and produce CGV-PLP, were able to generate CGV-PLP lysates capable of infecting target cell populations. DETAILED DESCRIPTION OF THE INVENTION

[0017] According to the present invention, for example, it is possible to culture on a large scale and to obtain a useful phage population on a large scale ( In the use of cells useful for propagation and growth (e.g., commercial scale), bacterial cells artificially modifying the receptor expressed by the The advantages of such a profile (selecting cells according to their profile) are recognized. The phage may encode HM-crRNA or gRNA as described in US20160333348, The phages can infect humans, animals, plants, food, beverages, cosmetics, and the environment (e.g., soil, waterways, reservoir or crude oil production environment), e.g., US20160333348, the disclosure of which is incorporated herein by reference. These applications are described in the literature (including those described in the literature) and are effective in killing host bacterial cells. It is useful.

[0018] The proteinaceous receptors are primarily outer membrane proteins; the sugar moieties are located in the cell wall, periclinal membrane, and pericytes. For example, the receptors of the present invention include those that comprise teichoic acid, teichoic acid, and LTA. Either one is selected.

[0019] The infection process begins with the adsorption of bacteriophages. through a series of interactions between the binding proteins of the phage and receptors on the bacterial cell surface. The virus recognizes a potentially susceptible host, and then the virus DNA is released Therefore, phage adsorption is a crucial step in the infection process. Not only are they the primary pathogenic vectors, but they also represent the starting point of contact between the virus and the host, specifically defining the host range. There are also some that are

[0020] Bacteriophage adsorption generally involves three steps: initial contact, reversible binding, and irreversible binding. The first step consists of reverse adhesion (Duckworth 1987). The second step is Brownian motion, dispersion, diffusion or flow. are involved in random collisions between phages and hosts triggered by In a reversible process, binding to bacterial surface components is not final, but rather occurs after Phages can detach from the host. This process, first identified by Garen and Puck (1951), allows phages to bind to specific receptors. It may play a role in keeping the phage close to the cell surface to search for the scepter (K (Okjohn and Miller 1992). Specific connections between bacterial receptors and phage binding domains. This process allows the transfer of genetic material to the host. (More specifically, see Molineux and Pan on the mechanism of phage genome release.) (See review by ja (2013)) induces conformational rearrangements in other phage molecules. do.

[0021] Numerous review studies have identified a wide range of targets that bacteriophages target during adsorption. Host-associated receptors (proteins, sugars, and cell surface structures) have been emphasized (Lindberg 1977 ;Schwartz 1980;Wright, McConnell and Kanegasaki 1980;Heller 1992;Frost 1993;Henni ng and Hashemolhosseini 1994;Vinga et al. 2006;Rakhuba et al. 2010;Chaturongakul and Ounja i 2014). The nature and location of the host cell receptor recognized by bacteriophages These vary greatly depending on the phage and host. They range from peptide sequences to polysaccharide moieties. In fact, bacteriophages range from Gram-positive (Xia et al. 2011) to Gram-negative ( both cell walls of bacterial colonies (Marti et al. 2013), bacterial capsules or slime layers (Fehmel et al. 1975), and Located in appendages [e.g., fimbriae (Guerrero-Ferreira et al. 2011) and flagella (Shin et al. 2012)] It has been shown to bind to the receptors involved and the structure of this Diversity is a key factor in overcoming the evolutionary strategies adopted by these counterparts. This is a testament to the diversity of mechanisms deployed by different hosts and their various hosts. There are numerous possibilities to consider given the diversity and staggering abundance of phages that are predicted to inhabit the border. It is not surprising that sexual encounters occur (Clokie et al. 2011). In both cases, adsorption is thought to involve either components of the bacterial cell wall or protrusion structures. Therefore, in one embodiment, the receptor of the present invention is It may be any such receptor mentioned in the paragraph or elsewhere in this disclosure.

[0022] Optionally, the receptor is lipopolysaccharide (LPS), a heptose moiety, or a glycosylated portion of the host. Wall teichoic acid (WTA), YueB, or phage tail fiber protein or phages It contains a receptor recognized by gp21 of the IgG.

[0023] Receptors in the cell wall of Gram-positive bacteria Peptidoglycan, or murein, is an important component of the bacterial cell wall and It is often involved in phage adsorption. It is a multi-unit amino acid and sugar derivative. - A polymer composed of N-acetylglucosamine and N-acetylmuramic acid. These sugar components are connected via glycosidic bonds and held together via amino acid bridges. The cross-linking is carried out by diaminopimelic acid (Aminopimelic acid) to form a glycan tetrapeptide sheet. via a peptide bond between the amino acid analogue and D-alanine, or via a short peptide intercalation These peptide interbridges are formed via ridges in Gram-positive bacteria. They are more numerous, resulting in their characteristic thicker cell walls.

[0024] Another major component of the Gram-positive bacterial cell wall that may be involved in phage adsorption is glycerol. ribitol phosphate and amino acids. These are bound to the muramic acid of peptidoglycan. When bound to lipids, these are called lipoteichoic acids (LTA). For further details on this topic, see Tortora, Funke, and Case (2007) and Willey, Sherwood, and Woolvert (2009). on (2008), Pommerville (2010) and Madigan et al. (2012).

[0025] Most of the receptors identified so far are based on peptidoglycan or teichoic acid structures. The gram-positive bacteria reported in Table 1 are related to either Of the 30 phages, only 10 utilize other structures for adsorption. Among the phage species, nine species were found to bind to either teichoic acid (phage SPP1) or peptidoglycan (phage SPP2) due to reversible binding. It interacts with any of the residues in the phage 5, 13, c2, h, ml3, kh, L, and p2. These structures may play an important role in the adsorption of phages to Gram-positive bacteria. Emphasize.

[0026] Optionally, the receptor of the present invention is a peptide, a murein, a teichoic acid or a lipoprotein. Optionally, the phage is a phage of a family listed in Table 1. phage (optionally, the host is a host for a phage listed in Table 1) , and / or the receptor is a receptor for a phage listed in Table 1). In certain embodiments, the host and second cells are Gram-positive bacteria. The host and / or second cell are of the species or strains listed in Table 1 (the host and second cell (if different species or strains). Preferably, when the host is a Gram-positive bacterium, the receptor is Alternatively, preferably, when the host is a gram-positive bacterium, The base is a teichoic acid.

[0027] [Table 1A]

[0028] [Table 1B]

[0029] [Table 1C]

[0030] [Table 1D]

[0031] [Table 1E]

[0032] Receptors in the cell wall of Gram-negative bacteria In Gram-negative bacteria, the peptidoglycan layer is relatively thin and is the main component of the cell wall. These two layers are connected by Brown's lipoproteins. The outer membrane is decorated with proteins, polysaccharides and lipids (the latter two molecules originating from the LPS layer). LPS is a complex structure composed of a lipid bilayer. It consists of three parts: lipid A, a core polysaccharide, and Lipid A is a complex of O-polysaccharides. Lipid A is generally composed of glucosamine phosphate disaccha- The core polysaccharide consists of fatty acids attached to a ketodeoxyoctonate linker. The core polysaccharide and O-polysaccharide (O-chain or O-antigen) are attached to the outer membrane via a Contains several units of sugar residues extending outwards. Contains all three components of LPS. Cells with O-polysaccharide moieties are designated as smooth (S) type, and those lacking O-polysaccharide moieties are recognized as rough (R) type. Generally, the sugars that make up the O-antigen are very diverse, and the core polysaccharides are classified into several species. Therefore, phages specific only to S-type strains target the O-polysaccharide. tend to be more targeted and therefore generally more likely to be adsorbed to R-type cells. and thus have a narrower host range (Rakhuba et al. 2010).

[0033] Table 2(a) shows the cell wall-interacting phage receptor-binding proteins (RBPs). Interestingly, coliphages are able to collect receptors on Gram-negative bacteria. There is no preference for proteinaceous or polysaccharide receptors: some phages bind to cells Some adsorb to the wall proteins, some to the sugar moieties, and others require both structures for adsorption. In the case of Salmonella phages, the situation is not very different: some Some use proteins, some use sugar moieties, and some use both types of receptors On the other hand, Pseudomonas phages usually adsorb onto polysaccharide receptors. Although no definitive conclusions can be drawn from such a small sample size, Pseudomonas has two LPS moieties: a short chain LPS called the A band and a longer B band. It should be noted that the serovars may have bands of LPS (Beveridge and Graham 1991).

[0034] Optionally, the receptor is a host cell wall protein. Optionally, the receptor is Optionally, the receptor is an O-antigen, LPS lipid A, or LPS core polysaccharide. In some instances, the receptor is smooth LPS or rough LPS. Optionally, the host cell is Optionally, the host cell is an S-type bacterium and the receptor comprises the host O-antigen. The receptor contains the host's LPS lipid A.

[0035] Optionally, the receptor is a host cell wall protein. Optionally, the receptor is Optionally, the receptor is an O-antigen, LPS lipid A, or LPS core polysaccharide. In some instances, the receptor is smooth LPS or rough LPS. Optionally, the host cell is Optionally, the host cell is an S-type bacterium and the receptor comprises the host O-antigen. The receptor contains the host's LPS lipid A.

[0036] In one example, the host is E. coli, the phage is a coliphage, and The receptor is a polysaccharide receptor and / or a host cell wall protein. The cells express an E. coli polysaccharide receptor and / or an E. coli cell wall protein receptor. Optionally, the E. coli is of the same strain as the host cell. do.

[0037] In one example, the host is Salmonella and the receptor is a polysaccharide receptor. In some instances, the second cell is a Salmonella sp. Genetically engineered to express sugar receptors and / or Salmonella cell wall protein receptors. It is genetically engineered and, optionally, the Salmonella is of the same strain as the host cell.

[0038] In one example, the host is a Pseudomonas bacterium and the receptor is a polysaccharide receptor. In some instances, the second cell expresses a Pseudomonas polysaccharide receptor. Optionally, the Pseudomonas is genetically engineered to grow in the same strain as the host cell. It is of the type.

