EP3898953A1 - Branched receptor binding multi-subunit protein complexes for use in bacterial delivery vehicles - Google Patents

Branched receptor binding multi-subunit protein complexes for use in bacterial delivery vehicles

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Publication number
EP3898953A1
EP3898953A1 EP19829637.8A EP19829637A EP3898953A1 EP 3898953 A1 EP3898953 A1 EP 3898953A1 EP 19829637 A EP19829637 A EP 19829637A EP 3898953 A1 EP3898953 A1 EP 3898953A1
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EP
European Patent Office
Prior art keywords
rbp
seq
virus
terminal
terminal domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP19829637.8A
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German (de)
English (en)
French (fr)
Inventor
Jesus FERNANDEZ-RODRIGUEZ
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Eligo Bioscience
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Eligo Bioscience
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Priority claimed from PCT/EP2019/082640 external-priority patent/WO2020109339A1/en
Application filed by Eligo Bioscience filed Critical Eligo Bioscience
Publication of EP3898953A1 publication Critical patent/EP3898953A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10342—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates generally to bacterial delivery vehicles for use in efficient transfer of a desired payload into a target bacterial cell.
  • Bacteriophages are parasites that infect and multiply in bacteria. In general, the infection process can be divided in several stages: (i) adsorption corresponding to recognition and binding to the bacterial cell; (ii) injection of the DNA genome into the bacterial cell cytoplasm; (iii) production of a set of viral proteins that can lead to insertion in the host target genome (lysogenic phages) or to the production of infective particles (lytic phages) and (iv) release of mature virions from the infected cell, usually by controlled lysis [1]
  • a bacteriophage encodes two main sets of proteins that are involved in the recognition process.
  • the first set is able to attach to the bacteriophage’s primary receptor on the cell surface, an event that triggers DNA ejection into the cytoplasm and is usually viewed as an“irreversible” binding process [3].
  • Different bacteriophage genera differ in the organization of this set of proteins, and hence the naming can be different. In some Siphovirus, for example, they are called the “central tail fiber” or“tail tip”, which binds irreversibly to the LamB receptor in Escherichia coli.
  • the“central tail fiber” or“tail tip” is composed of the protein gpj [4].
  • a protein located at the very tip of the tail mediates this process.
  • pb5 recognizes the FhuA receptor [5]. This type of protein can be found in many other bacteriophages.
  • Myoviruses like T4, the irreversible binding to the primary receptor or to the cell surface in general is mediated by the“short tail fibers”, which are also located at the end of the tail tube [5].
  • the second set of proteins in the bacteriophage (herein referred to as“receptor binding proteins”) encodes recognition and binding activities to the so-called“secondary receptor” on the bacterium.
  • This secondary receptor allows for transient binding of the phage particle on the cell surface in order to scan the surface and position the first set of proteins in contact with the primary receptor. Since this binding is reversible, it allows the phage to“walk” on the cell surface until a primary receptor is found and the infection process starts.
  • These protein complexes are sometimes referred to as“L-shape fibers”, such as in T5,“side tail fibers” such as in lambda,“long tail fibers” as in T4, or tailspikes such as in phage P22 [5]— [8] .
  • the presence of this second set of proteins is necessary for the infection process to occur, such as T4 [5].
  • this second set of proteins is not strictly necessary for the infection process to happen, but it may allow for a more efficient binding to the target cell [7].
  • bacteria can develop multiple ways to avoid being recognized by a bacteriophage. For example, they can mutate the primary or secondary receptor to which the bacteriophage binds; they can mask this receptor by attaching proteins to it (receptor masking); or they can grow physical barriers around them in the form of bacterial capsules, thus blocking any access to the cell surface [9]. Bacteria can produce many different types of extracellular polymeric capsules [10]. In turn, bacteriophages have evolved different strategies to bypass these defense mechanisms. For instance, mutating the tail tip proteins allows them to use a different receptor [11].
  • the host range of a packaged phagemid tends to be larger than that of the parental bacteriophage it derives from.
  • novel bacterial delivery vehicles designed for the efficient delivery of exogenous DNA payload into target strains, it is of utmost importance to be able to engineer delivery vehicles with desired host ranges as well as the ability to bypass bacterial mechanisms that can lead to unsuccessful binding of the packaged phagemid to the bacterial cell surface.
  • a bacteriophage encodes sets of proteins that are involved in the bacterial cell recognition process. Described herein are novel approaches to engineering synthetic bacterial delivery vehicles with desired target host ranges.
  • synthetic bacterial delivery vehicles are provided that are characterized by a chimeric receptor binding protein (RBP), wherein the chimeric RBP comprises a fusion between an N-terminal domain of a RBP from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different bacteriophage RBP.
  • RBP chimeric receptor binding protein
  • Such bacteriophage RBPs include, for example, and depending on phages families,“L-shape fibers”,“side tail fibers (stfs)”,“long tail fibers” or“tailspikes.”
  • a lambda-like bacteriophage receptor binding protein such as a stf protein
  • RBP lambda-like bacteriophage receptor binding protein
  • the disclosure relates to bacterial delivery vehicles with desired host ranges based on the presence of an engineered branched receptor binding multi-subunit protein complex (“branched- RBP”).
  • branched- RBP comprises two or more associated receptor binding proteins derived from bacteriophages, wherein said RBPs contain“interaction domains” (IDs) that mediate association of the different subunits.
  • IDs “interaction domains”
  • the association of one subunit to another can be non-covalent or covalent.
  • the two or more associated RBPs include, but are not limited to, the chimeric receptor binding proteins (RBPs) described herein that comprise a fusion between the N-terminal domain of a RBP derived from a lambda-like, or lambda bacteriophage and the C- terminal domain of a different RBP.
  • RBPs chimeric receptor binding proteins
  • the chimeric receptor binding protein is one wherein the chimeric RBP comprises a fusion between an N-terminal domain of a RBP derived from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different RBP wherein said N-terminal domain of the RBP is fused to said C-terminal domain of a different RBP within one of the amino acids regions selected from positions 1-150, 320- 460, or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO: 1) or a similar region of a RBP having homology with one or more of three amino acid regions ranging from positions 1-150, 320-460, and 495-560 of the RBP with reference to the lambda stf sequence.
  • the different RBP domain of the chimeric receptor binding protein is derived from any bacteriophage or from any bacteriocin.
  • the RBP from the lambda-like bacteriophage, or the lambda bacteriophage, or the different RBP contains homology in one or more of three amino acid regions ranging from positions 1-150, 320- 460, or 495-560 of the N-terminal RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO: 1).
  • the homology between the lambda-like bacteriophage, the lambda bacteriophage, or the different RBP and the one or more of three amino acids regions is around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, and around 90% identity for 18 amino acids or more with reference to the lambda bacteriophage stf sequence (SEQ ID NO:l). Determination of homology can be performed using alignment tools such as the Smith- Waterman algorithm (Smith et ah, 1981, J. Mol. Biol 147:195-197) or EMBOSS Matcher (Rice, Longden, Bleasby 2000 EMBOSS Trends in Genetics 16: 276-277),
  • the chimeric RBP comprises the N-terminal domain of a RBP fused to the C-terminal domain of a different RBP within one of the amino acid regions selected from positions 80-150, 320-460, or 495-560 of the N- terminal RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO:l).
  • the chimeric RBP comprises an N-terminal domain and a C-terminal domain fused within one of the amino acids regions selected from positions 1-150, 320-460 or 495-560 at an insertion site having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with an insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 190), ADAKKS (SEQ ID NO: 191), MDETNR (SEQ ID NO: 192), S AS AAA (SEQ ID NO: 193), GAGENS (SEQ ID NO: 194), ATLKQI (SEQ ID NO: 195), IIQLED (SEQ ID NO: 196), GNIIDL (SEQ ID NO: 197), IATRV (SEQ ID NO: 198), TPGEL (SEQ ID NO: 199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN
  • the chimeric RBP comprises an N-terminal domain and a C-terminal domain fused within one of the amino acids regions selected from positions 1-150, 320-460 or 495-560 at an insertion site having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with an insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 190), ADAKKS (SEQ ID NO: 191), MDETNR (SEQ ID NO: 192), S AS AAA (SEQ ID NO: 193) and GAGENS (SEQ ID NO: 194)
  • SAGDAS SEQ ID NO: 190
  • ADAKKS SEQ ID NO: 191
  • MDETNR SEQ ID NO: 192
  • S AS AAA SEQ ID NO: 193
  • GAGENS SEQ ID NO: 194
  • the chimeric RBP comprises the N-terminal domain of a RBP fused to the C-terminal domain of different RBP wherein the different RBP is a protein or group a different proteins that confers an altered host range.
  • the different RBP is a T4-like or T4 long tail fiber composed of a proximal tail fiber and a distal tail fiber (DTF), and the C-terminal domain of a T4-like or T4 RBP is the distal tail fiber (DTF).
  • the N-terminal domain of a RBP is fused to the T4-like or T4 distal tail fiber at an insertion site within the T4-like or T4 DTF having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with an insertion site selected from the group consisting of amino acids ATLKQI (SEQ ID NO: 195), IIQLED (SEQ ID NO: 196), GNIIDL (SEQ ID NO: 197), IATRV (SEQ ID NO: 198), TPGEL (SEQ ID NO: 199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:202), and VDRAV (SEQ ID NO:203).
  • the N-terminal domain of a RBP is fused to the T4-like or T4 distal tail fiber within a region from amino acid 1 to 90, with a preferred region from amino acids ATLKQI (S
  • the disclosure provides specific chimeric RBPs.
  • SEQ ID NOS 2-61, 135-165, 215-242, 271, 273, 282 and 283 disclose the amino acid sequences of such chimeric RBPs as well as, in some instances, their corresponding natural chaperone proteins (designated“AP”). Such AP proteins assist in the folding of the chimeric RBPs.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 135 to 144, 147, 150, 151, 154, 157, 160, 163, 215, 216, 219, 221, 223, 225, 227, 229, 232, 325, 237, 239, 241, 282 or 283.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56 or 59.
  • nucleotide sequences encoding for the chimeric RBPs disclosed herein are depicted in SEQ ID NOS 62-120, 166-189, 206-212, 243-270, 272, 274 and 284.
  • the nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, 119, 166, 167, 168, 171, 174, 175, 178, 181, 184, 187, 206, 207, 208, 209, 210, 211, 212, 243, 244, 247, 249, 251, 253, 255, 257, 260, 263, 265, 267, 269 or 284.
  • the nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116 or 119.
  • the different RBP domain of the chimeric RBP comprises enzymatic activity such as depolymerase activity against an encapsulated bacterial strain.
  • the depolymerase is an endosialidase such as, for example, a K1F or K5 endosialidase.
  • the present disclosure provides for engineered branched-RBPs, as well as bacterial delivery vehicles, with desired host ranges and/or specific biological functions, based on the presence of an engineered branched receptor binding multi subunit protein complex (“branched-RBP”).
  • the engineered branched-RBP comprises two or more associated receptor binding proteins, derived from bacteriophages that associated with one another based on the presence of interaction domains (IDs).
  • IDs interaction domains
  • Each of the protein complex subunits contain IDs that function as“anchors” for association of one subunit RBP with another.
  • the association of one subunit with another can be non- covalent or covalent.
  • the engineered branched RBP may comprise non-covalent association of the different subunits; in some instance, the engineered branched RBP may comprise covalent association of the different subunits; and in further instances, the engineered branched RBP may comprise both covalent and non-covalent associations of the different subunits.
  • the protein subunits are assembled into the engineered branched-RBP as separate protein subunits each having their own ID.
  • the engineered branched-RBP may exist as a single fusion protein comprising different protein domains of interest fused to two or more ID domains.
  • the branched-RBP may comprise multiple RBP subunits, including, for example, two, three, four, etc. subunits.
  • Each of the RBP subunits may bring different biological functions to the overall branched-RBP.
  • Such functions include, but are not limited to, host recognition and enzymatic activity.
  • enzymatic activity includes depolymerase activity.
  • IDs amino acid sequences that are able to function as interaction domains (IDs).
  • IDs are those amino acid sequences that provide for association of one subunit to another thereby providing for assembly of the engineered branched-RBPs.
  • the IDs may be naturally occurring bacteriophage IDs, IDs derived from non-bacteriophage polypeptides, or recombinantly derived IDs.
  • the two or more of the associated receptor binding proteins of the engineered branched-RBP may be any bacteriophage RBP, or a functional domain of a bacteriophage RBP, e.g.
