Phage and transduction particles
AssigneeSNIPR Biome ApS
InventorJakob Krause HAABER
Life of this patent
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Abstract
The invention relates to the production of phage and non-replicative transduction particles using DNAs (eg, plasmids and helper phage, mobile genetic elements (MGEs) or plasmids with chromosomally integrated helper phage genes), as well as the phage, helper phage, kits, compositions and methods involving these. The non-replicative transduction particles can be used to deliver antibacterial agents comprising a guided nuclease system.
Claims119
A composition for use in antibacterial treatment of bacteria, the composition comprising an engineered mobile genetic element (MGE) that is capable of being mobilised in a first bacterial host cell of a first species or strain, the cell comprising a first phage genome, wherein in the cell the MGE is mobilised using proteins encoded by the phage and replication of first is inhibited, wherein the MGE encodes an antibacterial agent or encodes a component of such an agent.
The composition of any preceding claim, wherein the first phage is a temperate phage.
The composition of any preceding claim, wherein the first cell comprises the first phage as a prophage.
The composition of any preceding claim, wherein in the presence of a first phage the mobilisation of the MGE causes host cell lysis.
The composition of any preceding claim, wherein the MGE is capable of being packaged in transduction particles that comprise some, but not all, structural proteins of the first phage.
The composition of any preceding claim, wherein mobilisation of the MGE comprises packaging of copies of the MGE or nucleic acid encoding the agent or component into transduction particles that are capable of transferring the copies into target bacterial cells for antibacterial treatment of the target cells.
The composition of any preceding claim, wherein the MGE is devoid of genes encoding phage structural proteins.
The composition of any preceding claim, wherein the MGE is a modified version of a MGE that is naturally found in bacterial cells of the first species or strain.
The composition of any preceding claim, wherein the MGE comprises a modified genomic island.
The composition of any preceding claim, wherein the MGE comprises a modified pathogenicity island.
The composition of any preceding claim, wherein the MGE comprises a pacA gene of the Enterobacteriaceae bacteriophage P1.
The composition of any preceding claim, wherein the MGE comprises a packaging initiation site sequence, optionally a packaging initiation site sequence of P1.
The composition of any preceding claim, wherein the MGE comprises a nucleotide sequence that is beneficial to cells of the first species or strain, optionally encoding a protein that is beneficial to cells of the first species or strain.
The composition of any preceding claim, wherein the MGE is devoid of rinA.
The composition of any preceding claim, wherein the MGE is is devoid of terL.
The composition of any preceding claim, wherein the MGE comprises a terS or a homologue thereof, and optionally is devoid of any other terminase gene.
The composition of any preceding claim, wherein the first phage is a pac-type phage operable with a pac comprised by the MGE.
The composition of any preceding claim, wherein the plasmid or MGE comprises a pac and/or cos sequence or a homologue thereof.
The composition of any preceding claim, wherein the plasmid or MGE comprises a terS or a homologue thereof and optionally devoid of terL.
The composition of any preceding claim, wherein the MGE is a modified SaPIbov1 or SaPIbov5 and is devoid of a terS.
The composition of any preceding claim, wherein the first phage is devoid of a functional packaging signal sequence and the MGE comprises a packaging signal sequence operable with proteins encoded by the first phage for producing transduction particles that package copies of the MGE or copies of a nucleic acid encoding the agent or component.
The composition of any preceding claim, wherein the MGE or plasmid comprises a Ppi or homologue, which is capable of complexing with first phage TerS, thereby blocking function of the TerS.
The composition of any preceding claim, wherein the MGE comprises a morphogenesis (cpm) module.
The composition of any preceding claim, wherein the MGE comprises cpmA and/or cpmB.
The composition of any preceding claim, wherein the MGE or first phage comprises one, more or all genes cp1, cp2, and cp3.
The composition of any preceding claim, wherein the MGE or first phage encodes a HNH nuclease.
The composition of any preceding claim, wherein the MGE or first phage comprises an integrase gene that encodes an integrase for excising the MGE and integrating the MGE into a bacterial cell genome.
The composition of any preceding claim, wherein the MGE is devoid of a functional integrase gene, and the first phage or host cell genome (eg, bacterial chromosome or a bacterial episome) comprises a functional integrase gene.
The composition of any preceding claim, wherein the transcription of MGE nucleic acid is under the control of a constitutive promoter, for transcription of copies of the agent or component in a host cell.
The composition of any preceding claim comprising a nucleic acid that is separate from the MGE, wherein the nucleic acid comprises all genes necessary for producing first phage particles.
The composition of any preceding claim, wherein the agent is a guided nuclease system or a component thereof, wherein the agent is capable of recognising and cutting host cell DNA (eg, chromosomal DNA).
The composition of any preceding claim, wherein the first species or strain is a gram positive species or strain.
The composition of any preceding claim, wherein the first species or strain is selected from Table 1.
The composition of any preceding claim, wherein the first species or strain is selected from Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter.
A nucleic acid vector comprising a MGE integrated therein, wherein the MGE is according to any preceding claim and the vector is capable of transferring the MGE or a copy thereof into a host bacterial cell.
A non-self replicative transduction particle comprising said MGE or vector of any preceding claim.
The MGE, vector, particle, composition or kit of any preceding claim for medical use in a human or animal patient.
The MGE, vector, particle, composition or kit of any preceding claim for treating or preventing an infection by target bacterial cells in a human or animal patient, wherein the antibacterial agent is toxic to the target cells.
The MGE, vector, particle, composition or kit of any preceding claim for treating or preventing an infection by target bacterial cells in a human or animal patient, wherein in the presence of the antibacterial agent
- (i) target cells are killed by the antibacterial agent;
- (ii) growth or proliferation of target cells is reduced;
- and/or
- (iii) target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
The composition, vector, particle, kit or method of any preceding claim, wherein the cell, host cell or target cell is selected from a Staphylococcal, Vibrio, Pseudomonas, Clostridium, E coli, Helicobacter, Klebsiella and Salmonella cell.
A plasmid comprising
- (i) A nucleotide sequence encoding an antibacterial agent or component thereof for expression in target bacterial cells;
- (ii) A constitutive promoter for controlling the expression of the agent or component;
- (iii) An optional terS nucleotide sequence;
- (iv) An origin of replication (ori);
- and
- (v) A phage packaging sequence (optionally pac, cos or a homologue thereof);
- and
- the plasmid being devoid of
- (vi) All nucleotide sequences encoding phage structural proteins necessary for the production of a transduction particle (optionally a phage), or the plasmid being devoid of at least one of such sequences;
- and
- (vii) Optionally terL.
A method of making a plurality of transduction particles, the method comprising
- (i) Producing host cells whose genomes comprise nucleic acid encoding structural proteins necessary to produce transduction particles that can package first DNA, wherein the genomes are devoid of a phage packaging signal, wherein the expression of the proteins is under the control of inducible promoter(s);
- (ii) Producing first DNA encoding an antibacterial agent or a component thereof (eg, as defined in any preceding claim), wherein the DNA comprises a phage packaging signal;
- (iii) Introducing the DNA into the host cells;
- (iv) Inducing production of the structural proteins in host cells, whereby transduction particles are produced that package the DNA;
- (v) Optionally isolating a plurality of the transduction particles;
- and
- (vi) Optionally formulating the particles into a pharmaceutical composition for administration to a human or animal for medical use.
Dependencies are inferred from the claim text.