[0039] Optionally, the phage is a phage of a family listed in Table 2 (optionally In the alternative, the host is a host for a family listed in Table 2 and / or Sceptors are receptors for the phages listed in Table 2. Optionally, The phage is a phage listed in Table 2 (optionally, the host is a phage listed in Table 2). The host and / or receptor for the families listed in Table 2 (These are receptors for the phages listed in Table 1).

[0040] In one embodiment, the host cell and the second cell are Gram-negative cells. The second cell is an E. coli cell. Optionally, the host cell and / or the second cell is a cell of E. coli. 2 (Table 2) (wherein the host cell and the second cell are of different species or strains).

[0041] In one example, the host is E. coli, the phage is an E. coli phage, and the receptor The receptors are polysaccharide receptors and / or host cell wall protein receptors. In an example, the second cell is a cell that expresses an E. coli polysaccharide receptor and / or an E. coli cell wall protein receptor. The E. coli is genetically engineered to express the protein, and optionally the E. coli is grown in the same medium as the host cell. It is a stock.

[0042] In some instances, the host is a Salmonella species and the receptor is a polysaccharide receptor and / or In some instances, the second cell is a Salmonella multiforme. Genetically engineered to express sugar receptors and / or Salmonella cell wall protein receptors. It is genetically engineered and, optionally, the Salmonella is of the same strain as the host cell.

[0043] In some instances, the host is a Pseudomonas bacterium and the receptor is a polysaccharide receptor. In some instances, the second cell is genetically engineered to express a Pseudomonas polysaccharide receptor. The Pseudomonas is engineered and, optionally, the Pseudomonas is of the same strain as the host cell.

[0044] Optionally, the phage is a phage of a family listed in Table 2 (optionally In the alternative, the host is a host for a family listed in Table 2 and / or Sceptors are receptors for the phages listed in Table 2. Optionally, The phage is a phage listed in Table 2 (optionally, the host is a phage listed in Table 2). The host and / or receptor for the families listed in Table 2 (These are receptors for the phages listed in Table 1).

[0045] In one embodiment, the host cell and the second cell are Gram-negative cells. The second cell is an E. coli cell. Optionally, the host cell and / or the second cell is a cell of E. coli. 2 (Table 2) (wherein the host cell and the second cell are of different species or strains).

[0046] Table 2(b) shows that phages not only adsorb to the bacterial surface but also have a specific function in the O-chain structure. We report here the case of enzymatic degradation of the sugar moiety. All of these phages are podoviruses. It should be noted that it belongs to the family Podoviridae.

[0047] [Table 2A]

[0048] [Table 2B]

[0049] [Table 2C]

[0050] [Table 2D]

[0051] [Table 2E]

[0052] [Table 2F]

[0053] [Table 2G]

[0054] [Table 2H]

[0055] [Table 2I]

[0056] [Table 2J]

[0057] [Table 2K]

[0058] [Table 2L]

[0059] [Table 2M]

[0060] [Table 2N]

[0061] Receptors in other structures of Gram-negative bacteria This section discusses bacterial structures other than the cell wall that also act as receptors for phages. These include structures such as flagella, pili, and capsules. It can be found in species derived from both the ram strains (see, for example, Table 3).

[0062] Optionally, the receptor of the present invention is a flagella, pili, or capsule component (e.g., Table 3( in the species listed in Table 3) or in hosts of a species different from the listed species Optionally, the phage may be from a family listed in Table 3. (Optionally, the host is a host for a phage listed in Table 3) and / or the receptor is a receptor for a phage listed in Table 3 Optionally, the phage is a phage listed in Table 3 (optionally, the host is a host and / or receptor for the phages listed in Table 3. le 3 (which is a receptor for the phages listed in Table 3).

[0063] Flagella are long, thin helical structures that give cells their motility. It consists of a flagellar hook and a flagellar filament made up of flagellin proteins. (Willey, Sherwood, and Woolverton 2008). Table 3(a) shows the amino acid sequence of the flagellar proteins. We report on the attachment of phages to filamentous structures. The spiral movement of the flagellum allows the phage to move along its surface until it reaches the bacterial cell wall. They then attach to receptors located on the bacterial surface near the base of the flagellum. , irreversible adsorption occurs (Schade, Adler, and Ris 1967; Lindberg 1973; Guerrero-Ferreira Interestingly, some phages (φCbK and φCb13) have a reversible binding mechanism to the host flagellum. It has been observed that phage contain filaments protruding from the capsid that are responsible for specific binding. Irreversible adsorption occurs when the phage tail interacts with the tip of a pilus at the cell pole. In these phages, the interaction between the phage and the flagellum occurs only once (Guerrero-Ferreira et al. 2011). Even if they do work, irreversible adsorption occurs at the pili, so they are not compatible with the pili and mating partners. Focusing on the phage interacting with the forming structures, reported in Table 3(b) .

[0064] Pili are rod-shaped filamentous appendages used in bacterial conjugation (Lindberg 1973). The pilus elongates from the donor cell and attaches to receptors on the cell wall of the recipient cell. Polymerization causes contraction, which brings the cells closer together. This is achieved through binding proteins on the cell surface; genetic material is transferred from this junction. Adsorption to fimbriae has been reported in the Caudovirales (Madigan et al. 2012). ales and phages belonging to different orders (Table 3b). Indeed, Frost (1993 According to the WHO, the families Cystoviridae and Inoviridae Li constitute the majority of phages that adsorb to pilus structures. may be selective for a particular part of the pilus. Adsorption occurs only at the tip of the pilus. This is the case for phages of type φ6 (Click and Webster 1998). Attachment occurs on the side (shaft) of the structure (Daugelavicius et al. 2005).

[0065] [Table 3A]

[0066] [Table 3B]

[0067] [Table 3C]

[0068] [Table 3D]

[0069] The capsule is a flexible cementitious substance that extends radially from the cell wall. They act as binders between substrates and / or between cells and substrates (Beveridge and Graham 1991 The mucus layer is similar to the capsule but is more easily deformed. Both are released by the bacteria. The common components of these adhesive substances are polysaccharides or proteins (Madig 2012). Phage adsorption to the capsule or mucus layer is due to the enzymatic action of the exopolysaccharides that make up the layer. Hydrolysis of the layer is a reversible process, whereas irreversible bonds are This is achieved by binding to receptors on the phage cell wall (Rakhuba et al. 2010). As can be seen in Table 3(c), it is also found that the RBP that recognizes exopolysaccharides is present. The few phages identified are mostly of the Podoviridae morphology.

[0070] In some instances, the host is a Salmonella species (e.g., S. enterica Serovar Typhi murium)) and the receptor is the flagellum, vitamin B12 Imported outer membrane proteins, BtuB and In some instances, the receptor is selected from the group consisting of a lipopolysaccharide-associated O-antigen, a flagellum, or BtuB, The phage is a phage of the family Siphoviridae. is an O-antigen of LPS, and the phage is a phage of the Podoviridae family. The receptor is the FliC host receptor or the FljB receptor.

[0071] Optionally, the host is Salmonella enterica or Pseudomonas aeruginosa. Optionally, the receptor is a host receptor such as those listed in Table 4.

[0072] [Table 4]

[0073] The O-antigen structure of Salmonella O66 has been established and is composed of one linkage (most likely The only difference is the O-unit linkage and O-acetylation, which are known to be related to the O-antigen of E. coli O166. It has been reported that the O-antigen gene clusters of Salmonella O66 and Escherichia coli O166 differ from those of the O-antigen gene clusters of Salmonella O66 and Escherichia coli O166. The star indicates that only Salmonella O66 has the wzy gene replaced by a non-coding region. The function of the wzy gene of E. coli O166 was found to be similar to that of the wzy gene of E. coli O166. The activity of the enzyme was confirmed by the construction and analysis of deletion and transcomplementation mutants. Outside the O-antigen gene cluster, as previously reported in serogroups A, B, and D1 The functional wzy gene located in is proposed to be involved in O-antigen biosynthesis of Salmonella O66. The corresponding genes between the O-antigen gene clusters of Salmonella O66 and E. coli O166 are The sequence homology between them is 64 to 70%, indicating that they are derived from a common ancestor. After species diversification, Salmonella O66 lacked the wzy gene located in the O-antigen gene cluster. Activation and the expression of two new genes (wzy genes) both located outside the O-antigen gene cluster The acquisition of the specific O-antigen (the gene for the O-acetyl modification and the prophage gene for the O-acetyl modification) It seems to have taken shape.

[0074] In some instances, the second cell comprises an expressible E. coli (e.g., E. coli O166) wzy gene. In some instances, the second cell is an expression-competent E. coli (e.g., Optionally, the host cell does not contain the wzy gene of E. coli or Salmonella spp. For example, Salmonella spp. O66) cells.

[0075] In some instances, the phage or particle comprises a phage genome or phagemid, e.g., A genome or phagemid is a DNA that encodes one or more proteins or nucleic acids of interest; For example, to target the host cell genome or antibiotics to kill the host cell. Contains crRNA.

[0076] Alternatively, instead of bacteria, the host cell and the second cell (propagator cell) are archaea. Instead, the disclosure herein relating to bacteria makes the necessary modifications to archaea. You can also read more.

[0077] The bacterial target host strain or species recognizes and cleaves or otherwise destroys the invading nucleic acid. or a restriction-modification system, such as a RM containing a restriction endonuclease, which can degrade or The host DNA may contain a methylation system (RM system). The host DNA methylates the host DNA and transfers it to the RM system. Therefore, RM is protected by the action of methyltransferases that protect against The genome does not contain one or more regulatory systems encoded by the host cell. providing a second bacterial cell (propagator cell) lacking the nucleic acid encoding the restriction endonuclease; Additionally or alternatively, the second cell may be provided by the host cell. one or more methyltransferases encoded by the host cell, optionally All or substantially all of the encoded methyltransferases (e.g., For example, at least 50, 60, 70, 80, or 90% of the total cell mass is comprised of a second cell. Optionally, the second cell is a host cell. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or at least 1, 2, 3, 4, 5, 6) , 7, 8, 9 or 10) of the methyltransferases.