  • the associated receptor binding proteins may include, but are not limited to, chimeric receptor binding proteins (RBPs) described herein that comprise of a fusion between the N-terminal domain of a RBP derived from a lambda-like, or lambda bacteriophage and the C-terminal domain of a different RBP wherein said chimeric RBP also comprises an ID.
  • RBPs chimeric receptor binding proteins
  • nucleic acid molecules encoding the chimeric RBPs disclosed herein, as well as the two or more subunit RBPs of the engineered branched-RBP are provided.
  • Such nucleic acids may be included in vectors such as bacteriophages, plasmids, phagemids, viruses, and other vehicles which enable transfer and expression of the chimeric RBP encoding nucleic acids.
  • Bacterial delivery vehicles are provided which enable transfer of a nucleic acid payload, encoding a protein or nucleic acid of interest, into a desired target bacterial host cell.
  • Such bacterial delivery vehicles are characterized by having a chimeric RBP comprising a fusion between the N-terminal domain of a RBP from a lambda- like bacteriophage, or lambda bacteriophage, and the C-terminal domain of a different RBP.
  • the bacterial delivery vehicles contain a chimeric RBP comprising a fusion between an N-terminal domain of a RBP derived from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different RBP wherein said N-terminal domain of the chimeric RBP is fused to said C- terminal domain of a different RBP within one of the amino acids regions selected from positions 1-150, 320-460, or 495-560 of the N-terminal domain with reference to the lambda stf sequence (SEQ ID NO: 1).
  • the RBP from the lambda-like bacteriophage, the lambda bacteriophage, and the different RBP contain homology in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO: 1).
  • the homology is around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, or around 90% identity for 18 amino acids or more within the one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO:l).
  • the different RBP domain of the chimeric receptor binding protein is derived from a bacteriophage or a bacteriocin.
  • the chimeric RBP comprises an N-terminal domain of a RBP fused to a C-terminal domain of a RBP within one of the amino acids regions selected from positions 80-150, 320-460, or 495- 560 of the N-terminal RBP domain with reference to the lambda stf sequence (SEQ ID NO:l).
  • the chimeric RBP comprises an N- terminal domain of a RBP and a C-terminal domain of a RBP fused within a site of the N-terminal RBP domain having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with a site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 190), ADAKKS (SEQ ID NO: 191), MDETNR (SEQ ID NO: 192), S AS AAA (SEQ ID NO: 193), GAGENS (SEQ ID NO: 194), ATLKQI (SEQ ID NO: 195), IIQLED (SEQ ID NO: 196), GNIIDL (SEQ ID NO: 197), IATRV (SEQ ID NO: 198), TPGEL (SEQ ID NO: 199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:
  • the disclosure provides a bacterial delivery vehicle comprising a chimeric RBP.
  • SEQ ID NOS 2-61, 135-165, 215-242, 271, 273, 282 and 283 disclose the amino acid sequences of such chimeric RBPs and in addition, in some instances, their corresponding natural chaperone proteins (designated“AP”). Such AP proteins assist in the folding of the chimeric PBPs.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 135 to 144, 147, 150, 151, 154, 157, 160, 163, 215, 216, 219, 221, 223, 225, 227, 229, 232, 325, 237, 239, 241, 282 or 283.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56 or 59.
  • the present disclosure also provides nucleotide sequences encoding for the chimeric RBPs disclosed herein.
  • nucleic acids encoding such chimeric RBPs, as well as corresponding AP proteins are depicted in SEQ ID NOS 62-120, 166-189, 206-212, 243-270, 272, 274 and 284.
  • the nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, 119, 166, 167, 168, 171, 174, 175, 178, 181, 184, 187, 206, 207, 208, 209, 210, 211, 212, 243, 244, 247, 249, 251, 253, 255, 257, 260, 263, 265, 267, 269 or 284.
  • the nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, or 119.
  • the different RBP domain of the chimeric RBP comprises a domain having depolymerase activity against an encapsulated bacterial strain.
  • the depolymerase is an endosialidase, such as for example, a K1F or K5 endosialidase.
  • bacterial delivery vehicles which enable transfer of a nucleic acid payload, encoding a protein or nucleic acid of interest, into a desired target bacterial host cell wherein said bacterial delivery vehicles are characterized by having a branched-RBP as disclosed herein.
  • the bacterial delivery vehicles provided herein enable transfer of a nucleic acid payload, encoding a protein or nucleic acid of interest, into a desired target bacterial host cell.
  • the nucleic acid of interest is selected from the group consisting of a Cas nuclease gene, a Cas9 nuclease gene, a guide RNA, a CRISPR locus, a toxin gene, a gene expressing an enzyme such as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane protein, a structural protein, a secreted protein, a gene expressing resistance to an antibiotic or to a drug in general, a gene expressing a toxic protein or a toxic factor, and a gene expressing a virulence protein or a virulence factor, or any of their combination.
  • the bacterial delivery vehicle enables the transfer of a nucleic acid payload that encodes a nuclease that targets cleavage of a host bacterial cell genome or a host bacterial cell plasmid.
  • the cleavage occurs in an antibiotic resistant gene.
  • the nuclease mediated cleavage of the host bacterial cell genome is designed to stimulate a homologous recombination event for insertion of a nucleic acid of interest into the genome of the bacterial cell.
  • the present invention also provides pharmaceutical or veterinary compositions comprising one or more of the bacterial delivery vehicles disclosed herein and a pharmaceutically-acceptable carrier. Also provided is a method for treating a disease or disorder caused by bacteria, preferably a bacterial infection, comprising administering to a subject having a disease or disorder caused by bacteria, preferably a bacterial infection, in need of treatment the provided pharmaceutical or veterinary composition.
  • the present invention also relates to a pharmaceutical or veterinary composition as disclosed herein for use in the treatment of a disease or disorder caused by bacteria, preferably a bacterial infection. It further relates to the use of a pharmaceutical or veterinary composition as disclosed herein for the manufacture of a medicament for treating a disease or disorder caused by bacteria, preferably a bacterial infection.
  • a method for reducing the amount of virulent and/or antibiotic resistant bacteria in a bacterial population comprising contacting the bacterial population with the bacterial delivery vehicles disclosed herein.
  • the method may be an in vivo or in vitro method.
  • the present invention also relates to a pharmaceutical or veterinary composition as disclosed herein for use in reducing the amount of virulent and/or antibiotic resistant bacteria in a bacterial population, in particular in a subject having a bacterial infection. It further relates to the use of a pharmaceutical or veterinary composition as disclosed herein for the manufacture of a medicament for reducing the amount of virulent and/or antibiotic resistant bacteria in a bacterial population, in particular in a subject having a bacterial infection.
  • FIG. 1 demonstrates delivery in wild-type E. coli strains with lambda and OMPF-lambda packaged phagemids. Lambda packaged phagemids were diluted 1:5 in
  • FIG. 2 depicts wild-type lambda and lambda-stf-KlF chimeric delivery vehicles on K1+ strains.
  • Lambda packaged phagemids were sequentially diluted 10X in LB plus 5mM CaCh and 10 pL added in each well. Cells grown to an ODeoo of around 0.5 were then added to each phagemid dilution, incubated for 30 min at 37°C and 10 pL plated on LB supplemented with chloramphenicol. Top panel, strain UTI89; bottom panel, strain S88. Left plates, wild type lambda packaged phagemids; right plates, stf-KlF lambda packaged phagemids.
  • FIG. 3 depicts wild-type lambda and lambda-stf-K5 chimeric delivery vehicles on a K5+ strain.
  • Lambda packaged phagemids were sequentially diluted 10X in LB plus 5mM CaCh and 10 pL added in each well.
  • ECOR55 grown to an ODeoo of around 0.5 were then added to each phagemid dilution, incubated for 30 min at 37°C and 10 pL plated on LB supplemented with chloramphenicol.
  • Left panel wild type lambda packaged phagemids
  • right panel stf-K15 lambda packaged phagemids.
  • FIG. 4 depicts wild-type lambda, lambda-stf-AG22 and lambda-stf-SIEAl l chimeric delivery vehicles on a variety of encapsulated strains (O and K capsules).
  • Lambda phagemids were diluted 1:5 in LB plus 5mM CaCh and 10 pL added in each well. 90 pL of cells grown to an ODeoo of around 0.5 were then added to each phagemid- containing well, incubated for 30 min at 37°C and 10 pL spotted on LB-agar supplemented with chloramphenicol. Left panel, wild type lambda phagemids; middle panel, lambda stf-SIEAl l variant; right panel, lambda-stf-AG22 variant. Circles show strains with modified delivery as compared to lambda wild-type.
  • FIG. 5A-C demonstrates delivery of wild-type lambda and stf chimeras with different insertion sites on a variety of encapsulated strains (O and K capsules).
  • Lambda packaged phagemids were diluted 1:5 in LB plus 5mM CaCh and 10 pL added in each well. 90 pL of cells grown to an ODeoo of around 0.5 were then added to each phagemid- containing well, incubated for 30 min at 37°C and 10 pL spotted on LB-agar supplemented with chloramphenicol.
  • FIG. 5A demonstrates delivery of wild-type lambda and stf chimeras with different insertion sites on a variety of encapsulated strains (O and K capsules).
  • Lambda packaged phagemids were diluted 1:5 in LB plus 5mM CaCh and 10 pL added in each well. 90 pL of cells grown to an ODeoo of around 0.5 were then added
  • FIG. 5B Left panel, wild type lambda packaged phagemids; rest of panels, three different ADAKKS (SEQ ID NO:191)-stf variants.
  • FIG. 5B Left panel, wild type lambda packaged phagemids; rest of panels, three different SASAAA (SEQ ID NO: 193)-stf variants.
  • FIG. 5C Left panel, wild type lambda packaged phagemids; rest of panels, three different MDETNR (SEQ ID NO:192)-stf variants. For all panels, red circles show strains with improved delivery efficiency as compared to lambda wild-type.
  • FIG. 6 depicts a phmmer search that was performed with a 50aa sliding window (step 10) on the representative proteome database (rp75). The number of significant hits
  • FIG. 7A-B depicts branched stf architectures with 2 subunits.
  • FIG. 7A is a schematic view of a delivery vehicle with a 2 subunits branched stf architecture. ID: “Interaction Domain”.
  • FIG. 7B is a schematic view of the genetic architecture of an engineered lambda stf construct.
  • FIG. 8 demonstrates delivery of branched lambda stf packaged phagemids.
  • Lambda packaged lambda- stf- WW 11.1 stf, lambda-stf-KlF or the branched construct shown in FIG. 7 (WW11.1-K1F) were produced and titrated against 057 and K1 strains.
  • FIG. 9A-B depicts branched stf architectures with 4 subunits.
  • FIG. 9 A is a schematic view of a delivery vehicle with a 4 subunits branched stf architecture. Actual interactions among different ID may be different in the biological assembly from the graph depicted here.
  • FIG. 9B depicts a genetic circuit encoding the 4 subunits branched stf under the control of an inducible promoter.
  • FIG 10. depicts architecture of the engineered lambda stf-T4-like DTF chimera.
  • the semicircles denote RBS sites; the T sign, a transcriptional terminator; the arrow, a promoter.
  • FIG. 11 shows screening of phagemid particles with chimeric lambda stf-T4- like DTFs.
  • Left panel wild- type lambda stf
  • middle panel chimeric lambda-stf-WW13
  • right panel chimeric lambda-stf-PP-1.
  • FIG 12. demonstrates screening of phagemid particles with chimeric lambda stf- T4-like DTFs.
  • Left panel wild-type lambda stf; middle panel, chimeric lambda- stf-WW55; right panel, chimeric lambda-stf-WW34.
  • FIG. 13 depicts screening of phagemid particles with chimeric lambda stf-T4- like DTFs. All points shown refer to the universal insertion site of the DTF, located within amino acid range from position 1 to 90 with reference to WW13 amino acid sequence.
  • FIG. 14. depicts dot scoring system to quantify delivery efficiency. Density 0, 5 or fewer colonies; density 1, more than 5 colonies but not enough to define a clear circular drop; density 2, several colonies, but the background is clearly visible and some colonies are still separated; density 3, many colonies, the background is still visible but the colonies are hardly discernible as separate; density 4, spot almost completely dense, the background can only be seen faintly in some parts of the drop; density 5, spot looks completely dense, background cannot be seen.
  • FIG. 15 depicts raw dot titrations of delivery particles with chimeric stf in 40 human strains of the ECOR collection. Below each panel, the name of the chimeric stf. Above each dot, the 1-2 letter code used to identify strains.
  • FIG. 16 represents bar- formatted delivery data of Figure 15. From 0 (no entry, grey background) to 5 (maximum delivery). The bar length is proportional to the entry score from 1 (smallest bars) to 5 (longest bars).