[0078] Advantageously, to produce phage or transducing particles that target specific bacterial host populations, To do this, phages or particles are produced in bacterial strains related to the target host cells, e.g., Evading cellular defense mechanisms, such as RM systems or restriction endonuclease action It may be beneficial to produce the nucleic acid (e.g., DNA) of a phage or particle capable of carrying out the procedure. Thus, optionally, the host cell and the second cell (propagator cell) are of the same species (or except that the second cell contains one or more genetic modifications not found in the genome of the host cell. strains of the same species; such modifications can be deletions of one or more protospacer sequences, e.g. For example, the host cell contains such a sequence and the phage or particle recognizes the sequence in the host cell. (The gene expresses a crRNA that recognizes the protospacer sequence and guides Cas to modify it.) For example, Then, when the phage or particle infects a target host cell (the latter shares a common methyltransferase with the second cell), a second bacterial methyltransferase), and a second bacterial phage or particle Such modifications of DNA may be useful to shield the DNA against restriction modifications. By employing (or selecting) a second cell according to the present invention that exhibits the surface receptor Thus, the present invention next provides beneficial DNA modifications to restriction and modification by the target host bacterium. Advantageously, the production of phages or particles in strains capable of transfecting the target host cells is achieved. In bacteria, protospacer targeted by crRNA encoded by phage or particle The sequence may be deleted or not naturally occurring in the genome of the second cell. and resulting Cas-mediated cleavage of the second cell's genome, resulting in the production of phage or particles. It doesn't happen between.

[0079] A heterologous methyltransferase (MTase) is used to convert the production bacteria (the propagator -bacteria or second cells) to give a methylation pattern similar to that of the target host bacterium For example, a production strain containing the desired MTase for use in the present invention can be identified by WO201620, which is incorporated herein by reference, provides an illustration of a method for providing a medicament for the treatment of rhesus macular degeneration. In bacteria and archaea, some DNA methyltransferases are known to modify Depending on their location and the type of reaction they catalyze, they can be separated into three distinct classes: N6-methyladenine (m6A) and N4-methylcytosine (m4C) are adenine and cytosine, respectively. 5-methylcytosine (m5C) is derived from the methylation of the amino moiety of cytosine, while 5-methylcytosine (m5C) is derived from the methylation of the amino moiety of cytosine. This is the result of methylation at the C5 position of .

[0080] Non-limiting examples of DNA MTases useful in the context of the present invention include type II MTases in lactococci. It contains LlaPI from phage Φ50, which can be introduced to protect against HIV-1 (Hill et al., 2003). J Bacteriol. 173(14):4363-70 (1991)). Optionally, the producing bacterium is, for example, N6- One or more DNAs that catalyze the methylation of adenine to produce methyladenine (m6A) Optionally, the production bacteria encodes and expresses an A-modifying enzyme, e.g., N4-methylcytosine. one that catalyzes the methylation of cytosine to produce (m4C) or 5-methylcytosine (m5C) or multiple DNA modifying enzymes. Optionally, the production bacterium encodes and expresses adenine residues. The R- The M system is sensitive to adenine methylation. A polypeptide that acetimidates the adenine residues in the DNA fragments is known to be effective against such systems. Acetoimidation of adenine residues in the producing host bacteria will protect the Non-limiting examples of polypeptides that can be prepared include those in which adenine residues are converted to N(6)-methyladenine, Thereby, phage Mu and Mu-like profiles protect against adenine-sensitive restriction enzymes. Examples include the mom gene derived from the Rd gene sequence of Haemophilus influenzae. (FluMu), Neisseria meningitidis type A strain Z2491 (Pnmel) and Koch-Weeks (See H. influenzae biotype aegyptius ATCC 111 16). The methylation patterns conferred by methylase were analyzed by Pacbio SMRT sequencing. We used established DNA sequencing techniques (O'Loughlin et al. PLoS One. 2015:e0118533) Once generated, the production strain can be used to deliver DNA to the target strain. bacteriophage particles of

[0081] Bacterial "restriction-modification systems" (RM systems) (1) methylate DNA at specific sequences; (2) methyltransferases that regulate methylation and / or (3) unmethylated (types I, II, and III) or The RM system contains restriction enzymes that cut methylated (type IV) DNA. Bacterial DNA with a pattern is cut at multiple positions by restriction enzymes in the RM system. They constitute a defense system. Most bacteria contain more than one RM system. Roberts , RJ et al. Nucleic Acids Res. 31, pp. 1805-1812 (2003). Type I methyltransferase Type II and III enzymes require the presence of a corresponding specific protein for functionality. This methyltransferase does not require any additional proteins to function. Therefore, methyltransferases and restriction enzymes (modifying or inhibiting) useful in the present invention or expressed in the producing bacteria, thereby providing a target for the production of RM systems. either as a heterologous polypeptide modifying the stem) can be expressed by bacterial restriction-modification systems (e.g. any methyltransferase or restriction enzyme in the ribosomal DNA (e.g., type I, II, III, or IV) Thus, in some instances, the production bacteria (second cells or propagator cells) ) genome encodes a type I methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in some instances, the production bacteria (second cells or propagator cells) The genome encodes a type II methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in some instances, the production bacteria (second cells or propagator cells) may be cultured in a broth containing 100% ethanol. The genome encodes a type III methyltransferase that is also encoded by the host bacterium. Additionally or alternatively, in some instances, the production bacteria (second cells or propagator cells) may be cultured in a broth containing 100% ethanol. Nom encodes a type IV methyltransferase that is also encoded by the host bacterium .

[0082] In some instances, two or more genes encoding enzymes of the endogenous restriction-modification system of the producing bacterium are The nucleic acid sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) disrupted or altered in nature (e.g., reduced or eliminated in activity).

[0083] The producing bacteria (i.e., the second or propagator cells) may be Gram-positive or Gram-negative. Thus, for example, the producing bacteria can be Escherichia coli, Bacillus subtilis, ), Lactobacillus rhamnosus, Salmonella enterica (S almonella enteria, Streptococcus thermophilus s), Listeria, Campylobacter or Staphylococcus aureus In some examples, the producing bacteria is E. coli strains MG1655, Nissle, BW25113, BL21, TOP 10, or MG1655 Δdam Δdcm ΔhsdRMS.

[0084] The enzymatic activity of the endogenous RM system can be determined by methods known in the art or by the function and activity of the polypeptide. These methods can be used to destroy the activity of the ATP-binding protein. including, but not limited to, point mutations (e.g., missense or nonsense or a single base pair insertion or deletion resulting in a frameshift), and / or cleavage. In some embodiments, polypeptide inhibitors are used. By using the enzymes, it is possible to destroy or inhibit the enzymatic activity of the bacterial restriction-modification system (RM system). Such polypeptide inhibitors are known in the art. The biter may be, for example, encoded within the DNA of the phage or particle and / or may be present on the phage or particle. For example, the P1 phage may be packaged as a protein in the colon. Encoding two polypeptide inhibitors that inhibit type I restriction enzymes found in bacteria (Loboc ka et al. J. Bacteriol. 186, 7032-7068 (2004)). In some embodiments, the endogenous RM system The system is a polypeptide inhibitor, i.e., a methylation inhibitor that promotes and protects the delivered DNA. The activity of the host methyltransferase is inhibited or destroyed by the introduction of a polypeptide that stimulates the activity of the host methyltransferase. can be.

[0085] Inhibitors of enzymes in the RM system include, but are not limited to, REases (restriction enzymes). nuclease), thereby allowing the host's RM enzyme system to degrade the phage or particle These proteins prevent DNA cleavage. Enzyme activity of endogenous bacterial RM systems Non-limiting examples of RM enzyme inhibitors that can be used in accordance with this invention include those that disrupt or modify the As a rule, (a) we produce the protein ArdA, which inhibits all major classes of type I RM systems. (b) orf18 from Enterococcus faecalis, which produces the mitochondrial β-glucanase; and (b) the type I RM enzyme EcoKI. Examples of such proteins include gp0.3 from bacteriophage T7, which produces the protein Ocr that captures the RM signal. Additional non-limiting examples of proteins that can be used to block the enzymatic activity of the stem include: Examples of masking proteins include the masking proteins in the head of the phage. Upon DNA injection, the phage DNA is bound to the phage DNA, thereby forming the RM Non-limiting examples of masking proteins useful in the context of this invention include: Examples include the DarA and DarB proteins (Iida et al., Virology. 1 57(1):156-66 (1988) 7) These proteins are expressed by P1 bacteriophage during the lytic cycle. Upon DNA injection into the host bacterium, these are transformed into type I R- It binds to and masks the recognition site of M.

[0086] Additionally or alternatively, the endogenous RM system of the producing bacterium may contain at least one heterologous methyltransferase. The methylation pattern of the producing host bacterium can be altered / modified by the expression of the transferase. The endogenous methylation of the producing host bacterium is regulated so that the methylation pattern is substantially similar to that of the target host bacterium. Any methyltransferase that alters the methylation pattern may be used in accordance with the present invention. Heterologous methyltransferases can induce methylation patterns that are substantially similar to those of the target bacterial strain. They may be derived from the same or different organisms, as long as they provide a turn to the production host bacterium. Non-limiting examples of DNA MTases useful for protecting against the type II RM system of Lactococcus include An example of a gene that can be introduced to target the phage is LlaPI from Φ50 (McGrath et al. Appl. Lied Environmental Microbiology. 65:1891-1899 (1999)). The methylation patterns conferred by the transferase were analyzed by Pacbio SMRT sequencing. Evaluate the expression of genomic DNA using established DNA sequencing techniques (O'Loughlin et al., 2014). One. 2015:e0118533). Once generated, the production strain can be used for DNA delivery into the target host strain. The phage or particle is produced.

[0087] Further heterologous DNA-modifying enzymes are known to interact with the RM system of the producing bacterium and the RM system of the target host bacterium. It can be expressed in the production bacterium so as to be substantially similar to An example of such a DNA modifying enzyme is the enzyme that converts adenine residues in DNA to acetamidoadenine. Examples of the DNA of a phage or particle include those encoding polypeptides that convert the The polypeptide that converts the adenine residue to acetamidoadenine is adenine methylation. This protects the DNA against restriction enzymes that are sensitive to the enzyme. Non-limiting examples of polypeptides that can convert adenine residues in For example, adenine residues are converted to N(6)-methyladenine, thereby increasing the adenine-sensitive The mom gene from phage Mu and Mu-like prophage sequences protects against endonuclease-dependent restriction enzymes. Examples include Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnmel), and Koch-Williams See Streptomyces cerevisiae ATCC 111 16; (Drozdz et al. Nucleic Acids Res. 40(5):2119-30 (2012)). (This refers to lighting.)