  • the synthetic bacterial delivery vehicles are characterized by a chimeric receptor binding protein (RBP), wherein the chimeric RBP comprises a fusion between the N-terminal domain of a RBP from a lambda-like bacteriophage, or lambda bacteriophage, and the C-terminal domain of a different RBP.
  • RBP chimeric receptor binding protein
  • branched-RBP engineered branched receptor binding multi-subunit protein complex
  • the engineered branched-RBP comprises two or more associated receptor binding proteins, derived from bacteriophages, which associate with one another based on the presence of interaction domains (IDs).
  • IDs interaction domains
  • Each of the polypeptide subunits are engineered to contain IDs that function as “anchors” for association of one subunit RBP with another.
  • the association of one subunit with another can be non-covalent or covalent.
  • the branched-RBP may comprise multiple RBP subunits, including, for example, two, three, four, etc. subunits.
  • a receptor binding protein or RBP is a polypeptide that recognizes, and optionally binds and/or modifies or degrades a substrate located on the bacterial outer envelope, such as, without limitation, bacterial outer membrane, LPS, capsule, protein receptor, channel, structure such as the flagellum, pili, secretion system.
  • the substrate can be, without limitation, any carbohydrate or modified carbohydrate, any lipid or modified lipid, any protein or modified protein, any amino acid sequence, and any combination thereof.
  • a lambda-like bacteriophage refers to any bacteriophage encoding a RBP having amino acids sequence homology of around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, or around 90% identity for 18 amino acids or more in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 with reference to the lambda bacteriophage stf sequence of SEQ ID NO: 1, independently of other amino acids sequences encoded by said bacteriophage.
  • the present disclosure provides a chimeric receptor binding protein (RBP), wherein the chimeric RBP comprises a fusion between an N-terminal domain of a RBP from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different bacteriophage RBP.
  • RBP chimeric receptor binding protein
  • Such bacteriophage RBPs include, for example,“L-shape fibers”,“side tail fibers (stfs)”,“long tail fibers” or“tailspikes.”
  • a lambda- like bacteriophage receptor binding protein such as a stf protein
  • RBP lambda- like bacteriophage receptor binding protein
  • stfs bacteriophage receptor binding protein
  • specific fusion positions in the RBPs have been identified which allow one to obtain a functional chimeric RBP.
  • Such chimeric RBPs include those having an altered host range and/or biological activity such as, for example, depolymerase activity.
  • the chimeric receptor binding protein is one wherein the chimeric RBP comprises a fusion between an N-terminal domain of a RBP derived from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different RBP wherein said N-terminal domain of the RBP is fused to said C-terminal domain of a different RBP within one of the amino acids regions selected from positions 1-150, 320- 460, or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO: 1) or a similar region of a RBP having homology with one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the RBP with reference to the lambda stf sequence (SEQ ID NO:l).
  • the different RBP of the chimeric receptor binding protein (RBP) is derived from any bacteri
  • the RBP from the lambda-like bacteriophage, the lambda bacteriophage, or the different RBP contain homology with one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO:l).
  • the homology between the lambda-like bacteriophage, the lambda bacteriophage, or the different RBP and the one or more amino acids regions is around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, and around 90% identity for 18 amino acids or more.
  • the chimeric RBP comprises the N-terminal domain of the chimeric RBP fused to the C-terminal domain of the chimeric RBP within one of the amino acids regions selected from positions 80- 150, 320-460, or 495-560 with reference to the lambda bacteriophage stf sequence (SEQ ID NO: 1).
  • the chimeric RBP comprises an N- terminal domain and a C-terminal domain fused within one of the three amino acids regions at an insertion site having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with an insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 190), ADAKKS (SEQ ID NO: 191), MDETNR (SEQ ID NO: 192), S AS AAA (SEQ ID NO: 193), GAGENS (SEQ ID NO: 194), ATLKQI (SEQ ID NO: 195), IIQLED (SEQ ID NO: 196), GNIIDL (SEQ ID NO: 197), IATRV (SEQ ID NO: 198), TPGEL (SEQ ID NO: 199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:202), and VDRAV (SEQ ID NO: 190),
  • IDs may be inserted at such insertion sites thereby acting to fuse the N-terminal domain to the C-terminal domain.
  • an ID domain may be fused to either an N-terminal domain, or C-terminal domain, of a bacteriophage RBP, to provide a non-chimeric protein subunit of an engineered branched RBP.
  • the N-terminal domain, or C-terminal domain may be chosen depending on the desired function of the domain, e.g. host range or biological function. Where such non-chimeric protein subunits are utilized for production of an engineered branched-RBP, the ID domain may be fused at the preferred insertion sites disclosed herein, or alternatively, at insertion sites that permit maintainance of the function of the chosen domain.
  • the disclosure provides chimeric RBPs.
  • Such chimeric RBPs may function as protein subunits of an engineered branched-RBP protein complex.
  • SEQ ID NOS 2-61, 135-165, 215-242, 271, 273, 282 and 283 disclose the amino acid sequences of such chimeric RBPs and in addition, in some instances, their corresponding natural chaperone proteins (designated“AP”).
  • AP proteins assist in the folding of the chimeric RBPs.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59,135 to 144, 147, 150, 151, 154, 157, 160, 163, 215, 216, 219, 221, 223, 225, 227, 229, 232, 325, 237, 239, 241, 282 or 283.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56 or 59.
  • the present disclosure also provides nucleotide sequences encoding for the chimeric RBPs disclosed herein.
  • nucleic acids encoding such chimeric RBPs, as well as corresponding AP proteins are depicted in SEQ ID NOS 62-120, 166-189, 206-212, 243-270, 272, 274 and 284.
  • the nucleic acids encoding the chimeric RBP comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, 119, 166, 167, 168, 171, 174, 175, 178, 181, 184, 187, 206, 207, 208, 209, 210, 211, 212, 243, 244, 247, 249, 251, 253, 255, 257, 260, 263, 265, 267, 269 or 284.
  • the nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, or 119.
  • said chimeric RBPs may be further engineered to contain ID domains that act to mediate the association of the various engineered branched-RBP protein subunits with one another.
  • the different RBP domain of the chimeric RBP comprises depolymerase activity against an encapsulated bacterial strain.
  • the depolymerase is an endosialidase such as, for example, a K1F or K5 endosialidase
  • any of the chimeric RBPs disclosed herein may be used as RBP subunits, wherein said RBPs may be further engineered to contain IDs.
  • RBPs may be further engineered to contain IDs.
  • engineering branched-RBPs can alter the host range of the resulting delivery particle.
  • Nucleic acid molecules encoding the chimeric RBPs and branched-RBPs, disclosed herein are provided. Such nucleic acids may be included in vectors such as bacteriophages, plasmids, phagemids, viruses, and other vehicles which enable transfer and expression of the chimeric RBP encoding nucleic acids.
  • Bacterial delivery vehicles are provided which enable transfer of a nucleic acid payload, encoding a protein or nucleic acid of interest, into a desired target bacterial host cell.
  • Such bacterial delivery vehicles are characterized by having a chimeric RBP comprising a fusion between the N-terminal domain of a RBP from a lambda- like bacteriophage, or lambda bacteriophage, and the C-terminal domain of a different RBP.
  • the bacterial delivery vehicles contain a chimeric RBP comprising a fusion between an N-terminal domain of a RBP derived from a lambda-like bacteriophage, or lambda bacteriophage, and a C-terminal domain of a different RBP wherein said N-terminal domain of the chimeric RBP is fused to said C- terminal domain of a different RBP within one of the amino acids regions selected from positions 1-150, 320-460, or 495-560 of the N-terminal domain RBP with reference to the lambda stf sequence (SEQ ID NO: 1).
  • the RBP from the lambda-like bacteriophage, the lambda bacteriophage, and the different RBP contain homology in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the N-terminal RBP with reference to the lambda bacteriophage stf sequence.
  • the homology is around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, or around 90% identity for 18 amino acids or more within the one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the N-terminal RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO: 1).
  • the different RBP domain of the chimeric receptor binding protein is derived from a bacteriophage or a bacteriocin.
  • the chimeric RBP comprises an N-terminal domain of a RBP fused to a C-terminal domain of a RBP within one of the amino acids regions selected from 80-150, 320-460, or 495- 560 of the RBPs with reference to the lambda stf sequence (SEQ ID NO: 1).
  • the chimeric RBP comprises an N-terminal domain of a RBP and a C-terminal domain of a RBP fused within a site of the N-terminal RBPs having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%, identity with a site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 190), ADAKKS (SEQ ID NO: 191), MDETNR (SEQ ID NO: 192), S AS AAA (SEQ ID NO: 193), GAGENS (SEQ ID NO: 194), ATLKQI (SEQ ID NO: 195), IIQLED (SEQ ID NO: 196), GNIIDL (SEQ ID NO: 197), IATRV (SEQ ID NO: 198), TPGEL (SEQ ID NO: 199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:
  • the disclosure provides a bacterial delivery vehicle comprising a chimeric RBP.
  • SEQ ID NOS 2-61, 135-165, 215-242, 271, 273, 282 and 283 disclose the amino acid sequences of such chimeric RBPs and in addition, in some instances, their corresponding natural chaperone proteins (designated“AP”). Such AP proteins assist in the folding of the chimeric RBPs.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 135 to 144, 147, 150, 151, 154, 157, 160, 163, 215, 216, 219, 221, 223, 225, 227, 229, 232, 325, 237, 239, 241, 282 or 283.
  • the RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56 or 59.
  • the present disclosure also provides nucleotide sequences encoding for the chimeric RBPs disclosed herein.
  • nucleic acids encoding such chimeric RBPs, as well as corresponding AP proteins are depicted in SEQ ID NOS 62-120, 166-189, 206-212, 243-270, 272, 274 and 284.
  • the nucleic acids encoding the chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, 119, 166, 167, 168, 171, 174, 175, 178, 181, 184, 187, 206, 207, 208, 209, 210, 211, 212, 243, 244, 247,
  • nucleic acids encoding such chimeric RBPs comprise the nucleotide sequence of SEQ ID NO: 62, 64, 67, 69, 72, 75, 77, 80, 83, 84, 85, 87, 89, 91, 93, 95, 97, 99, 101, 102, 104, 106, 107, 108, 109, 110, 111, 112, 113, 116, or 119.
  • the different RBP domain of the chimeric RBP comprises a domain having depolymerase activity against an encapsulated bacterial strain.
  • the depolymerase is an endosialidase, such as for example, a K1F or K5 endosialidase.
  • the present disclosure provides synthetic bacterial delivery vehicles that are characterized by the presence of an engineered branched receptor binding multi-subunit protein complex (“branched-RBP”).
  • the engineered branched-RBP comprises two or more associated receptor binding proteins, derived from bacteriophages, which associate with one another based on the presence of interaction domains (IDs).
  • IDs interaction domains
  • the association of one subunit with another can be non-covalent or covalent.
  • Each of the polypeptide subunits contain IDs that function as“anchors” for association of one subunit RBP with another.
  • the branched-RBP may comprise multiple RBP subunits, including, for example, two, three, four, etc. subunits.
  • the individual RBP subunit may bring different biological functions to the overall engineered branched-RBP. Such functions include but are not limited to host recognition and enzymatic activity. Such enzymatic activity includes depolymerase activity.
  • ID polypeptides that are able to function as ID polypeptides.
  • IDs for purposes of the present invention, are those amino acid sequences that provide for non-covalent or covalent association of one receptor binding protein to another.
  • An interaction domain is a polypeptide whose function mediates the association of one biological molecule, e.g., a protein, to another biological molecule.
  • the biological molecule can be a protein, a part of a protein, a carbohydrate, a lipid and a nucleic acid.
  • the IDs may be naturally occurring bacteriophage IDs, IDs derived from non bacteriophage polypeptides that naturally associate with one another, or recombinantly derived IDs that function to mediate non-covalent or covalent association of two proteins or polypeptide domains.
  • the two or more associated receptor binding proteins of the branched-RBP include, but are not limited to, chimeric receptor binding proteins (RBPs) described herein that comprise a fusion between the N-terminal domain of a RBP derived from a lambda-like, or lambda bacteriophage and the C-terminal domain of a different RBP wherein said chimeric RBP further comprises an ID domain.
  • RBPs chimeric receptor binding proteins
  • RBPs receptor binding proteins
  • An RBP subunit of the branched-RBP may be a polypeptide that recognizes, and optionally binds and/or modifies or degrades a substrate located on the bacterial outer envelope, such as, without limitation, bacterial outer membrane, LPS, capsule, protein receptor, channel, structure such as the flagellum, pili, secretion system.