[0088] In some embodiments, the polypeptide inhibitors and other DNA modifying enzymes described herein The polynucleotide encoding the gene is then transferred to the target host bacterium to protect the delivered DNA from the RM system. can be directly introduced into the genome of a phage or particle for use in protecting against .

[0089] Thus, in some embodiments, the present invention provides a method for the production of a heterologous nucleic acid of interest by incorporating the heterologous nucleic acid into a bacteriophage. or a method for increasing the efficiency of transduction into a target host bacterium via a transduction particle, the method comprising the steps of: at least one heterologous nucleic acid of the formula (I) is introduced into the DNA of the phage or particle before introduction into the production bacterium. a process for transforming a production host bacterium into a bacterium that disrupts at least one enzyme of an endogenous RM system; and / or a polynucleotide encoding at least one heterologous methyltransferase. modified to include a methyltransferase, thereby methylating the DNA of the phage or particle. and modified methylation patterns (as compared to the production bacteria without the altered methylation activity). at least one variant of interest (compared to the DNA of the phage or particle produced in generating DNA for a phage or particle containing a seed nucleic acid; and producing a phage or particle containing said recombinant DNA comprising said bacteriophage or a step of infecting a target host bacterium with the particles, wherein the target host bacterium is an acidobacterium. Identical, similar, or substantially similar methylation patterns (or RM systems) and whereby the control production bacterium (the control production host bacterium) is the same as that of the target host bacterium. having the same, similar or substantially similarly altered methylation activity bacteriophage grown in a medium containing a target nucleic acid (e.g., a phage grown in a medium containing a target nucleic acid) increasing the efficiency of introducing the heterologous nucleic acid of interest into the target host bacterium compared to In some aspects, after infection with the phage or particle, the production Live bacteria are modified to alter their RM systems (e.g., to alter at least one of the endogenous RM systems). disrupt one enzyme and / or encode at least one heterologous methyltransferase The polynucleotide may include a polynucleotide that is a

[0090] In some embodiments, the heterologous nucleic acid of interest is delivered to the target host via a phage or transduction particle. A method for increasing the efficiency of introducing into bacteria at least one enzyme of an endogenous RM system. methyltransferases by disruption of the enzyme and / or expression of at least one heterologous methyltransferase The producing bacteria with altered enzyme activity are then transfected with bacteria containing DNA containing at least one heterologous nucleic acid of interest. infecting the modified phage or particle, thereby methylating the DNA; containing the DNA of a bacteriophage or particle with a specific methylation pattern, producing a bacteriophage containing / encoding a heterologous nucleic acid; and methylation patterns (or RMs) that are identical to, similar to, or substantially similar to those of the bacteria a target host bacterium having a virulence factor receptor agonist (VPR) ... The control production bacteria (the control production host bacteria is the target host bacteria described herein) methylation activity identical to, similar to, or modified to be substantially similar to that of bacteriophage or particles produced in a laboratory (which did not have the ability to compared to introducing the heterologous nucleic acid into the target host bacterium. and increasing the efficiency of the bacteriophage. or after infection by the particle, modifying the producing bacteria to alter their RM systems (e.g., disrupting at least one enzyme of the causative RM system and / or introducing at least one heterologous methyltransferase The polypeptide may include a polynucleotide encoding a transferase.

[0091] In some instances, the target host bacterium interacts substantially with the restriction-modification system (RM system) of the producing host bacterium. These are selected based on having similar DNA methylation patterns.

[0092] Methylation patterns are present in bacteria, as are specific sequences that are methylated (e.g., GmATC). The difference between methylation patterns is determined by the type of methylation present (e.g., m4C). The level of similarity (whether natural or the result of modification) refers to the frequency with which a target site has the appropriate type of methylation. A methylation pattern is defined as a pattern of at least two nucleotides between target sites that have the appropriate type of methylation as described herein. Similarity of approximately 20% or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, or more, or any range therein Thus, in some embodiments, the methylation pattern is Between about 20% and 99% or more similarity between the bacterium and the target bacterium, about 30% to 99% or more similar, about 40% to 99% or more similar, about 50% to about 60% to 99% or more similar; about 70% to 99% or more similar; 0% to 99% or more similar, approximately 80% to 99% or more similar , about 85% to 99% or more similar, about 90% to 99% or more similar It may be similar to or about 95% to 99% or more similar to the target host bacterium. The methylation patterns of the introduced DNA (the DNA of the modified bacteriophage or particle) Qualitative similarity means that the introduced DNA has a methylation pattern that is substantially similar to that of the target host bacterium. This means that the introduced DNA is not as degraded as the introduced DNA that does not share the same DNA. In this case, the methylation patterns of the producing and target bacteria may be identical.

[0093] In some embodiments, the present invention provides a method for the detection of RM systems that are identical to or similar to the RM systems of target host bacteria. or a DNA containing a modified DNA methylation pattern substantially similar thereto, At least one heterologous nucleic acid of interest is incorporated into the DNA (genome) of the bacteriophage or particle. Thus, for example, a bacteriophage or particle containing a modified methylation protein is provided. Bacteria with turn (a methylation pattern substantially similar to the RM system of the target host bacteria) The DNA of the phage or transduction particle contains (1) a polynucleotide encoding a CRISPR array; or (2) (a) a polynucleotide encoding a Cas9 polypeptide; (b) a polynucleotide encoding a CRISPR array. and / or (c) a type II CRISPR-Cas system containing a tracr nucleic acid. In some embodiments, the CRISPR array (a) and the polynucleotide encoding the tracr nucleic acid (c) can be The nucleotides can be fused to each other. In a further embodiment, the modified methyl rification pattern (identical to, similar to, or substantially similar to the RM system of the target host bacterium) The DNA of a bacteriophage or particle with a methylation pattern (which corresponds to the DNA fragment) is then (1) subjected to CRISPR array analysis. or (2) a polynucleotide encoding (a) a CRISPR array; and / or (b) at least one polypeptide encoding one or more type I CRISPR polypeptides. The recombinant type I CRISPR-Cas system may include a recombinant type I CRISPR-Cas system containing nucleotides. In this embodiment, at least one heterologous nucleic acid of interest is inserted into the target cell at a site of integration that is unnecessary or a site of integration that is complementary. The vector can be integrated into the DNA (e.g., genome) of the bacteriophage or particle.

[0094] As used herein, a "discarded site" refers to a portion of the genome of a bacteriophage or particle. DNA not necessary for maintenance, phage or particle production, and delivery of the packaged DNA or a portion of the genome that is not required to perform such function. Any site in the genome of the bacteriophage or particle can be used to integrate a nucleic acid of interest. Some exemplary unwanted sites include: These include, but are not limited to, (a) phage-encoded restriction-modification systems (e.g., P1 phage) (b) genes that block superinfection (e.g., simABC); (c) restriction-modification (d) inhibitors of the decoration system (e.g., darA in P1 phage), (e.g., insertion sequence elements ( (e) addition systems (e.g., phd / do in P1 phage) c) or (f) any combination thereof.

[0095] "Capturing site" or "capturing site," as used herein, refers to a bacteriophage Maintenance of the genome of the phage or particle, production of the phage or particle, and distribution of the packaged DNA Nucleic acids that are necessary for delivery but are destroyed by integration of the nucleic acid of interest (complementary site of integration) bacteriophage that can be complemented by a complementing polynucleotide encoding Complementary polynucleotide refers to an essential portion of the DNA or genome of a particle. can be integrated into the genome of the production bacterium or can be contained on a plasmid in the production bacterium Thus, the nucleic acid of interest is incorporated into the complementary site of the DNA of the bacteriophage or particle. Upon receipt of the DNA fragment, the producing bacteria encodes a complement to the complement site on the bacteriophage or particle's DNA. The encoding polynucleotide can be contained on a plasmid or within its genome. Complementary sites include, but are not limited to, (a) lytic cycle activators (e.g., , coi in P1 phage), (b) lytic genes (e.g., kilA in P1 phage), (c) tR NA (e.g., tRNA1,2 in P1 phage), (d) particle components (e.g., (e) any combination thereof), or .

[0096] In one embodiment, the methylation pattern of a production strain, e.g., E. coli MG1655 or B. subtilis 168 The endogenous restriction-modification system was deleted and a heterologous methyltransferase was introduced as follows. The restriction-modification gene is modified by introducing a restriction enzyme gene. Nucleic Acids Res 43:D298-D299. http: / / dx.doi.org / 10.1093 / nar / gkul046). These restriction-modification systems utilize standard recombineering strategies known in the art. Once deleted, the foreign methyltransferase gene can be deleted using It is inserted into a plasmid and recombines into the host genome under the control of a constitutive or inducible promoter. These genes are either codon-optimized for the native sequence or for the production host. Alternatively, a heterologous methyltransferase can be used to directly derive the target strain. The gene can be used to give a similar methylation pattern as the target strain. The methylation patterns conferred by individual methyltransferases were analyzed using PacBio S Established DNA sequencing techniques such as MRT sequencing (O'Loughlin et al. PLoS One. 2 Once generated, the production strain is used to test for D1 expression in the target host strain. Produce bacteriophages or transduction particles for NA delivery.

[0097] promoter: Promoters can be used in, for example, recombinant nucleic acid constructs, polynucleotides, expression cassettes, and and for use in preparing vectors containing the polynucleotides and recombinant nucleic acid constructs of the present invention. These promoters include constitutive, inducible, temporally regulated, developmentally regulated, and chemically regulated promoters. These various types of promoters are known in the art.

[0098] Thus, in some embodiments, suitable vectors that are functional in an organism of interest are prepared in accordance with the present invention. Constitutive, inducible, temporally regulated, developmentally regulated, and chemically regulated promoters are available. The expression of the present invention can be achieved using a recombinant nucleic acid construct of the present invention operably linked to a target. In an exemplary embodiment, for example, an inducible promoter that is functional in the organism of interest can be used. Using a recombinant nucleic acid construct of the present invention operably linked to a motor, the inhibition can be made reversible. It can be done.

[0099] The choice of promoter depends on the quantitative, temporal and spatial requirements for expression, as well as the type of promoter. Promoters for many different organisms are known in the art. Based on the extensive knowledge existing in the art, the specific host organism of interest can be Thus, for example, a suitable promoter for Much is known about the upstream promoters of constitutively expressed genes, and Such knowledge can be easily evaluated and implemented in other systems as appropriate.

[0100] Exemplary promoters useful in the context of this invention include promoters that are functional in bacteria. Useful promoters for bacteria include, but are not limited to, , L-arabinose-induced (araBAD, P BAD ) promoter, any lac promoter, L-lamb North-induced (rhaP B A-D) promoter, T7 RNA polymerase promoter, trc promoter -, tac promoter, lambda phage promoter (P L , P L -9G-50), anhydrotetra Cyclin-inducible (tetA) promoter, trp, lpp, phoA, recA, proU, cst-1, cadA, na r, lpp-lac, cspA, T7-lac operator, T3-lac operator, T4 gene 32, T5-lac operator promoter, nprM-lac operator, Vhb, protein A, Corynebacterium-Escherichia coli-like promoter promoter, thr, hom, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb , alpha-amylase (Pamy), Ptms, P43 (two overlapping RNA polymerase σ factor recognition sites) positions, σA, and σB), Ptms, P43, rplK-rplA, ferrodoxin promoter, and / or xylose promoters. (K. Terpe Appl. Microbiol. Biotechn ol. 72:211-222 (2006); Hannig et al. Trends in Biotechnology 16:54-60 (1998); and See Srivastava Protein Expr Purif 40:221-229 (2005).

[0101] In some embodiments of the invention, an inducible promoter may be used. For example, chemically controlled promoters can be used to induce expression in organisms by application of exogenous chemical regulators. Chemically regulated promoters can modulate the expression of genes in Regulation of expression of the nucleotide sequences of the invention via the nucleotide sequences of the present invention allows the organism to express the nucleotide sequences, for example, by inducing chemicals. Only after the treatment can the RNA and / or polypeptide of the present invention be synthesized. Therefore, the promoter is a chemically inducible promoter, where application of a chemical induces gene expression. Alternatively, it may be a chemically repressible promoter in which the application of a chemical substance represses gene expression. In some embodiments, the promoter is photoinducible, where application of a particular wavelength of light induces gene expression. It may also contain a promoter of the type (Levskaya et al. 2005. Nature 438:441-442).

[0102] Description By way of example, the present invention provides the following description.

[0103] 1. A method for producing a population of phages, wherein the phages are introduced into cells (host cells) of a first bacterial species or strain. host cells) by binding to cell surface receptors contained in bacteria of said species or strains. , is of a first type capable of infecting, and the method comprises: (a) a second species or strain that contains a receptor on its surface and is different from the first species or strain; providing a second population of bacterial cells comprising: (b) infecting a second cell with the first type of phage; (c) propagating the phage in a second cell, thereby producing a population of phage. The process and (d) optionally isolating phages from said population and

[0104] Preferably, the second cell is a bacterial cell. Alternatively, the second cell is an archaeal cell; a eukaryotic cell. cells, yeast cells, CHO cells or HEK293 cells.

[0105] In one embodiment, the receptor is an exogenous nucleotide sequence that can be expressed (i.e., a second the exogenous sequence is a protein encoded by a non-wild-type sequence of a second bacterium For example, the nucleotide sequence may be a nucleotide sequence contained in the genome of a host cell. or at least 85, 90, 95, or 98% identical to the sequence.

[0106] In another embodiment, the receptor is activated by the action of one or more enzymes in the second bacterium. the genome of the second bacterium comprises a sugar moiety produced by the or a plurality of expressible exogenous nucleotide sequences (i.e., non-wild-type sequences of the second bacterium). For example, each nucleotide sequence is identical to a nucleotide sequence contained in a host cell. or at least 85, 90, 95 or 98% identical.

[0107] Optionally, the second species or strain does not naturally express the receptor. The host and / or second cell may be a non-naturally occurring bacterial cell. It may also be a live cell.

[0108] Optionally, the host cell contains an expressible exogenous nucleotide sequence encoding the receptor. (e.g., integrated into a chromosome).

[0109] Alternatively, instead of infecting the second cell with the phage in step (b), The encoding DNA can be introduced into a second cell by other means, such as electroporation. In some instances, step (c) comprises culturing the second cells in a culture vessel, such as a steel fermentor. This includes culturing.

[0110] The second cell contains the cellular machinery operable to replicate the DNA encoding the phage.

[0111] In some instances, the host cell is pathogenic to humans (e.g., the host cell is a clostridial C difficile cells) and / or the second cell is a human The second cell is a non-pathogenic cell of a commensal species in the gut (e.g., a Lactobacillus cell). Lactobacillus lactis (L lactis) or Lactobacillus reuteri (L r euteri). For example, the second cell can be a cell of, for example, a mammalian cell, as described in US20160333348 (the specific disclosure of which is incorporated herein by reference). are carrier cells described in the above, which are incorporated herein by reference. In one example, the present invention provides a method for treating a host cell infection in a human or animal subject (e.g., an infection of the intestine of a subject). a method of treating or preventing a tumor comprising administering to a subject the second population of cells; In order to populate the intestine of a subject, the cells are transfected with the first type of phage (e.g., prophage) and a carrier cell comprising the phage, wherein the phage is delivered to a host cell contained in the subject (e.g., the intestine of the subject). a cRNA or gRNA encoding a cRNA or gRNA targeting a protospacer sequence in a host cell containing the cRNA or gRNA; the second cell is a carrier for a phage that infects a host cell of interest, and the crRNA Alternatively, nucleic acid of the phage encoding the gRNA is produced in the host cell, thereby An active CRISPR / Cas system is formed in the cell, whereby Cas binds to the crRNA or gRNA is guided by the protospacer sequence contained in the genome of the host cell, modify (e.g., cleave) the DNA, thereby killing or disrupting the host cell The present invention provides a method for inhibiting the growth or proliferation of a bacterial cell, thereby treating or preventing infection. In some embodiments, such methods are for treating or preventing a disease or condition in a subject. and the disease or condition is associated with or caused by infection of a host cell; The disease or condition is thereby treated or prevented. It may be any such cell disclosed herein.

[0112] 2. Phages can target one or more protospacer nucleotide sequences. the bacterial Ca of said host cell strain or species to form an active CRISPR / Cas system that is capable of A crRNA (or single guide RNA) (or tracrRNA, if necessary) that can function together with s is used. Each target sequence is contained in the genome of the host cell and By this, the crRNA (or gRNA) guides the Cas of the host cell to modify the target sequence (e.g., cleavage). disrupting the host cell, thereby killing the host cell or reducing the growth of the host cell population. Method 1 described above.

[0113] In one example, the phage is a phage derived from a phage strain selected from the group consisting of phage strains described in US2016, the specific disclosure of which is incorporated herein by reference. 0333348, or a gRNA encoding a nucleotide sequence.

[0114] 3. When infected with the phage, the second cell does not contain the active CRISPR / Cas system. Method 2 described above.

[0115] For example, one or more Cas may be defective in a CRI that is not operable with a crRNA or gRNA. It is repressed, inactivated or knocked out in a second cell containing the SPR / Cas system.

[0116] In some instances, an active CRISPR / Cas system is provided, the specific disclosure of which is incorporated herein by reference. As disclosed in incorporated US20160333348.

[0117] 4. The method of statement 2 or 3, wherein the genome of each second bacterial cell does not contain said target sequence.

[0118] In some instances, the target sequence is selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 As disclosed in US Pat. No. 6,033,348.

[0119] 5. (a) a Cas (e.g., Cas3, 9, cpf1 and / or CASCADE Cas) of the second cell transfects the crRNA ( or gRNA), (b) the tracrRNA of the second cell is not operable with the crRNA; and / or (c) causing the second cell to produce the crRNA from a nucleotide sequence encoding the crRNA. is not operable to generate said gRNA from a nucleotide sequence encoding said gRNA (or (not operable to create a

[0120] 6. The crRNA (or gRNA) is transduced into a Cas (e.g., Cas3, 9, cpf1 and / or Cas) of the second cell. Any one of statements 2 to 5 containing a repeat sequence that is not operable with CADE Cas Law.

[0121] In one example, the repeat is a repeat of the formula (I) of US2016, the specific disclosure of which is incorporated herein by reference. As disclosed in US Pat. No. 6,033,348.

[0122] 7. The nucleotide sequence of the phage is identical to that of the crRNA (or gRNA) in bacteria of the host species or strain. a promoter for transcription of the crRNA (or gRNA) in said second species or strain, any of statements 2 to 6, operably linked to a promoter not for transcription of There is one way.

[0123] In some instances, the promoter is constitutively active in the second cell.

[0124] 8. The host species or strain of bacteria kills the first type of phage that infects the host bacteria. and a second bacterium containing an antiphage toxin or mechanism that inhibits or reduces the growth of the second bacterium. The method of any one of statements 1 to 7, which does not include a mechanism.

[0125] 9. The host species or strain of bacteria kills the first type of phage that infects the host bacteria. the second bacterium comprises a CRISPR / Cas system that is active in inhibiting or reducing the growth of the The method of any one of statements 1 to 8, which does not include said system.

[0126] 10. A second bacterial cell is genetically engineered to produce a receptor, said second species or the wild-type bacteria of the strain do not produce said receptor.

[0127] 11. The phage is operable in the second cell and in cells of the first species or strain. 11. The method of any one of statements 1 to 10, comprising an origin of replication which is

[0128] 12. The method of any one of statements 1 to 11, wherein the second cell is an E. coli cell.

[0129] For example, the second cell is not pathogenic to humans. Group 1 or 2 cells (e.g., cells of a species listed as Group 2 in the tables herein) etc.).