  • the substrate can be, without limitation, any carbohydrate or modified carbohydrate, any lipid or modified lipid.
  • the bacterial delivery vehicles provided herein enable transfer of a nucleic acid payload, encoding a protein or nucleic acid of interest, into a desired target bacterial host cell.
  • delivery vehicle refers to any means that allows the transfer of a payload into a bacterium.
  • delivery vehicles encompassed by the present invention including, without limitation, bacteriophage scaffold, virus scaffold, chemical based delivery vehicle (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes), protein-based or peptide-based delivery vehicle, lipid-based delivery vehicle, nanoparticle-based delivery vehicles, non- chemical-based delivery vehicles (e.g., transformation, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment, cell-penetrating peptides) or donor bacteria (conjugation). Any combination of delivery vehicles is also encompassed by the present invention.
  • chemical based delivery vehicle e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes
  • protein-based or peptide-based delivery vehicle e.g., lipid-based delivery vehicle, nanoparticle-based delivery vehicles,
  • the delivery vehicle can refer to a bacteriophage derived scaffold and can be obtained from a natural, evolved or engineered capsid.
  • the delivery vehicle is the payload as bacteria are naturally competent to take up a payload from the environment on their own.
  • Delivery vehicles as disclosed herein include packaged phagemids, as well as bacteriophage, comprising the chimeric and/or branched-RBPs disclosed herein.
  • the engineering of such delivery vehicles are well known to those skilled in the art. Such engineering techniques may employ production cell lines engineered to express the chimeric RBPs or branched-RBP disclosed herein.
  • Generation of packaged phagemids and bacteriophage particles are routine techniques well-known to one skilled in the art.
  • a satellite phage and/or helper phage may be used to promote the packaging of the payload in delivery vehicles of the present invention. Helper phages provide functions in trans and are well known to the man skilled in the art.
  • the helper phage comprises all the genes coding for the structural and functional proteins that are indispensable for the payload to be packaged, according to the invention (i.e. the helper phage provides all the necessary gene products for the assembly of the delivery vehicle).
  • the helper phage may contain a defective origin of replication or packaging signal, or completely lack the latter, and hence it is uncapable of self-packaging, thus only bacterial delivery particles carrying the payload or plasmid will be produced.
  • Helper phages may be chosen so that they cannot induce lysis of the host used for the delivery particle production.
  • One skilled in the art would understand that some bacteriophages are defective and need a helper phage for payload packaging.
  • sequences coding for one or more proteins or regulatory processes necessary for the assembly or production of packaged payloads may be supplied in trans.
  • the RBPs of the present dislcosure may be provided in a plasmid under the control of an inducible promoter or expressed constitutively.
  • the phage wild-type sequence may or not contain a deletion of the gene or sequence supplied in trans.
  • chimeric or modified phage sequences encoding a new function may be directly inserted into the desired position in the genome of the helper phage, hence bypassing the necessity of providing the modified sequence in trans.
  • Methods for both supplying a sequence or protein in trans in the form of a plasmid, as well as methods to generate direct genomic insertions, modifications and mutations are well known to those skilled in the art.
  • the term“payload” refers to any nucleic acid sequence or amino acid sequence, or a combination of both (such as, without limitation, peptide nucleic acid or peptide-oligonucleotide conjugate) transferred into a bacterium with a delivery vehicle.
  • the term“payload” may also refer to a plasmid, a vector or a cargo.
  • the payload can be a phagemid or phasmid obtained from natural, evolved or engineered bacteriophage genome.
  • the payload can also be composed only in part of phagemid or phasmid obtained from natural, evolved or engineered bacteriophage genome.
  • nucleic acid refers to a sequence of at least two nucleotides covalently linked together which can be single- stranded or double- stranded or contains portion of both single-stranded and double-stranded sequence.
  • Nucleic acids of the present invention can be naturally occurring, recombinant or synthetic.
  • the nucleic acid can be in the form of a circular sequence or a linear sequence or a combination of both forms.
  • the nucleic acid can be DNA, both genomic or cDNA, or RNA or a combination of both.
  • the nucleic acid may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine, hypoxathanine, isocytosine, 5-hydroxymethylcytosine and isoguanine.
  • bases including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine, hypoxathanine, isocytosine, 5-hydroxymethylcytosine and isoguanine.
  • modified bases that can be used in the present invention are detailed in Chemical Reviews 2016, 116 (20) 12655-12687.
  • nucleic acid also encompasses any nucleic acid analogs which may contain other backbones comprising, without limitation, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkage and/or deoxyribonucleotides and ribonucleotides nucleic acids. Any combination of the above features of a nucleic acid is also encompassed by the present invention.
  • Origins of replication known in the art have been identified from species -specific plasmid DNAs (e.g. CoIEl, Rl, pT181, pSClOl, pMBl, R6K, RK2, pl5a and the like), from bacterial vims (e.g. cpX174, M13, FI and P4) and from bacterial chromosomal origins of replication (e.g. oriC).
  • the phagemid according to the disclosure comprises a bacterial origin of replication that is functional in the targeted bacteria.
  • the plasmid according to the disclosure does not comprise any functional bacterial origin of replication or contain an origin of replication that is inactive in the targeted bacteria. Thus, the plasmid of the disclosure cannot replicate by itself once it has been introduced into a bacterium by the bacterial virus particle.
  • the origin of replication on the plasmid to be packaged is inactive in the targeted bacteria, meaning that this origin of replication is not functional in the bacteria targeted by the bacterial virus particles, thus preventing unwanted plasmid replication.
  • the plasmid comprises a bacterial origin of replication that is functional in the bacteria used for the production of the bacterial virus particles.
  • Plasmid replication depends on host enzymes and on plasmid-controlled cis and trans determinants. For example, some plasmids have determinants that are recognized in almost all gram-negative bacteria and act correctly in each host during replication initiation and regulation. Other plasmids possess this ability only in some bacteria (Kues, U and Stahl, U 1989 Microbiol Rev 53:491-516).
  • Plasmids are replicated by three general mechanisms, namely theta type, strand displacement, and rolling circle (reviewed by Del Solar et al. 1998 Microhio and Molec
  • Origins of replication used on the plasmid of the disclosure may be of moderate copy number, such as colEl ori from pBR322 (15-20 copies per cell) or the R6K plasmid (15-20 copies per cell) or may be high copy number, e.g. pUC oris (500-700 copies per cell), pGEM oris (300-400 copies per cell), pTZ oris (>1000 copies per cell) or pBluescript oris (300-500 copies per cell).
  • moderate copy number such as colEl ori from pBR322 (15-20 copies per cell) or the R6K plasmid (15-20 copies per cell) or may be high copy number, e.g. pUC oris (500-700 copies per cell), pGEM oris (300-400 copies per cell), pTZ oris (>1000 copies per cell) or pBluescript oris (300-500 copies per cell).
  • the bacterial origin of replication is selected in the group consisting of ColEl, pMBl and variants (pBR322, pET, pUC, etc), pl5a, ColA, ColE2, pOSAK, pSClOl, R6K, IncW (pSa etc), IncFII, pT181, PI, F IncP, IncC, IncJ, IncN, IncPl, IncP4, IncQ, IncHl l, RSF1010, CloDF13, NTP16, Rl, f5, pPSlO, pC194, pE194, BBR1, pBCl, pEP2, pWVOl, pLF1311, pAPl, pWKSl, pLSl, pLSl l, pUB6060, pJD4, pIJlOl, pSN22, pAMbetal, pIP501, pIP407, ZM6100(Sa), pCUl
  • the bacterial origin of replication is a E.coli origin of replication selected in the group consisting of ColEl, pMBl and variants (pBR322, pET, pUC, etc), pl5a, ColA, ColE2, pOSAK, pSClOl, R6K, IncW (pSa etc), IncFII, pT181, PI, F IncP, IncC, IncJ, IncN, IncPl, IncP4, IncQ, IncHl l, RSF1010, CloDF13, NTP16, Rl, f5 and pPSlO.
  • E.coli origin of replication selected in the group consisting of ColEl, pMBl and variants (pBR322, pET, pUC, etc), pl5a, ColA, ColE2, pOSAK, pSClOl, R6K, IncW (pSa etc), IncFII, pT181, PI, F IncP, IncC, IncJ, IncN, IncPl, IncP4,
  • the bacterial origin of replication is selected in the group consisting of pC194, pE194, BBR1, pBCl, pEP2, pWVOl, pFF1311, pAPl, pWKSl, pFSl, pFSl l, pUB6060, pJD4, pIJlOl, pSN22, pAMbetal, pIP501, pIP407, ZM6100(Sa), pCUl, RA3, pMOF98, RK2/RP4/RP1/R68, pBlO, R300B, pR01614, pRO1600, pECB2, pCMl, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.
  • the bacterial origin of replication is ColEl .
  • the delivered nucleic acid sequence according to the disclosure may comprise a phage replication origin which can initiate, with complementation of a complete phage genome, the replication of the delivered nucleic acid sequence for later encapsulation into the different capsids.
  • a phage origin of replication comprised in the delivered nucleic acid sequence of the disclosure can be any origin of replication found in a phage.
  • the phage origin of replication can be the wild-type or non-wildtype sequence of the M13, fl, cpX174, P4, lambda, P2, lambda-like, HK022, mEP237, HK97, HK629, HK630, mEP043, mEP213, mEP234, mEP390, mEP460, mEPxl, mEPx2, phi80, mEP234, T2, T4, T5, T7, RB49, phiX174, R17, PRD1 Pl-like, P2-like, P22, P22- like, N15 and N 15-like bacteriophages.
  • the phage origin of replication is selected in the group consisting of phage origins of replication of M13, fl, cpX174, P4, and lambda.
  • the phage origin of replication is the lambda or P4 origin of replication.
  • the delivered nucleic acid of interest comprises a nucleic acid sequence under the control of a promoter.
  • the nucleic acid of interest is selected from the group consisting of a Cas nuclease gene, a Cas9 nuclease gene, a guide RNA, a CRISPR locus, a toxin gene, a gene expressing an enzyme such as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane protein, a structural protein, a secreted protein, a gene expressing resistance to an antibiotic or to a drug in general, a gene expressing a toxic protein or a toxic factor, and a gene expressing a virulence protein or a virulence factor, or any of their combination.
  • the nucleic acid payload encodes a therapeutic protein.
  • the sequence of interest is a programmable nuclease circuit to be delivered to the targeted bacteria.
  • This programmable nuclease circuit is able to mediate in vivo sequence- specific elimination of bacteria that contain a target gene of interest (e.g. a gene that is harmful to humans).
  • Some embodiments of the present disclosure relate to engineered variants of the Type II CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRIS PR-associated) system of Streptococcus pyogenes.
  • programmable nucleases that can be used include other CRISPR-Cas systems, engineered TALEN (Transcription Activator-Like Effector Nuclease) variants, engineered zinc finger nuclease (ZFN) variants, natural, evolved or engineered meganuclease or recombinase variants, and any combination or hybrids of programmable nucleases.
  • the engineered autonomously distributed nuclease circuits provided herein may be used to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene (cf. WO2014124226).
  • sequences of interest can be added to the delivered nucleic acid sequence so as to be delivered to targeted bacteria.
  • sequence of interest added to the delivered nucleic acid sequence leads to cell death of the targeted bacteria.
  • nucleic acid sequence of interest added to the plasmid may encode holins or toxins.
  • sequence of interest circuit added to the delivered nucleic acid sequence does not lead to bacteria death.
  • sequence of interest may encode reporter genes leading to a luminescence or fluorescence signal.
  • sequence of interest may comprise proteins and enzymes achieving a useful function such as modifying the metabolism of the bacteria or the composition of its environment.
  • the nucleic sequence of interest is selected in the group consisting of Cas9, a single guide RNA (sgRNA), a CRISPR locus, a gene expressing an enzyme such as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane protein, a structural protein, a secreted protein, resistance to an antibiotic or to a drug in general, a gene expressing a toxic protein or a toxic factor and a gene expressing a virulence protein or a virulence factor.
  • sgRNA single guide RNA
  • CRISPR locus a gene expressing an enzyme such as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane protein, a structural protein,
  • the delivered nucleic acid sequence according to the disclosure comprises a nucleic acid sequence of interest that encodes a bacteriocin, which can be a proteinaceous toxin produced by bacteria to kill or inhibit growth of other bacteria.
  • Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. Such bacteriocin had been described from gram negative bacteria (e.g. microcins, colicin-like bacteriocins and tailocins) and from gram positive bacteria (e.g. Class I, Class II, Class III or Class IV bacteriocins).
  • the delivered nucleic acid sequence according to the disclosure further comprises a sequence of interest encoding a toxin selected in the group consisting of microcins, colicin-like bacteriocins, tailocins, Class I, Class II, Class III and Class IV bacteriocins.
  • the corresponding immunity polypeptide i.e. anti toxin
  • the corresponding immunity polypeptide may be used to protect bacterial cells (Cotter et ah, Nature Reviews Microbiology 11: 95, 2013) for delivered nucleic acid sequence production and encapsidation purpose but is absent in the pharmaceutical composition and in the targeted bacteria in which the delivered nucleic acid sequence of the disclosure is delivered.
  • the CRISPR system is included in the delivered nucleic acid sequence.
  • the CRISPR system contains two distinct elements, i.e. i) an endonuclease, in this case the CRISPR associated nuclease (Cas or "CRISPR associated protein") and ii) a guide RNA.
  • the guide RNA is in the form of a chimeric RNA which consists of the combination of a CRISPR (RNAcr) bacterial RNA and a RNAtracr (trans-activating RNA CRISPR) (Jinek et al., 2012, Science 337: 816-821).
  • the guide RNA combines the targeting specificity of the RNAcr corresponding to the "spacing sequences" that serve as guides to the Cas proteins, and the conformational properties of the RNAtracr in a single transcript.
  • the target genomic sequence can be permanently modified or interrupted. The modification is advantageously guided by a repair matrix.
  • the CRISPR system includes two main classes depending on the nuclease mechanism of action. Class 1 is made of multi-subunit effector complexes and includes type I, III and IV.
  • Class 2 is made of single-unit effector modules, like Cas9 nuclease, and includes type II (II-A,II-B,II-C,II-C variant), V (V-A,V-B,V-C,V-D,V-E,V-U1,V- U2,V-U3,V-U4,V-U5) and VI (VI-A,VI-B1,VI-B2,VI-C,VI-D)
  • the sequence of interest comprises a nucleic acid sequence encoding Cas protein.
  • CRISPR enzymes are available for use as a sequence of interest on the plasmid.
  • the CRISPR enzyme is a Type II CRISPR enzyme.
  • the CRISPR enzyme catalyzes DNA cleavage.
  • the CRISPR enzyme catalyzes RNA cleavage.
  • the CRISPR enzymes may be coupled to a sgRNA.
  • the sgRNA targets a gene selected in the group consisting of an antibiotic resistance gene, virulence protein or factor gene, toxin protein or factor gene, a bacterial receptor gene, a membrane protein gene, a structural protein gene, a secreted protein gene and a gene expressing resistance to a drug in general.
  • Non-limiting examples of Cas proteins as part of a multi- subunit effector or as a single-unit effector include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl l (SS), Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), C2c4, C2c8, C2c5, C2cl0, C2c9, Casl3a (C2c2), Casl3b (C2c6), Casl3c (C2c7), Casl3d, Csa5, Cscl, Csc2, Csel, Cse2, Csyl, Csy2, Csy3, Csfl, Csf2, Csl
  • the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any variants, homologs or orthologs thereof.
  • Cas9 is meant a protein Cas9 (also called Csnl or Csxl2) or a functional protein, peptide or polypeptide fragment thereof, i.e. capable of interacting with the guide RNA(s) and of exerting the enzymatic activity (nuclease) which allows it to perform the double-strand cleavage of the DNA of the target genome.
  • Cas9 can thus denote a modified protein, for example truncated to remove domains of the protein that are not essential for the predefined functions of the protein, in particular the domains that are not necessary for interaction with the gRNA(s).
  • Cas9 the entire protein or a fragment thereof
  • the sequence encoding Cas9 can be obtained from any known Cas9 protein (Fonfara et al., Nucleic Acids Res 42 (4), 2014; Koonin et al., Nat Rev Microbiol 15(3), 2017).
  • Cas9 proteins useful in the present disclosure include, but are not limited to, Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcus thermophilus (StlCas9, St3Cas9), Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9) and Neisseria meningitides (NmCas9).
  • Cpfl (Casl2a) (the entire protein or a fragment thereof) as used in the context of the disclosure can be obtained from any known Cpfl (Casl2a) protein (Koonin et al., Nat Rev Microbiol 15(3), 2017).
  • Cpfl(Casl2a) proteins useful in the present disclosure include, but are not limited to, Cpfl(Casl2a) proteins of Acidaminococcus sp, Lachnospiraceae bacteriu and Francisella novicida.
  • Casl3a (the entire protein or a fragment thereof) can be obtained from any known Casl3a (C2c2) protein (Abudayyeh et ah, 2017, Nature 550: 280-284) .
  • Casl3a (C2c2) proteins useful in the present disclosure include, but are not limited to, Casl3a (C2c2) proteins of Leptotrichia wadei (LwaCasl3a).
  • Casl3d (the entire protein or a fragment thereof) can be obtained from any known Casl3d protein (Yan et ah, 2018, Mol Cell 70: 327-339).
  • Casl3d proteins useful in the present disclosure include, but are not limited to, Casl3d proteins of Eubacterium siraeum and Ruminococcus sp.
  • the nucleic sequence of interest is a CRISPR/Cas9 system for the reduction of gene expression or inactivation of a gene selected in the group consisting of an antibiotic resistance gene, virulence factor or protein gene, toxin factor or protein gene, a gene expressing a bacterial receptor, a membrane protein, a structural protein, a secreted protein, and a gene expressing resistance to a drug in general.
  • the CRISPR system is used to target and inactivate a virulence factor.
  • a virulence factor can be any substance produced by a pathogen that alter host-pathogen interaction by increasing the degree of damage done to the host.
  • Virulence factors are used by pathogens in many ways, including, for example, in cell adhesion or colonization of a niche in the host, to evade the host's immune response, to facilitate entry to and egress from host cells, to obtain nutrition from the host, or to inhibit other physiological processes in the host.
  • Virulence factors can include enzymes, endotoxins, adhesion factors, motility factors, factors involved in complement evasion, and factors that promote biofilm formation.
  • such targeted virulence factor gene can be E.
  • coli virulence factor gene such as, without limitation, EHEC-HlyA, Stxl (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e) and Stx2f (VT2f), Stx2h (VT2h), fimA, fimF, fimH, neuC, kpsE, sfa, foe, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnfl, hra, sat, ire A, usp ompT, ibeA, malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aa
  • such targeted virulence factor gene can be Shigella dysenteriae virulence factor gene such as, without limitation, stxl and stx2.
  • such targeted virulence factor gene can be Yersinia pestis virulence factor gene such as, without limitation, yscF (plasmid-borne (pCDl) T3SS external needle subunit).
  • yscF plasmid-borne (pCDl) T3SS external needle subunit
  • such targeted virulence factor gene can be Francisella tularensis virulence factor gene such as, without limitation, fslA.
  • such targeted virulence factor gene can be Bacillus anthracis virulence factor gene such as, without limitation, pag (Anthrax toxin, cell-binding protective antigen).
  • such targeted virulence factor gene can be Vibrio cholera virulence factor gene such as, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator).
  • Vibrio cholera virulence factor gene such as, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator).
  • such targeted virulence factor gene can be Pseudomonas aeruginosa virulence factor genes such as, without limitation, pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdF, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and toxins (e.g., exoU, exoS and exoT).
  • pyoverdine e.g., sigma factor pvdS, bio
  • such targeted virulence factor gene can be Klebsiella pneumoniae virulence factor genes such as, without limitation, fimA (adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
  • Klebsiella pneumoniae virulence factor genes such as, without limitation, fimA (adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
  • such targeted virulence factor gene can be Acinetobacter baumannii virulence factor genes such as, without limitation, ptk (capsule polymerization) and epsA (assembly).
  • such targeted virulence factor gene can be Salmonella enterica Typhi virulence factor genes such as, without limitation, MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile tolerance, including efflux pump genes acrA, acrB and tolC.
  • such targeted virulence factor gene can be Fusobacterium nucleatum virulence factor genes such as, without limitation, FadA and TIGIT.
  • such targeted virulence factor gene can be Bacteroides fragilis virulence factor genes such as, without limitation, bft.
  • the CRISPR/Cas9 system is used to target and inactivate an antibiotic resistance gene such as, without limitation, GyrB, ParE, ParY, AAC(l), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA b-lactamase, mecA, Omp36, OmpF, PIB, bla (blal, blaRl) and mec (mecl, mecR
  • an antibiotic resistance gene
  • the CRISPR/Cas9 system is used to target and inactivate a bacterial toxin gene.
  • Bacterial toxin can be classified as either exotoxins or endotoxins. Exotoxins are generated and actively secreted; endotoxins remain part of the bacteria. The response to a bacterial toxin can involve severe inflammation and can lead to sepsis.
  • Such toxin can be for example Botulinum neurotoxin, Tetanus toxin, Staphylococus toxins, Diphteria toxin, Anthrax toxin, Alpha toxin, Pertussis toxin, Shiga toxin, Heat- stable enterotoxin (E.
  • the bacteria targeted by bacterial delivery vehicles disclosed herein can be any bacteria present in a mammal organism.
  • the bacteria are targeted through interaction of the chimeric RBPs and/or the branched-RBPs expressed by the delivery vehicles with the bacterial cell. It can be any commensal, symbiotic or pathogenic bacteria of the microbiota or microbiome.
  • a microbiome may comprise of a variety of endogenous bacterial species, any of which may be targeted in accordance with the present disclosure.
  • the genus and/or species of targeted endogenous bacterial cells may depend on the type of bacteriophages being used for preparing the bacterial delivery vehicles. For example, some bacteriophages exhibit tropism for, or preferentially target, specific host species of bacteria. Other bacteriophages do not exhibit such tropism and may be used to target a number of different genus and/or species of endogenous bacterial cells.
  • Examples of bacterial cells include, without limitation, cells from bacteria of the genus Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Streptococcus spp., Staphylococcus
  • bacterial delivery vehicles may target (e.g., specifically target) a bacterial cell from any one or more of the foregoing genus of bacteria to specifically deliver the payload of interest according to the disclosure.
  • the targeted bacteria can be selected from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Helicobacter spp., Vibrio spp, Salmonella spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Clostridium spp., Shigella spp., Enterococcus spp., Enterobacter spp., and Listeria spp.
  • bacterial cells of the present disclosure are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth).
  • Anaerobic bacterial cells include facultative anaerobic cells such as but not limited to Escherichia coli, Shewanella oneidensis and Listeria.
  • Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species.
  • anaerobic bacteria are most commonly found in the gastrointestinal tract.
  • the targeted bacteria are thus bacteria most commonly found in the gastrointestinal tract.
  • Bacteriophages used for preparing the bacterial virus particles, and then the bacterial virus particles may target (e.g., to specifically target) anaerobic bacterial cells according to their specific spectra known by the person skilled in the art to specifically deliver the plasmid.
  • the targeted bacterial cells are, without limitation, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphilococcus lugdunensis, Le
  • the targeted bacteria are Escherichia coli.
  • bacteriophages used for preparing the bacterial delivery vehicles, and then the bacterial delivery vehicles may target (e.g., specifically target) a bacterial cell from any one or more of the foregoing genus and/or species of bacteria to specifically deliver the payload of interest.
  • the targeted bacteria are pathogenic bacteria.
  • the targeted bacteria can be virulent bacteria.
  • the targeted bacteria can be antibacterial resistance bacteria, preferably selected from the group consisting of extended- spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumoniae , vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii, MDR Enterobacter spp., and a combination thereof.
  • the targeted bacteria can be selected from the group consisting of extended- spectrum beta-lactamase-producing (ESBL) Escherichia coli strains.
  • the targeted bacterium can be a bacterium of the microbiome of a given species, preferably a bacterium of the human microbiota.
  • the present disclosure is directed to bacterial delivery vehicle containing the payload as described herein.
  • the bacterial delivery vehicles are prepared from bacterial virus.
  • the bacterial delivery vehicles are chosen in order to be able to introduce the payload into the targeted bacteria.
  • Bacterial viruses from which the bacterial delivery vehicles having chimeric receptor binding proteins and/or branched-RBPs may be derived, are preferably bacteriophages.