[0130] 13. Any of statements 1 to 12, wherein the first and second cells are of the same species (e.g., E. coli strains). Or one way.

[0131] For example, the second cell is a genetically engineered version of the host cell, e.g., wherein the second cell comprises a defective CRISPR / Cas system as referred to herein; and / or expressing a toxin that does not contain the protospacer sequence and / or is expressed by a host cell do not.

[0132] 14. The host cell strain is a human pathogenic strain (e.g., Clostridium difficile) and the second The cell line is not a human pathogenic strain (e.g., Lactobacillus reuteri or Lactobacillus Lactobacillus bacteria such as Lactobacillus lactis) and 13 methods described.

[0133] 15. The second cell is, compared to cells of the host species or strain (e.g., hazard group 3 or 4): Any prior cells that are of a lower hazard category (e.g., Hazard Group 1 or 2) Any one of the methods described in 1 to 14 below.

[0134] 16. The receptor binds to lipopolysaccharide, teichoic acid (e.g., ManNAc attached to the C4 hydroxyl of the residue) 1 to 3 glycerol phosphates, and glycerol- or ribitol phosphates. ManNAc(β1→4)GlcNAc disaccharide followed by a long chain of ferate repeats, selected from proteins and flagella One of the methods from 1 to 15 is selected.

[0135] 17. The method of any one of statements 1 to 16, wherein the receptor comprises a host cell O-antigen.

[0136] 18. The phage is capable of replicating in the second cell, e.g., when the phage replicates in the second cell. 18. Any one of statements 1 to 17, wherein the vector is operable to express an endolysin or a holin. Two ways.

[0137] 19. Cells for propagating phages (propagator cells), wherein the phages are In cells (host cells) of a bacterial species or strain, a cell surface receptor contained in the bacteria of said species or strain is The first type is capable of infecting by binding to the the target cells contain a receptor on their surface and are of a second species or strain, and the second species or strain The first species or strain is different from the first species or strain, thereby allowing the propagator cells to a protease capable of being infected by said first type of phage for propagation of said protease; Paget cells.

[0138] In one example, the genome of the propagator cell (the second cell in the method of the present invention) is and the wild-type cells of this species or strain of cells contain an exogenous nucleotide sequence encoding a promoter. It does not contain the nucleotide sequence.

[0139] 20. The receptor is an expressible nucleotide sequence contained in the genome of the propagator cell. and a wild-type cell of the same species or strain as the propagator cell. 20. The propagator cell of statement 19, wherein the cell does not contain said expressible nucleotide sequence.

[0140] 21. The receptor is a sugar that is the product of the action of one or more enzymes in the propagator cell. and the genome of the propagator cell contains one or more sequences encoding said one or more enzymes. A wild-type vector containing multiple expressible nucleotide sequences of the same species or strain as the propagator cell. 20. The propagator cell of statement 19, wherein the cell does not comprise said expressible nucleotide sequence.

[0141] 22. The receptor is a protein that is the product of the action of one or more enzymes in the propagator cell. a methylamino acid moiety, and the genome of the propagator cell encodes said one or more enzymes. and a plurality of expressible nucleotide sequences of the same species or strain as the propagator cell. 20. The propagator cell of statement 19, wherein wild-type cells do not contain said expressible nucleotide sequence. .

[0142] 23. The enzymes are TarO, TarA, TarB, TarF, TarK, and TarL (or the host cell and / or the second cell) These homologs expressed by) are selected from the propagator cells of the description 22.

[0143] 24. The propagator of any one of statements 19 to 23 in combination with a phage of the first type. -Cell.

[0144] 25. The one or more prophage of the first type (e.g., integrated into the chromosome of a propagator cell) Any one of descriptions 19 to 24 containing a propagator cell (including a nucleus or nucleus containing ...

[0145] 26. The propagator cells are gram-negative bacterial cells, and optionally, the host cells are gram-negative A propagator cell of any one of statements 19 to 25 that is a bacterial cell.

[0146] 27. The propagator cells are gram-positive bacterial cells, and optionally, the host cells are gram-positive. A propagator cell of any one of statements 19 to 25 that is a bacterial cell.

[0147] 28. Optionally, culturing propagator cells to propagate the first type of phage. A population of propagator cells described in any one of statements 19 to 27 contained in a fermentation vessel. .

[0148] 29. Each propagator cell is a second cell defined in any one of statements 1 to 18. 19 to 28. Any one of propagator cells or populations.

[0149] 30. A method according to statements 19 to 28, each of which is a host cell as defined in any one of statements 1 to 18. Any one propagator cell or population.

[0150] 31. Any of statements 19 to 28, wherein the phage is a phage as defined in any one of statements 1 to 18. Any one propagator cell or population.

[0151] 32. A method for treating or preventing a disease or condition in a human or animal subject, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, ... the condition is mediated by host cells contained in the subject (e.g., contained in the intestine of the subject), and the method However, the method also includes administering propagator cells to a subject (e.g., to populate the intestine of the subject). The propagator cells are described in any one of statements 19 to 31, and the propagator cells - cells produce phages, which infect host cells in the patient (e.g., in their intestines), thereby killing the host cells or inhibiting the growth or proliferation of the host cells of interest; thereby treating or preventing a disease or condition.

[0152] 33. Propagator cells are cells of Lactobacillus bacteria (e.g., Lactobacillus reuteri). Cells, description 32 methods.

[0153] 34. Phages guide Cas in host cells to modify (e.g., cleave) the DNA of the host cells. and thereby performing said killing or inhibiting of anti-host cell crRNA. or encoding a gRNA, the method of description 32 or 33.

[0154] concept The present invention also provides the following concepts: 1. A method for producing a population of phages, wherein the phages are introduced into cells (host cells) of a first bacterial species or strain. host cells) by binding to cell surface receptors contained in bacteria of said species or strains. , is of a first type capable of infecting, and the method comprises: (a) a second species or strain that contains a receptor on its surface and is different from the first species or strain; providing a second population of bacterial cells comprising: (b) infecting a second cell with the first type of phage; (c) propagating the phage in a second cell, thereby producing a population of phage. The process and (d) optionally isolating phages from said population and

[0155] 2. Transducing particles containing nucleic acids packaged by phage coat proteins A method for producing a population, comprising: introducing particles into cells of a first bacterial species or strain (host cells) of said They cause infection by binding to cell surface receptors on certain species or strains of bacteria. It is possible, whereby the host cell is transduced with the nucleic acid and the method comprises: (a) a second species or strain that contains a receptor on its surface and is different from the first species or strain; providing a second population of bacterial cells containing DNA capable of producing copies of said nucleic acid; and (b) infecting the second cell by binding the phage to a receptor contained on the second bacterial cell; Therefore, the steps of infecting with phage and (c) propagating the phage in a second cell, which packages copies of said nucleic acid. The coat protein of the phages that bind to the phage is produced, thereby producing a population of particles. and (d) optionally isolating particles of said population and

[0156] In some instances, the nucleic acid contained in the particle is DNA. In some instances, the nucleic acid is RNA. In some instances, the vector is used to infect a second cell in step (b), and optionally, The transducing particle is different from the helper phage (if the transducing particle is a phage). Optionally, the helper phage is defective for self-replication in the second cell. do.

[0157] For example, the DNA contained in the second cells is contained in the chromosomal DNA of each second cell. The DNA is contained in one or more episomes (e.g., plasmids) contained in each second cell. can be.

[0158] A "transduction particle" may be a phage or may be smaller than a phage and may contain nucleic acid (e.g., (e.g., encoding components of the antibiotic or CRISPR array) into host bacterial cells. These are particles that can be transduced.

[0159] The particles are composed of phage coat proteins and, optionally, the phage used in step (b). Examples of structural proteins include the major phage structural proteins encoded by the phage structural proteins. Head and tail proteins, portal proteins, tail fiber proteins, and Phage proteins selected from one, several, or all of a small number of tail proteins It's quality.

[0160] The particles comprise nucleic acid (e.g., DNA, such as DNA encoding an array or an antibiotic), and the nucleic acid is Packaging the nucleic acid or its replica into transduction particles capable of infecting host cells In order to bind the phage, an operable package is prepared together with the phage-encoded protein of step (b). Contains a caging signal sequence.

[0161] In some instances, each transduction particle is a non-self-replicating transduction particle. "Injected particle" refers to a particle that is injected with a nucleic acid molecule (e.g., encoding an antibacterial agent or component) of the particle into a bacterial cell. It is capable of delivering a replicating genome but does not package its own replicating genome into transducing particles. non-coding particles (e.g., phage or phage-like particles; or genomic islands (e.g., For example, particles produced from Staphylococcus aureus pathogenicity islands (SaPIs) or modified versions thereof. This refers to the option.

[0162] Optionally, the nucleic acid of each particle comprises a modified genomic island. A bacterial island is an island that is naturally found in the bacterial cells of the host species or strain. In this study, the genomic islands are SaPI, SaPI1, SaPI2, SaPIbov1 and SaPibov2 genomic islands. Optionally, the nucleic acid of each particle is selected from the group consisting of a modified pathogenic island. Optionally, the pathogenicity island is a gene that is naturally found in bacterial cells of the first species or strain. islands, such as Staphylococcus SaPI or VibroPL. E or Pseudomonas aeruginosa pathogenicity islands (e.g., PAPI or PAGI, e.g., PAPI-1, PAGI-5, PAGI Optionally, the pathogenicity island is Sa PI (Staphylococcus aureus pathogenicity island).

[0163] Optionally, transcription of the nucleic acid of the transduction particle is mediated by the presence of an antibacterial agent or a constitutive component in the host cell. For transcription of a single copy of the array, the sequence is under the control of an inducible promoter. For example, in the environment (e.g., soil or water) or in industrial cultures or Switching on antimicrobial activity, for example, for use against target bacterial cells in fermentation vessels For example, the host cell may be a recombinant host cell. useful in industrial processes (e.g., for fermentation in the brewing or dairy industries) The introduction of antibacterial agents or crRNA against the host bacteria may be useful. This allows the process to be controlled (eg stopped or reduced).