  • the bacteriophage is selected from the Order Caudovirales consisting of, based on the taxonomy of Krupovic et al, 2015, Arch Virol, 161(1): 233- 247:
  • Bacteriophages may be selected from the family Myoviridae (such as, without limitation, genus Cp220virus, Cp8virus, Ea214virus, Felixolvirus, Mooglevirus, Suspvirus, Hplvirus, P2virus, Kayvirus, PlOOvirus, Silviavirus, Spolvirus, Tsarbombavirus, Twortvirus, Cc31virus, Jdl8virus, Js98virus, Kpl5virus, Moonvirus, Rb49virus, Rb69virus, S16virus, Schizot4virus, Spl8virus, T4virus, Cr3virus, Selvirus, V5virus, Abouovirus, Agatevirus, Agrican357virus, Ap22virus, Arvlvirus, B4virus, Bastillevirus, Bc431virus, Bcep78virus, Bcepmuvirus, Biquartavirus, Bxzlvirus, Cdl l9virus, Cp51virus, CvmlOvirus, Eah2virus, Elvirus, Hapun
  • Bacteriophages may be selected from the family Podoviridae (such as, without limitation, genus Frilvirus, Kp32virus, Kp34virus, Phikmvvims, Pradovirus, Sp6virus, T7virus, Cplvirus, P68virus, Phi29vims, Nona33vims, Pocjvirus, T12011virus, Bcep22virus, Bpplvirus, Cba41virus, Dfll2virus, Ea92virus, Epsilon 15vims, F116vims, G7cvims, Jwalphavims, Kflvims, Kpp25vims, Litlvims, Luz24virus, Luz7virus, N4vims, Nonanavims, P22virus, Pagevirus, Phieco32virus, Prtbvims, Sp58vims, Una961vims and Vp5vims)
  • Podoviridae such as, without limitation, genus Fril
  • - Bacteriophages may be selected from the family Siphoviridae (such as, without limitation, genus Camvirus, Likavims, R4vims, Acadianvims, Coopervirus, Pglvims, Pipefishvims, Rosebushvirus, Bmjitavirus, Che9cvims, Hawkeyevims, Plotvirus, Jerseyvirus, Klgvims, Sp31vims, Lmdlvims, Una4vims, Bongovirus, Reyvirus, Buttersvirus, Charlievirus, Redivims, Baxtervims, Nymphadoravirus, Bignuzvirus, Fishburnevirus, Phayoncevirus, Kp36virus, Roguelvirus, Rtpvims, Tlvirus, Tlsvirus, Abl8vims, Amigovirus, Anatolevims, Andromedavirus, Attisvirus, Bamyardvirus, Bemall3vims, Biseptimavims, Bron
  • Bacteriophages may be selected from the family Ackermannviridae (such as, without limitation, genus Ag3virus, Limestonevirus, Cbal20virus and Vilvirus)
  • the bacteriophage is not part of the order Caudovirales but from families with unassigned order such as, without limitation, family Tectiviridae (such as genus Alphatectivirus, Betatectivirus), family Corticoviridae (such as genus Corticovirus), family Inoviridae (such as genus Fibrovirus, Habenivirus, Inovirus, Lineavirus, Plectrovirus, Saetivirus, Vespertilio virus), family Cystoviridae(such as genus Cystovirus), family Leviviridae(such as genus Allolevivirus, Levivirus), family Microviridae (such as genus Alpha3 micro virus, G4microvirus, Phixl74microvirus, Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus) and family Plasmaviridae (such as genus Plasmavirus).
  • family Tectiviridae such as genus Alphatectivirus, Betatecti
  • the bacteriophage is targeting Archea not part of the Order Caudovirales but from families with Unassigned order such as, without limitation, Ampullaviridae, FuselloViridae, Globuloviridae, Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae, Salterprovirus and Bicaudaviridae.
  • chimeric RBPs and/or the branched RBPs and the bacterial delivery vehicles disclosed herein may be engineered, as non-limiting examples, from the following phages.
  • Synonyms and spelling variants are indicated in parentheses. Homonyms are repeated as often as they occur (e.g., D, D, d). Unnamed phages are indicated by "NN" beside their genus and their numbers are given in parentheses.
  • Bacteria of the genus Actinomyces can be infected by the following phages: Av-I, Av-2, Av-3, BF307, CT1, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.
  • Bacteria of the genus Bacillus can be infected by the following phages: A, aizl, Al-K-I, B, BCJA1, BC1, BC2, BLL1, BL1, BP142, BSL1, BSL2, BS1, BS3, BS8, BS15, BS18, BS22, BS26, BS28, BS31, BS104, BS105, BS106, BTB, B1715V1, C, CK-I, Coll, Corl, CP-53, CS-I, CSi, D, D, D, D5, entl, FP8, FP9, FSi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3, gl2, gl3, gl4, gl6, gl7, g21, g23, g24, g29, H2, kenl, KK-88, Ku
  • the following Bacillus- specific phages are defective: DLP10716, DLP-11946, DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, SPa, type 1 and m.
  • Bacteria of the genus Bacteriodes can be infected by the following phages: ad 12, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, FI, b ⁇ , fAI, fBGqI, fBG02, 11, 67.1, 67.3,
  • Bacteria of the genus Bordetella can be infected by the following phages: 134 and NN-Bordetella (3).
  • Bacteria of the genus Borrellia can be infected by the following phages: NN- Borrelia (1) and NN-Borrelia (2).
  • Bacteria of the genus Burkholderia can be infected by the following phages: CP75, NN-Burkholderia (1) and 42.
  • Bacteria of the genus Enterococcus can be infected by the following phages: DF78, FI, F2, 1, 2, 4, 14, 41, 867, Dl, SB24, 2BV, 182, 225, C2, C2F, E3, E62, DS96,
  • Bacteria of the genus Erysipelothrix can be infected by the following phage: NN- Eiysipelothrix (1).
  • Bacteria of the genus Fusobacterium can be infected by the following phages: NN-Fusobacterium (2), fv83-554/3, fv88-531/2, 227, fv2377, fv2527 and fv8501.
  • Bacteria of the genus Haemophilus can be infected by the following phages: HP1, S2 and N3.
  • Bacteria of the genus Helicobacter can be infected by the following phages: HP1 and AA -Helicobacter (1).
  • Bacteria of the genus Lepitospira can be infected by the following phages: LEI, LE3, LE4 and ⁇ NN-Leptospira (1).
  • Bacteria of the genus Morganella can be infected by the following phage: 47.
  • Bacteria of the genus Neisseria can be infected by the following phages: Group I, group II and NP1.
  • Bacteria of the genus Nocardia can be infected by the following phages: MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.
  • Bacteria of the genus Proteus can be infected by the following phages: Pm5, 13vir, 2/44, 4/545, 6/1004, 13/807, 20/826, 57, 67b, 78, 107/69, 121, 9/0, 22/608, 30/680, Pml, Pm3, Pm4, Pm6, Pm7, Pm9, PmlO, Pml 1, Pv2, p ⁇ , (pm, 7/549, 9B/2, 10A/31, 12/55, 14, 15, 16/789, 17/971, 19A/653, 23/532, 25/909, 26/219, 27/953, 32 A/909, 33/971, 34/13, 65, 5006M, 7480b, VI, 13/3a, Clichy 12, p2600, fc7, 1/1004, 5/742, 9, 12, 14, 22, 24/860, 2600/D52, Pm8 and 24/2514.
  • Bacteria of the genus Providencia can be infected by the following phages: PL25, PL26, PL37, 9211/9295, 9213/921 lb, 9248, 7/R49, 7476/322, 7478/325, 7479,
  • Bacteria of the genus Rickettsia can be infected by the following phage: NN- Rickettsia.
  • Bacteria of the genus Serratia can be infected by the following phages: A2P, PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, ⁇ DCP-3, ⁇ DCP-6, 3M, 10/la, 20A, 34CC, 34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228, SLP, SMP A, V.43, s, cpCWl, ⁇ DCP6-1, FOR6-2, ⁇ DCP6-5, 3T, 5, 8, 9F, 10/1, 20E, 32/6, 34B, 34CT, 34P, 37, 41, 56, 56D, 56P, 60P, 61/6, 74/6, 76/4, 101/8900, 226, 227, 228, 229F, 286, 289, 290F, 512, 764
  • Bacteria of the genus Treponema can be infected by the following phage: NN- Treponema (1).
  • Bacteria of the genus Yersinia can be infected by the following phages: H, H-I, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3/M 64-76, 5/G394-76, 6/C753-76, 8/C239-76, 9/F18167, 1701, 1710, PST, 1/F2852-76, D'Herelle, EV, H, Kotljarova, PTB, R, Y, YerA41, fUeG03-12, 3, 4/C1324-76, 7/F783- 76, 903, 1/M6176 and Yer2AT.
  • the bacteriophage is selected in the group consisting of Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus Phaxl, Salmonella virus 38, Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM 10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virus Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus Vil, Erwinia virus Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPtlO, Campylobacter virus IBB35, Campylobacter virus CP81, Campylobacter virus CP
  • Mycobacterium vims Manad Mycobacterium vims Oline, Mycobacterium virus Osmaximus, Mycobacterium virus Pgl, Mycobacterium vims Soto, Mycobacterium virus Suffolk, Mycobacterium virus Athena, Mycobacterium vims Bernardo, Mycobacterium vims Gadjet, Mycobacterium virus Pipefish, Mycobacterium vims Godines, Mycobacterium virus Rosebush, Mycobacterium virus Babsiella,
  • Mycobacterium virus Brujita, Mycobacterium virus Che9c, Mycobacterium vims Sbash, Mycobacterium virus Hawkeye, Mycobacterium virus Plot, Salmonella virus AG11, Salmonella vims Entl, Salmonella virus H8SE, Salmonella vims Jersey, Salmonella virus L13, Salmonella vims LSPA1, Salmonella vims SE2, Salmonella vims SETP3, Salmonella vims SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonella virus SS3e, Salmonella virus wksl3, Escherichia vims K1G, Escherichia virus K1H, Escherichia vims Klindl, Escherichia virus Klind2, Salmonella vims SP31, Leuconostoc vims Lmdl, Leuconostoc vims LN03, Leuconostoc vims LN04, Leuconostoc vims LN12, Leuconostoc vim
  • Mycobacterium virus Fruitloop, Mycobacterium virus Gumbie, Mycobacterium virus Ibhubesi, Mycobacterium virus Llij, Mycobacterium virus Mozy, Mycobacterium virus Mutaformal3, Mycobacterium virus Pacc40, Mycobacterium virus PMC, Mycobacterium virus Ramsey, Mycobacterium virus Rockyhorror, Mycobacterium virus SG4, Mycobacterium virus Shaunal, Mycobacterium virus Shilan, Mycobacterium virus Spartacus, Mycobacterium virus Taj, Mycobacterium virus Tweety, Mycobacterium virus Wee, Mycobacterium virus Yoshi, Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonella virus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244, Mycobacterium virus Bask21, Mycobacterium virus CJW1, Mycobacterium virus Eureka, Mycobacterium virus Kostya, Mycobacterium virus Porky, Mycobacterium virus
  • PHL082M03 Propionibacterium virus PHL092M00, Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01, Propionibacterium vims PHL112N00, Propionibacterium vims PHL113M01, Propionibacterium vims
  • Propionibacterium vims PHL308M00 Propionibacterium vims Pirate, Propionibacterium vims Procrassl, Propionibacterium vims SKKY, Propionibacterium vims Solid, Propionibacterium vims Stormbom, Propionibacterium vims Wizzo, Pseudomonas vims PaMx28, Pseudomonas vims PaMx74, Mycobacterium vims Patience, Mycobacterium vims PBI1, Rhodococcus vims Pepy6, Rhodococcus vims Poco6, Propionibacterium vims PFR1, Streptomyces vims phiBTl, Streptomyces vims phiC31, Streptomyces vims TGI, Caulobacter vims Karma, Caulobacter vims Magneto, Caulobacter vims phiCbK, Caulobacter vims
  • the bacterial vims particles target E. coli and includes the capsid of a bacteriophage selected in the group consisting of BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, 025, Phi-5, Pk, PSP3, PI, P1D, P2, P4, SI, Wcp, cpK13, ⁇ pl, q>2, q>7, cp92, 7 A, 8 ⁇ p, 9f, 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, E4, E7, E28, FI1, FI3, H, HI, H3, H8, K3, M, N, NO- 2, ND-3, ND4, ND-5, ND6, ND-7, Ox-I, Ox-2, Ox-3, Ox
  • a "prebiotic” refers to an ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that may confer benefits upon the host.
  • a prebiotic can be a comestible food or beverage or ingredient thereof.
  • a prebiotic may be a selectively fermented ingredient.
  • Prebiotics may include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition.
  • Prebiotics include, but are not limited to, amino acids, biotin, fructo-oligosaccharide, galacto-oligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carregenaan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans- galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber,
  • a“probiotic” refers to a dietary supplement based on living microbes which, when taken in adequate quantities, has a beneficial effect on the host organism by strengthening the intestinal ecosystem.
  • Probiotic can comprise a non- pathogenic bacterial or fungal population, e.g., an immunomodulatory bacterial population, such as an anti-inflammatory bacterial population, with or without one or more prebiotics. They contain a sufficiently high number of living and active probiotic microorganisms that can exert a balancing action on gut flora by direct colonisation.
  • probiotic is taken to mean any biologically active form of probiotic, preferably but not limited to lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria or saccharomycetes but even other microorganisms making up the normal gut flora, or also fragments of the bacterial wall or of the DNA of these microorganisms.
  • lactobacilli preferably but not limited to lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria or saccharomycetes but even other microorganisms making up the normal gut flora, or also fragments of the bacterial wall or of the DNA of these microorganisms.
  • Probiotics include, but are not limited to lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria, saccharomycetes, lactobacilli, bifidobacteria, or proteobacteria.
  • the antibiotic can be selected from the group consisting in penicillins such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; cephalosporins such as cefacetrile, cefadroxil, cephalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cef
  • the present invention provides pharmaceutical or veterinary compositions comprising one or more of the bacterial delivery vehicles disclosed herein and a pharmaceutically-acceptable carrier.
  • the bacterial delivery vehicles may be formulated as a pharmaceutical preparation or compositions comprising at least one bacterial delivery vehicles and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds.
  • Such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
  • Such administration forms may be solid, semi-solid or liquid, depending on the manner and route of administration.
  • formulations for oral administration may be provided with an enteric coating that will allow the synthetic bacterial delivery vehicles in the formulation to resist the gastric environment and pass into the intestines.
  • synthetic bacterial delivery vehicle formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract.
  • suitable suppositories may be used for delivery into the gastrointestinal tract.
  • Various pharmaceutically acceptable carriers, diluents and excipients useful in bacterial delivery vehicle compositions are known to the skilled person.
  • kits for treating a bacterial infection using the synthetic bacterial delivery vehicles disclosed herein include administering the synthetic bacterial delivery vehicles or compositions disclosed herein to a subject having a bacterial infection in need of treatment.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • the pharmaceutical or veterinary composition according to the disclosure may further comprise a pharmaceutically acceptable vehicle.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical or veterinary composition may be prepared as a sterile solid composition that may be suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • the pharmaceutical or veterinary compositions of the disclosure may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the particles according to the disclosure can also be administered orally either in liquid or solid composition form.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for enteral administration include sterile solutions, emulsions, and suspensions.
  • the bacterial delivery vehicles according to the disclosure may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
  • suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • the pharmaceutical or veterinary composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.
  • nasal sprays for transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used.
  • the active compounds can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
  • the present invention relates to a method for treating a disease or disorder caused by bacteria comprising administering a therapeutically amount of the pharmaceutical or veterinary composition as disclosed herein to a subject having such disease or disorder and in need of treatment. It also relates to the pharmaceutical or veterinary composition as disclosed herein for use in the treatment of a disease or disorder caused by bacteria. It further relates to the use of a pharmaceutical or veterinary composition as disclosed herein for the manufacture of a medicament for treating a disease or disorder caused by bacteria.
  • the diseases or disorders caused by bacteria may be selected from the group consisting of abdominal cramps, acne vulgaris, acute epiglottitis, arthritis, bacteraemia, bloody diarrhea, botulism, Brucellosis, brain abscess, chancroid venereal disease, Chlamydia, Crohn’s disease, conjunctivitis, cholecystitis, colorectal cancer, polyposis, dysbiosis, Lyme disease, diarrhea, diphtheria, duodenal ulcers, endocarditis, erysipelothricosis, enteric fever, fever, glomerulonephritis, gastroenteritis, gastric ulcers, Guillain-Barre syndrome tetanus, gonorrhoea, gingivitis, inflammatory bowel diseases, irritable bowel syndrome, leptospirosis, leprosy, listeriosis, tuberculosis, Lady Widermere syndrome, Legionaire’s disease, meningitis
  • the disease or disorder caused by bacteria may be a bacterial infection selected from the group consisting of skin infections such as acne, intestinal infections such as esophagitis, gastritis, enteritis, colitis, sigmoiditis, rectitis, and peritonitis, urinary tract infections, vaginal infections, female upper genital tract infections such as salpingitis, endometritis, oophoritis, myometritis, parametritis and infection in the pelvic peritoneum, respiratory tract infections such as pneumonia, intra-amniotic infections, odontogenic infections, endodontic infections, fibrosis, meningitis, bloodstream infections, nosocomial infection such as catheter-related infections, hospital acquired pneumonia, post-partum infection, hospital acquired gastroenteritis, hospital acquired urinary tract infections, and a combination thereof.
  • the infection according to the disclosure is caused by a bacterium presenting an antibiotic resistance.
  • the infection is caused by a bacterium as listed
  • the disease or disorder caused by bacteria may also be a metabolic disorder, for example, obesity and diabetes.
  • the disclosure thus also concerns a pharmaceutical or veterinary composition as disclosed herein for use in the treatment of metabolic disorder including, for example, obesity and diabetes. It further concerns a method for treating a metabolic disorder comprising administering a therapeutically amount of the pharmaceutical or veterinary composition as disclosed herein, and the use of a pharmaceutical or veterinary composition as disclosed herein for the manufacture of a medicament for treating a metabolic disorder.
  • the disease or disorder caused by bacteria may also be a pathology involving bacteria of the human microbiome.
  • the disclosure concerns a pharmaceutical or veterinary composition as disclosed herein for use in the treatment of pathologies involving bacteria of the human microbiome, such as inflammatory and auto-immune diseases, cancers, infections or brain disorders. It further concerns a method for treating a pathology involving bacteria of the human microbiome comprising administering a therapeutically amount of the pharmaceutical or veterinary composition as disclosed herein, and the use of a pharmaceutical or veterinary composition as disclosed herein for the manufacture of a medicament for treating a pathology involving bacteria of the human microbiome.
  • the present disclosure relates also to modulating microbiome composition to improve the efficacy of immunotherapies based, for example, on CAR-T (Chimeric Antigen Receptor T) cells, TIL (Tumor Infiltrating Lymphocytes) and Tregs (Regulatory T cells) also known as suppressor T cells.
  • CAR-T Chimeric Antigen Receptor T
  • TIL Tumor Infiltrating Lymphocytes
  • Tregs Regulatory T cells
  • Modulation of the microbiome composition to improve the efficacy of immunotherapies may also include the use of immune checkpoint inhibitors well known in the art such as, without limitation, PD-1 (programmed cell death protein 1) inhibitor, PD-L1 (programmed death ligand 1) inhibitor and CTLA-4 (cytotoxic T lymphocyte associated protein 4).
  • immune checkpoint inhibitors well known in the art such as, without limitation, PD-1 (programmed cell death protein 1) inhibitor, PD-L1 (programmed death ligand 1) inhibitor and CTLA-4 (cytotoxic T lymphocyte associated protein 4).
  • Some bacteria of the microbiome can also secrete molecules that will affect the brain.
  • a further object of the disclosure is a method for controlling the microbiome of a subject, comprising administering an effective amount of the pharmaceutical composition as disclosed herein in said subject.
  • the disclosure also relates to a method for personalized treatment for an individual in need of treatment for a bacterial infection comprising: i) obtaining a biological sample from the individual and determining a group of bacterial DNA sequences from the sample; ii) based on the determining of the sequences, identifying one or more pathogenic bacterial strains or species that were in the sample; and iii) administering to the individual a pharmaceutical composition according to the disclosure capable of recognizing each pathogenic bacterial strain or species identified in the sample and to deliver the packaged plasmid.
  • the biological sample comprises pathological and non-pathological bacterial species, and subsequent to administering the pharmaceutical or veterinary composition according to the disclosure to the individual, the amount of pathogenic bacteria on or in the individual are reduced, but the amount of non-pathogenic bacteria is not reduced.
  • the disclosure concerns a pharmaceutical or veterinary composition according to the disclosure for use in order to improve the effectiveness of drugs.
  • some bacteria of the microbiome without being pathogenic by themselves, are known to be able to metabolize drugs and to modify them in ineffective or harmful molecules.
  • composition according to the invention may further comprise at least one additional active ingredient, for instance a prebiotic and/or a probiotic and/or an antibiotic, and/or another antibacterial or antibiofilm agent, and/or any agent enhancing the targeting of the bacterial delivery vehicle to a bacteria and/or the delivery of the payload into a bacteria.
  • additional active ingredient for instance a prebiotic and/or a probiotic and/or an antibiotic, and/or another antibacterial or antibiofilm agent, and/or any agent enhancing the targeting of the bacterial delivery vehicle to a bacteria and/or the delivery of the payload into a bacteria.
  • the disclosure concerns the in-situ bacterial production of any compound of interest, including therapeutic compound such as prophylactic and therapeutic vaccine for mammals.
  • the compound of interest can be produced inside the targeted bacteria, secreted from the targeted bacteria or expressed on the surface of the targeted bacteria.
  • an antigen is expressed on the surface of the targeted bacteria for prophylactic and/or therapeutic vaccination.
  • the present disclosure also relates to a non-therapeutic use of the bacterial delivery particles.
  • the non-therapeutic use can be a cosmetic use or a use for improving the well-being of a subject, in particular a subject who does not suffer from a disease, and in particular from a disease or disorder caused by bacteria.
  • the present disclosure also relates to a cosmetic composition or a non- therapeutic composition comprising the bacterial delivery particles of the disclosure.
  • a significative portion of a lambda receptor binding protein e.g. the stf protein
  • RBP lambda receptor binding protein
  • specific fusion positions in the lambda RBP have been identified which allow one to obtain a functional chimeric RBP.
  • the data demonstrate in a non-limiting embodiment that in the case of packaged phagemids derived from bacteriophage lambda, modifying the side tail fiber protein results in an expanded host range.
  • K1+ strains are immune to T7 infection because the capsule forms a physical barrier that prevents attachment of the phage, but if purified K1F enzyme is added to the cells before infection, T7 is able to lyse them [21], confirming that the presence of bacterial capsules is a powerful mechanism to avoid recognition by bacteriophages.
  • K1+ strains are immune to T7 infection because the capsule forms a physical barrier that prevents attachment of the phage, but if purified K1F enzyme is added to the cells before infection, T7 is able to lyse them [21], confirming that the presence of bacterial capsules is a powerful mechanism to avoid recognition by bacteriophages.
  • sequence of K1F tail fiber (SEQ ID NO: 121) is:
  • the stf gene was modified to include the K1F endosialidase at its C-terminus using a Cas9-mediated gene exchange protocol [22] and resulted in a lambda-KlF chimeric stf of nucleotide sequence SEQ ID NO: 106 and aminoacid sequence SEQ ID NO: 46.
  • Lambda-KIF packaged phagemids were produced as in [23] and titrated against some K1+ strains, specifically E. coli UTI89 and S88.
  • a delta-stf lambda production strain was produced with the lambda-K5 stf fusion gene expressed in trans under the control of an inducible promoter.
  • FIG. 3 there was some residual delivery using the wild- type lambda-stf, probably due to the presence of some cells with a thinner K5 capsule.
  • the addition of lambda-stf-K5 chimeras allows for an improvement in delivery of more than 10 6 fold.
  • side tail fibers can be found that have some degree of homology to lambda stf, although no crystal structure is available.
  • the insertion site was designed as the last stretch of amino acids with identity to lambda stf.
  • the predicted side tail fiber proteins are as follows:
  • the addition of a chimeric stf allows the lambda-based packaged phagemid to show increased delivery efficiency in 25 out of 96 strains tested (more than 25% of the collection). In some cases, the increase is modest; in others, it allows for very good delivery efficiency in strains that had no or very low entry with wild- type lambda packaged phagemids. It is also worth noting that AG22 belongs to the Siphovirus_ family, like lambda, but SIEA11 is a P2-like phage. This highlights the significant observation that stf modules can be exchanged across bacteriophage genera.
  • the lambda stf protein consists of 774 aminoacids.
  • the insertion sites can be found closer to the N-terminus (amino acid 131 , insertion point ADAKKS (SEQ ID NO: 191)) or closer to the C-terminus (amino acid 529, insertion point GAGENS (SEQ ID NO: 194)).
  • Stf chimeras with aminoacid sequences of SEQ ID NO: 2-45 and 48-61 and corresponding nucleotide sequences of SEQ ID NO: 62- 105 and 108- 120 were engineered using these insertion sites.
  • stf chimeras for the insertion sites ADAKKS (SEQ ID NO: 191), SASAAA (SEQ ID NO: 193) and MDETNR (SEQ ID NO: 192).