[0164] 3. The method of concept 2, wherein the particle is a non-replicating transducing particle or a phage.

[0165] 4. Phages or particles target one or more protospacer nucleotide sequences of said host cell strain or species to form an active CRISPR / Cas system capable of Nucleotides encoding crRNAs (or single guide RNAs) that can operate with bacterial Cas Each target sequence is contained in the genome of the host cell, thereby or gRNA) guides the host cell's Cas to modify (and, optionally, cleave) the target sequence, Thus, any preceding concept that kills host cells or reduces the growth of the host cell population Please select one of the methods 1 to 3.

[0166] 5. When infected with the phage, the second cell does not contain the active CRISPR / Cas system. Nen 4 method.

[0167] 6. The method of concepts 4 or 5, wherein the genome of each second bacterial cell does not contain the target sequence.

[0168] 7. (a) a Cas (optionally Cas3, 9, cpf1 and / or CASCADE Cas) of said second cell induces said cr Not operable with RNA (or gRNA) (b) the tracrRNA of the second cell is not operable with the crRNA; and / or (c) causing the second cell to produce the crRNA from a nucleotide sequence encoding the crRNA. is not operable to generate said gRNA from a nucleotide sequence encoding said gRNA (or 7. The method of any one of concepts 4 to 6.

[0169] 8. The crRNA (or gRNA) is transduced into a Cas (optionally Cas3, 9, cpf1 and / or Any one of concepts 4 to 7, wherein the repeat sequence is not operable with CASCADE (Cas). Two ways.

[0170] 9. The nucleotide sequence is capable of directing transcription of crRNA (or gRNA) in bacteria of the host species or strain. a promoter for transcription of the crRNA (or gRNA) in said second species or strain; 10. The method of any one of concepts 4 to 8, wherein the method is operably linked to a promoter that is not intended for the method.

[0171] 10. (a) The phage or particle targets one or more protospacer nucleotide sequences. strain or species of said host cell to form an active CRISPR / Cas system capable of Nucleotides encoding crRNA (or single guide RNA) that can operate with the bacterial Cas each target sequence is contained in the genome of said host cell, thereby A (or gRNA) guides the host cell's Cas to modify (and optionally cleave) the target sequence, which killing the host cells or reducing the growth of the host cell population by (b) the host cell and the second cell are of the same species (optionally, an E. coli strain); (c) the genome of each second bacterial cell does not contain said target sequence, and the first and second cells are of the same species. The method of any one of concepts 1 to 9, wherein the different strains are

[0172] 11. The host species or strain of bacteria transmits the first type of phage or particle that infects the host bacteria. the second bacterium contains an antiphage toxin or mechanism that kills or reduces its growth, 11. The method of any one of concepts 1 to 10, wherein the method does not include a toxin or mechanism.

[0173] 12. The host species or strain of bacteria transmits the first type of phage or particle that infects the host bacteria. a CRISPR / Cas system that is active in killing or reducing the proliferation of the second 12. The method of any one of concepts 1 to 11, wherein the bacterium does not comprise said system.

[0174] 13. A second bacterial cell is genetically engineered to produce a receptor, said second species or the wild-type bacteria of the strain do not produce said receptor.

[0175] 14. The phage or particle in the second cell and in the cell of the first species or strain. 14. The method of any one of concepts 1 to 13, comprising an operable origin of replication.

[0176] 15. The method of any one of concepts 1 to 14, wherein the second cell is an E. coli cell.

[0177] 16. Any of concepts 1 to 15, wherein the first and second cells are of the same species (optionally, E. coli strain). Either one way.

[0178] 17. Concept 16, where the host cell strain is a human pathogenic strain and the second cell strain is not a human pathogenic strain. method.

[0179] 18. The second cells are more resistant to the pathogen than cells of the host species or strain (optionally hazard group 3 or 4). and cells of a lower hazard category (optionally, hazard group 1 or 2), Any one of concepts 1 to 17.

[0180] 19. The receptor binds to lipopolysaccharide, teichoic acid (optionally attached to the C4 hydroxyl of the ManNAc residue) 1 to 3 glycerol phosphates, and glycerol or ribitol ManNAc(β1→4)GlcNAc disaccharide followed by a long chain of phosphate repeats, from proteins and flagella Any one of the methods from concepts 1 to 18 is selected from the above.

[0181] 20. The method of any one of concepts 1 to 19, wherein the receptor comprises a host cell O-antigen.

[0182] 21. The phage or particle optionally replicates in a second cell. In the case of Just remember to do one of the 20 methods.

[0183] 22. Forms containing nucleic acids packaged by phage or phage coat proteins Cells for propagating transducing particles (propagator cells), in which phages or particles The cells of a first bacterial species or strain (host cells) are then transfected with cell surface proteins contained in the bacteria of said species or strain. The first type is capable of infecting by binding to the scepter, The propagator cells contain receptors on their surfaces and are of a second species or strain, and the second The species or strain is different from the first species or strain, whereby the propagator cells for the propagation of the phages or particles in the first type, Propagator cells capable of being infected by the virus.

[0184] 23. The receptor is an expressible nucleotide sequence contained in the genome of the propagator cell. and a wild-type cell of the same species or strain as the propagator cell. 23. The propagator cell of claim 22, wherein the cell does not contain said expressible nucleotide sequence.

[0185] 24. The receptor is a sugar that is the product of the action of one or more enzymes in the propagator cell. and the genome of the propagator cell contains one or more sequences encoding said one or more enzymes. A wild-type vector containing multiple expressible nucleotide sequences of the same species or strain as the propagator cell. The propagator cell of Concept 22, wherein the cell does not comprise said expressible nucleotide sequence.

[0186] 25. The receptor is a protein that is the product of the action of one or more enzymes in the propagator cell. a methylamino acid moiety, and the genome of the propagator cell encodes said one or more enzymes. and a plurality of expressible nucleotide sequences of the same species or strain as the propagator cell. The propagator cell of Concept 22, wherein the wild-type cell does not contain said expressible nucleotide sequence. .

[0187] 26. The enzymes are TarO, TarA, TarB, TarF, TarK, and TarL (or the host cell and / or the second cell) These homologs expressed by the concept 25 propagator cells selected from.

[0188] 27. Any of concepts 22 to 26 in combination with the first type of phage or the transduction particle. or one propagator cell.

[0189] 28. The one or more prophage of the first type (optionally, the chromosome of the propagator cell) incorporated into the transduction particle), or DNA capable of producing a copy of said nucleic acid ( Optionally, integrated into the chromosome of a propagator cell) One propagator cell.

[0190] 29. The propagator cells are gram-negative bacterial cells, and optionally, the host cells are gram-negative The propagator cell of any one of Concepts 22 to 28, which is a sexually active bacterial cell.

[0191] 30. The propagator cells are gram-positive bacterial cells, and optionally, the host cells are gram-positive. The propagator cell of any one of Concepts 22 to 28, which is a sexually active bacterial cell.

[0192] 31. Optionally, culturing propagator cells and injecting said first type of phage or said transducing 31. The process of any one of concepts 22 to 30, contained in a fermentation vessel for growing particles. A population of paget cells.

[0193] 32. The concept wherein each propagator cell is a second cell as defined in any one of concepts 1 to 21. 22 to 31. A propagator cell or population of any one of

[0194] 33. Concepts 22 to 31, each host cell being a host cell as defined in any one of concepts 1 to 21. Any one propagator cell or population.

[0195] 34. The phage or particle is a phage or particle as defined in any one of concepts 1 to 21. The propagator cell or population of any one of concepts 22 to 31.

[0196] 35. A method for treating or preventing a disease or condition in a human or animal subject, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, ... the condition is mediated by host cells contained in the subject (optionally contained in the intestine of the subject); The method comprises administering propagator cells to a subject (optionally to populate the intestine of the subject). the propagator cell is as described in any one of Concepts 22 to 34, The pagator cells produce phages or transducing particles, which are then delivered to the patient's (any) selectively infect host cells (in the gut) thereby killing the host cells or targeting the target inhibiting the growth or proliferation of a host cell of the How to do it.

[0197] 36. Propagator cells are cells of Lactobacillus bacteria (e.g., Lactobacillus reuteri). Cellular, Concept 35 method.

[0198] 37. The phage guides Cas in the host cell and modifies the DNA of the host cell (optionally, cleavage) thereby effecting said killing or inhibition of anti-host cells The method of concept 35 or 36, encoding rRNA or gRNA. [Example]

[0199] Genetic engineering of producer cells to make them sensitive to helper phage overview: The inventors genetically engineered bacterial production strains (in this case, E. coli production cells) to produce the first The receptor was expressed in the strain, rendering it susceptible to infection with helper phage. , carrying a vector containing a CRISPR array and a phage packaging site, resulting in The vector is packaged in cells infected with the helper (infected cells). (It is not packaged in cells that do not have this gene), thereby The present inventors further discovered that the use of bacteria as a production strain for HIV-like particles is now possible. The lysate produced by such production strains contains phage-like particles and can be used to identify other demonstrated that CRISPR arrays can be delivered to relevant E. coli target populations. We refer to these vectors as CRISPR Guided Vectors (CGV™).

[0200] Advantageously, CGV-loaded phage-like particles (CGV-PLPs) are produced that target specific bacterial populations. To achieve this, it is necessary to produce CGV-PLP in a strain related to the target strain, e.g., in the target strain. It may be beneficial to produce CGV-PLPs that evade the host's defense mechanisms. For example, P LP is a method for detecting bacterial phenotypes in which the species or strains of the producing and target bacteria are the same or closely related (or any In the producing bacteria, DNA modification of CGV-PLP by methyltransferases in the CGV-PLP shields DNA against restriction modification. It may be useful to employ a production strain according to the present invention that exhibits a surface receptor. By doing so, the present invention then provides a method for modifying DNA that is beneficial to restriction and modification by the target host bacterium. Advantageously, PLP production is enabled in strains capable of expressing P in the target bacterium. The protospacer sequence targeted by the LP crRNA is deleted in the genome of the producing bacterium. may be absent or not naturally occurring, resulting in a Cas-mediated transcriptional response in the genome of the producing bacterium. The cleavage does not occur during the production of PLP.