  • SEQ ID NO: 191 SEQ ID NO: 191
  • SASAAA SEQ ID NO: 193
  • MDETNR SEQ ID NO: 192
  • the results described herein show that it is possible to build chimeric tail fibers that combine the part of one tail fiber that attaches to the capsid of one phage (usually the N-terminus of the protein) with the part of another fiber that interacts with the bacterium (usually the C-terminus of the protein). Stretches of homology between the sequences of different tail fibers can be considered as preferable recombination sites.
  • phages contain a single stf protein, which is a very important factor determining their host specificity. However, there are also several examples of phages encoding more than one stf gene, which is a beneficial trait since, presumably, each of them recognizes a different host.
  • phages have found different solutions to achieve this feature: some of them, like phi92, encode up to 6 stfs that bind to different parts of the baseplate/viral particle, and probably to other stfs [29]; others, like CBA120 [30], encode 4 stfs that form a tetrameric structure in which one of the stfs attaches to the phage particle while the other three attach to the first one through interaction; and others, like DT57C, contain an stf that binds the particle and a second one that attaches to the first through an interaction domain (branched stfs) [32] (FIG. 7A).
  • branched stfs branched stfs
  • an engineered lambda stf was constructed based on a branched architecture.
  • a phage referred to as WW11, contains two stfs of SEQ ID NO: 124 and 125 that follow the same order and contain homology to phage DT57C, which has been suggested to have branched stfs.
  • the interaction domains of stf- 1 and stf-2 in phage WW11 have been identified and used as modules to attach to the lambda stf.
  • the final construct contains the N-terminal part of the lambda stf of SEQ ID NO: 1 up to the GAGENS insertion site SEQ ID NO: 194 fused to WW11 stf- 1 interaction domain ID1 of SEQ ID NO: 280 and WW11 stf- 1 proper; after this, a synthetic RBS was inserted and immediately after, the stf-2 interaction domain ID2 of SEQ ID NO: 281 was fused to the C-terminal part of the K1F tail fiber of SEQ ID NO: 121 (see bold sequence of section [198] (FIG. 7B).
  • the GAGENS insertion site of SEQ ID NO: 194 was chosen as the insertion site.
  • Both chimeric proteins were transcribed from a polycistronic mRNA. This construction resulted in a final branched stf WW11-K1F of aminoacid sequence SEQ ID NO: 282 and 283 and of nucleotide sequence of SEQ ID NO: 284.
  • the K1F stf in the branched architecture seems to be less efficient than in the particle containing only one stf, further engineering such as, for example, (i) choosing a more efficient RBS between both stfs,(ii) increasing the length or (iii) introducing flexible linkers between the interaction domains and the fusion stfs and/or (iv) increasing the translation rate of the first stf in the polycistron (since this is known to affect the translation rate of the second CDS in a polycistronic message).
  • An advantage of this approach is also that the stfs are present in the phagemid particle in a 1:1 ratio, assuming proper expression of both components, which may be important for regulatory purposes.
  • CBA120 is a phage with 4 fibers (called stf; See, FIG. 9). Their sequences are below:
  • Stf4 also called TSP4 of FIG. 9 (orf213, protein ID YP_004957867.1) (SEQ ID NO: 127), stf3 (also called TSP3) of FIG. 9 (orf212, protein ID YP_004957866.1) (SEQ ID NO: 128), stf2 (also called TSP2) of FIG. 9 (orf211, protein ID YP_004957865.1) (SEQ ID NO: 129), and stfl(also called TSP1) of FIG. 9 (orf210, protein ID YP_004957864.1) (SEQ ID NO: 130).
  • the N-termini have homology to phages G7C/WW11/DT57C, which is a strong indication that these stfs associate to form a tetrameric complex.
  • the first stf to be translated, according to the TSP operon in the CBA120 genome, is TSP4, then TSP3, then TSP2, then TSP1. From this information, it was assumed that the main stf is TSP4, since it’s the one to be expressed first. It may encode an ID domain to which more than one TSP attaches.
  • the N-terminal domains also share homology to one another, which helps in the identification of the presumable ID domains.
  • TSP4 ID4 Of FIG. 9 SEQ ID NO: 131
  • ID4 seems to have an extra domain at the N-terminus that may be involved in capsid binding, so it will be left out of the final construct
  • TSP3 ID3 of FIG. 9 SEQ ID NO: 132
  • TSP2 ID2 of FIG. 9 SEQ ID NO: 133
  • TSP1 ID1 of FIG. 9 SEQ ID NO: 134.
  • Recoding is a very common process, and the objective is two-fold.
  • First recoding is done to avoid codon bias based on the fact that the codons used for a given amino acid vary depending on the organism. Accordingly, to avoid expression problems, which can lead to truncated, mutated or misfolded proteins, sequences were recoded with the codon usage of the host organism (in this case E. coli).
  • Second, removal of unknown layers of regulation may be advantageous.
  • operons phages and in general any other sequences with high "density information", like a phage genome; they have a limited amount of DNA that can be packaged, so several signals and functions may be encoded within a region, and this may impact the ability to use a genetic part in the desired way.
  • Plasmid pPhlF-Tetra STF Bsal cloning (pSClOl 37C, KanR) has been cloned, containing a fusion to Lambda stf GAGENS of SEQ ID NO: 194 with TSP4 interaction domain SEQ ID NO: 131 to 134.
  • the other TSP interaction domains are preceded by a synthetic RBS to avoid including unknown layers of regulation from the phage.
  • This plasmid allows the TypellS cloning (BSal) of 4 stfs fused to each of the TSP interaction domains.
  • the final architecture for expression of the tetrameric is depicted in FIG. 9B, although such an expression scheme may be modified, since the plasmid will be large (>16 kb) and the length of the transcribed mRNA is >9kb.
  • T4-like phages are a very diverse family of bacteriophages that share a common long tail fiber architecture: a proximal tail fiber that attaches to the phage particle and a distal tail fiber (DTF) that encodes host specificity linked by proteins acting as“hinge connectors” (Desplats and Krisch, 2003, Res. Microbiol. 154:259-267; Bartual et al. 2010, Proc. Natl. Acad. Sci. 107: 20287-20292). It is thought that the main host range determinants of the tail fiber reside in the distal part.
  • DTF distal tail fiber
  • T4-like phage which are known to be very broad, to any other phage or packaged phagemid of interest.
  • the distal tail fiber (C-terminal domain of the T4-like long tail fiber) of several T4-like phages were screened for possible functional insertion sites, several fusions with the Lambda stf gene were generated and their host range screened.
  • a synthetic linker encoding a RBS was designed to replace the natural DNA linker between the DTF gene and the adhesin; immediately downstream, another synthetic RBS preceding the chaperone gp57A was added, hence creating a polycistronic mRNA encoding for all the functions needed for the proper folding of the DTF (FIG. 10).
  • This construct was built in a plasmid under the control of an inducible promoter and complemented in trans in a strain producing lambda-based phagemids.
  • FIG 10. depicts the architecture of an engineered lambda stf-T4-like DTF chimera.
  • the semicircles denote RBS sites; the T sign, a transcriptional terminator; the arrow, a promoter.
  • Several parts of the C-terminus of the DTF were screened and fused to the lambda stf gene at the GAGENS (SEQ ID NO: 194) insertion site.
  • Several variants of the chimera lambda stf-WW13 were functional, as assessed by production of phagemid particles and transduction of a chloramphenicol marker in a collection of E. coli strains. The functional chimeras shown in FIG.
  • T4-like phages like PP-1, sharing sequence homology with WW13 were also tested and verified to produce functional chimeras (FIG. 11). These functional chimeras show a IATRV (SEQ ID NO: 198) insertion site at the beginning of PP-1 DTF part.
  • FIG. 11 depicts screening of phagemid particles with chimeric lambda stf-T4- like DTFs and in particular chimeric lambda stf-WW13 and chimeric lambda stf-PPl of aminoacids sequences SEQ ID NO: 142 to 149 and nucleotide sequences of SEQ ID NO: 166 to 173 including their respective chaperones proteins.
  • Left panel represents wild-type lambda stf; the middle panel represents chimeric lambda-stf- WW13; and the right panel, represents chimeric lambda- stf-PP-1.
  • the insertion sites found for WW13 do not always exist in a given T4-like DTF, thereby complicating the analysis.
  • Another functional insertion site without homology to WW13 was discovered for a second phage (WW55, FIG. 12).
  • the same TPGEL (SEQ ID NO: 199) insertion site could be found in a subset of T4-like phages and proven to yield functional chimeras with at least one of them, WW34 (FIG. 12), and at MDETNR (SEQ ID NO: 192) insertion site in lambda stf.
  • FIG. 12. shows screening of phagemid particles with chimeric lambda stf-T4-like DTFs and in particular chimeric lambda stf-WW55 and chimeric lambda stf-WW34 of aminoacids sequences SEQ ID NO: 150 to 156 and nucleotide sequences of SEQ ID NO: 174 to 180 including their respective chaperones proteins.
  • the left panel represents wild-type lambda stf; the middle panel represents chimeric lambda-stf-WW55; and the right panel represents chimeric lambda-stf-WW34.
  • T4-like DTF proteins may or may not share common sites for insertion, attempts were made to identify a universal insertion site that exists in all T4-like DTFs. When several T4-like DTFs are aligned, no homology along the whole DTF gene present in all the sequences exists, except for the N-terminus which is well conserved. The N-terminus of the DTF is thought to interact with the hinge connectors for attachment to the main phage particle.
  • chimeric lambda stf-WW14 chimeric lambda stf- WW170 and chimeric lambda stf-202 of aminoacids sequences SEQ ID NO: 157 to 165 and nucleotide sequences of SEQ ID NO: 181 to 189 including their respective chaperones proteins.
  • the present disclosure is useful for the generation of phage and phagemid particles with altered host ranges, since it provides a practical framework for the construction of chimeras using the DTFs from any T4-like phage, highlighting its modularity and translatability.
  • the human microbiome comprises different zones of the body, including gut, skin, vagina and mouth [29].
  • the microbiota in these areas is composed of differentcommunities of microorganisms, such as bacteria, archaea and fungi [29]-[31]. While numerous studies have been made that try to elucidate the specific composition of these communities, it is becoming clear that while there may exist a "core microbiome", there are many variations in the relative content of each microorganism depending on several factors, such as geographical location, diet or age [32]-[35].
  • packaged phagemids are of great interest, since they do not kill the host (unless their payload carries genes aimed at killing the host), payload does not replicate and expand and does not contain any endogenous phage genes.
  • a diagnostic study would be needed to identify the specific serotypes/variants of bacteria that exist in the patient before the treatment in order to find or design a packaged phagemid that allows for delivery of a payload adding a function to the target bacteria without killing them.
  • engineered delivery vehicles that are able to recognize a large number of strains belonging to different serotypes and phylogenetic groups (i.e., engineered particles having a "broad host range"), with a focus on Escherichia coli.
  • a therapeutic delivery approach does not need a priori to reach a large percentage of bacteria; the delivery needs to be high enough for the therapeutic payload to be expressed at the correct levels, which may be highly variable depending on the application.
  • the payload can be expressed by different serotypes or phylogenetic groups. This approach increases the chance that the particle will deliver a payload expressed in vivo in the majority of patients.
  • the ECOR collection is a set of strains isolated from different sources that is thought to represent the variability of this bacterium in Nature [42]. Some phage have been shown to have a broad host range against this collection (for instance, about 53% of the ECOR strains can be lysed with phage AR1 [43] and about 60% with phage SU16 [44]). As opposed to this, a single phage is able to infect 95% of Staphylococcus aureus strains [40].
  • the density of the dots was scored from 0 to 5, with 0 being no transductants and 5 being a spot with very high density [FIG. 14].
  • the density of the spots is directly related to the delivery efficiency of the packaged phagemids, since it corresponds to the number of bacteria that have received a payload containing a chloramphenicol acetyltransferase gene.
  • stf s can be considered as broad host range because the delivery efficiency in the selected ECOR strains is significantly higher than when using the wild type stf. For example, for stf EB6 or stf 68B, about 50% of the strains show medium to high delivery efficiencies, as compared to 17.5% of the strains with the wild type stf. These stf are good candidates for in vivo delivery, since they are able to deliver in different phylogenetic groups as well as serotypes. At the bottom of the Table in FIG.
  • a bar- formatted representation for density scores higher than 3 is shown, where the threshold for a broad host range stf is set at an increase of at least 2X compared to the basal line of the wild type stf; this is, stf that are able to deliver with scores of 3 and higher in at least 35% of the strains.
  • Other stf also show an increased delivery as compared to the wild type stf, so a less stringent threshold was set for stf able to deliver with scores 3 or higher with at least a 50% increase compared to the number of strains delivered with the wild- type stf (this is, delivery with scores of 3 and higher in at least 26.25% of the strains).

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