[0201] Methods and Results: As a production strain, the present inventors used a receptor for the helper phage M13KO7 (Figure 1_X). The E. coli strain MG1655 was transformed with a plasmid expressing the receptor (Figure 1Y). The receptor was obtained from New England Biolabs. The F-pilus was expressed from the plasmid pCJ105. Both strains were transformed with CGV (Fig. 1_3), and the CGV -PLP production was achieved by infection with helper phage M13KO7.

[0202] In line X, the presence of the receptor led to the production of CGV-PLP lysates, whereas in line Y, the lysis No lysate was produced (Fig. 1_4). The resulting lysate was related to the production strain and contained phage receptors. We demonstrated that CGV can be delivered to different target populations carrying the HIV-1 virus (Figures 1-5 and 2). ). The control strain did not produce CGV-PLP and was unable to deliver CGV to the target population (Figure 1). 2).

[0203] Table 5

[0204] Table 6A

[0205] Table 6B

[0206] Table 6C

[0207] Table 6D

[0208] Table 6E

[0209] Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15 Table 7-16 Table 7-17 Table 7-18 Table 7-19 Table 7-20 Table 7-21 Table 7-22 Table 7-23 Table 7-24 Table 7-25 Table 7-26 Table 7-27

[0210] [References] TIFF0007766653000058.tif232169TIFF0007766653000059.tif232169TIFF0007766653000060.tif238168TIFF0007766653000061.tif242168 TIFF0007766653000062.tif231168TIFF0007766653000063.tif241168TIFF0007766653000064.tif228168TIFF0007766653000065.tif217168

Claims

1. 1. A method for producing a population of phages, the phages being of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding to a cell surface receptor contained in bacteria of said species or strain, the method comprising: (a) providing a population of second bacterial cells that contain a receptor on their surface and that can be infected by a first type of phage, the phage being of a second species or strain different from the first species or strain; (b) infecting a second cell with said first type of phage; (c) propagating the phage in a second cell, thereby producing a population of phage; and infecting the first bacterial species or the second bacterial species with a phage to form an active CRISPR / Cas system capable of targeting one or more protospacer nucleotide sequences. the target sequence is comprised in the genome of the cell of said first bacterial species or strain, whereby the crRNA or gRNA guides the Cas of the cell of the first bacterial species or strain to cleave the target sequence, thereby modifying the genome of the cell of the first bacterial species or strain; and (i) the second cell does not contain an active CRISPR / Cas system when infected with the phage; or (ii) (1) the Cas of the second cell is not operable with the crRNA or gRNA; (2) the tracrRNA of the second cell is not operable with the crRNA; (3) the second cell is not operable to produce the crRNA from a nucleotide sequence encoding the crRNA or to produce the gRNA from a nucleotide sequence encoding the gRNA; (4) the crRNA or gRNA contains a repeat sequence that is not operable with the Cas of the second cell; and / or (5) The method, wherein the nucleotide sequence is operably linked to a promoter for transcription of crRNA or gRNA in the first species or strain of bacteria, but not a promoter for transcription of crRNA or gRNA in the second species or strain.

2. 10. The method of claim 1, further comprising the step of (d) isolating phage from said population.

3. 1. A method for producing a population of transduction particles comprising a nucleic acid packaged by a phage coat protein, the particles being capable of infecting cells of a first bacterial species or strain (host cells) by binding to a cell surface receptor contained on bacteria of said species or strain, thereby transducing the host cells with the nucleic acid, the method comprising: (a) providing a population of second bacterial cells that can be infected by a first type of phage, the second bacterial cell comprising a receptor on its surface, the second bacterial cell being of a second species or strain different from the first species or strain, and the second bacterial cell comprising DNA capable of producing copies of said nucleic acid; (b) infecting a second cell with the phage by allowing the phage to bind to a receptor contained on the second bacterial cell; (c) propagating the phage in a second cell, wherein a phage coat protein that packages copies of the nucleic acid is produced, thereby producing a population of particles; the particle comprises a nucleotide sequence encoding a crRNA or single guide RNA operable with a bacterial Cas of a cell of said first bacterial species or strain to form an active CRISPR / Cas system capable of targeting one or more protospacer nucleotide sequences, each target sequence being contained in the genome of the cell of said first bacterial species or strain, whereby the crRNA or gRNA guides the Cas of the first bacterial species or strain cell to cleave the target sequence, thereby modifying the genome of the cell of the first bacterial species or strain; and (i) the second cell does not contain an active CRISPR / Cas system when infected with the phage; or (ii) (1) the Cas of the second cell is not operable with the crRNA or gRNA; (2) the tracrRNA of the second cell is not operable with the crRNA; (3) the second cell is not operable to produce the crRNA from a nucleotide sequence encoding the crRNA or to produce the gRNA from a nucleotide sequence encoding the gRNA; (4) the crRNA or gRNA contains a repeat sequence that is not operable with the Cas of the second cell; and / or (5) The nucleotide sequence is a promoter for transcription of crRNA or gRNA in the bacterium of the first species or strain, and is a promoter for transcription of crRNA or gRNA in the second species or strain. operably linked to a promoter that is not for transcription of the gRNA; The method.

4. 4. The method of claim 3, further comprising the step of (d) isolating phage from said population.

5. The method of claim 3, wherein the particle is a non-replicative transducing particle or a phage.

6. 6. The method of any one of claims 1 to 5, wherein the bacteria of the first bacterial species or strain contain a CRISPR / Cas system that is active in modifying the first type of phage or particle that infects the first bacteria, and the second bacteria do not contain the system, the method further comprising genetically engineering the second bacterial cell to produce a receptor, wherein the second species or strain of bacteria contain a CRISPR / Cas system that is active in modifying the first type of phage or particle that infects the first bacteria, and the second bacteria do not contain the system, The method, wherein wild-type bacteria of the strain do not produce the receptor, and / or the phage or particle comprises an origin of replication that is operable in the second cell and in cells of the first species or strain, and / or the second cell is an E. coli cell, and / or the first and second cells are of the same species and the second cell strain is not a human pathogenic strain.

7. 7. The method of any one of claims 1 to 6, wherein the second cell is a cell of a lower hazard category compared to the cell of the first species or strain, and / or the receptor is selected from lipopolysaccharide, teichoic acid, protein and flagellum, and / or the receptor comprises an O-antigen of the first cell, and / or the phage or particle is operable to express an endolysin or holin in the second cell.

8. 8. The method of any one of claims 1 to 7, wherein the crRNA or gRNA guides the Cas of the cells of the first bacterial species or strain to cleave the target sequence, thereby killing the cells of the first bacterial species or strain or reducing the growth of a cell population of the first bacterial species or strain.

9. a cell (propagator cell) containing a phage or transduction particle, the phage or transduction particle containing nucleic acid packaged by a phage coat protein; A propagator cell, wherein the phage or particle is of a first type capable of infecting cells (host cells) of a first bacterial species or strain by binding to a cell surface receptor contained in bacteria of said species or strain, and the propagator cell is of a second species or strain that contains a receptor on its surface, the second species or strain being different from the first species or strain, whereby the propagator cell is capable of being infected by the first type of phage or particle for the propagation of the phage or particle, respectively, therein, and the phage or particle is a phage or particle described in any one of claims 1 to 8, and each propagator cell is a second cell defined in any one of claims 1 to 8.

10. the receptor comprises a protein encoded by an expressible nucleotide sequence contained in the genome of the propagator cell, and wild-type cells of the same species or strain as the propagator cell do not contain said expressible nucleotide sequence; and / or the receptor comprises a sugar moiety that is the product of the action of one or more enzymes in the propagator cell, the genome of the propagator cell comprises one or more expressible nucleotide sequences encoding said one or more enzymes, and wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequences; and / or the receptor comprises a teichoic acid moiety that is the product of the action of one or more enzymes in a propagator cell, the genome of the propagator cell comprises one or more expressible nucleotide sequences encoding said one or more enzymes, and wild-type cells of the same species or strain as the propagator cell do not comprise said expressible nucleotide sequences; The propagator cell of claim 9.

11. 11. The propagator cell of claim 9 or 10, in combination with said first type of phage or said transduction particle; and / or or the cell comprises one or more prophages of said first type, or DNA capable of generating copies of said nucleic acid of a transduction particle; or The propagator cell, wherein the propagator cell is a gram-positive bacterial cell.

12. A population of propagator cells described in any one of claims 9 to 11 for culturing propagator cells and propagating the first type of phage or the transduction particles, wherein a fermentation vessel contains the population of propagator cells.

13. 13. A propagator cell or population according to any one of claims 9 to 12, wherein each first cell is a first cell as defined in any one of claims 1 to 8.

14. 14. A method of treating or preventing a disease or condition in an animal subject (excluding humans), wherein the disease or condition is mediated by cells of a first bacterial species or strain contained in the subject, the method comprising administering to the subject propagator cells, the propagator cells being as defined in any one of claims 9 to 13; The method, wherein the propagator cells produce phages or transduction particles, and the phages or particles, respectively, infect cells of a first bacterial species or strain in the patient, thereby modifying the genome of the first bacterial species, thereby treating or preventing a disease or condition.

15. 14. A composition for use in a method of treating or preventing a disease or condition in a human or animal subject, wherein the disease or condition is mediated by cells of a first bacterial species or strain contained in the subject, said method comprising the step of administering to the subject propagator cells, the propagator cells being as defined in any one of claims 9 to 13, wherein the propagator cells produce phages or transduction particles, respectively, which infect cells of the first bacterial species or strain in the patient, thereby modifying the genome of the cells of the first bacterial species or strain; The composition thereby treating or preventing a disease or condition.

16. 15. The method of claim 14, wherein the phage or particle, respectively, infects cells of the first bacterial species or strain in the patient, thereby killing cells of the first bacterial species or strain or inhibiting the growth or proliferation of cells of the first bacterial species or strain in the subject.

17. 16. The composition for use of claim 15, wherein the phage or particle, respectively, infects cells of the first bacterial species or strain in the patient, thereby killing cells of the first bacterial species or strain or inhibiting the growth or proliferation of cells of the first bacterial species or strain in the subject.

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