Modifying bacteriophage
A method for modifying the genome of a lytic target phage, uses of the method and products thereof are described. Compositions comprising such phage are also described. The compositions may be formulated as a medicament, which are useful for human treatment and may treat various conditions, including bacterial infections.
1. A method for modifying the genome of a lytic target phage, wherein the target phage genome includes a first target sequence and a second target sequence, which method comprises:
(a) providing a vector which contains a phage-targeting region comprising a host range determinant of a marker phage, different from the host range determinant of the target phage, wherein the phage-targeting region is flanked by first and second flanking sequences homologous to the first and second target sequences of the target phage genome, and wherein the phage-targeting region of the vector further comprises an exogenous DNA sequence for incorporation into the genome of the target phage;
(b) introducing the vector into a first host cell, which host cell is a host for the target phage;
(c) infecting the first host cell with the target phage;
(d) allowing replication of the phage and recombination between the target phage genome and vector to take place whereby the genome of the target phage is modified;
(e) propagating resultant phage on a second host cell, which host cell is a host for the marker phage and not the target phage, wherein said propagating indicates that said resultant phage comprise said exogenous DNA sequence; and
(f) harvesting the resultant phage.
2. The method according to claim 1 , wherein the exogenous DNA comprises a gene which encodes an antibacterial protein.
3. The method according to claim 2 , wherein the gene is under the control of a constitutive promoter.
4. The method according to claim 2 , for the production of a modified bacteriophage capable of infecting a plurality of different target bacteria, which modified bacteriophage includes a SASP which is toxic to the target bacteria; wherein the modified bacteriophage is non-lytic.
5. The method according to claim 4 , wherein the modified bacteriophage expresses a plurality of different host range determinants or tail fibre proteins and wherein each host range determinant has a different bacterial host specificity; or wherein the bacteriophage expresses a host range determinant protein or tail fibre protein which comprises an amino acid sequence from a plurality of different bacteriophages.
6. The method according to claim 5 , wherein the bacterial host specificity of the host range determinants are within the same bacterial species.
7. The method according to claim 1 , wherein the first and second target sequences of the target phage genome are non-contiguous.
8. The method according to claim 7 , wherein the first and second target sequences of the target phage genome flank a phage gene, a lysis gene, or part thereof, such that the recombination between the target phage genome and vector inactivates the gene.
9. The method according to claim 1 , wherein at least one of the first and second flanking sequences contains a mutation as compared with the first and second target sequences of the target phage genome.
10. The method according to claim 1 , wherein the host range determinant of the marker phage encodes a tail fibre protein or region thereof.
11. The method according to claim 1 , wherein the host range determinant of the marker phage encodes a tail fibre protein which comprises a receptor binding region for binding to the second host cell and a region linking the receptor binding region to the body of the phage.
12. The method according to claim 11 , wherein the receptor binding region is a C-terminal receptor binding region and the region linking the C-terminal receptor binding region to the body of the phage is an N-terminal region.
13. The method according to claim 12 , wherein the C-terminal region has no more than 96% amino acid sequence identity with the C-terminal region of bacteriophage Phi33.
14. The method according to claim 13 , wherein the C-terminal region is from any one of bacteriophage Phi33, LBL3, SPM-1, F8, PR1, KPP12, LMA2, SN, 14-1, JG024, NH-4, PTP47, C36, PTP93 and PTP92.
15. The method according to claim 13 , wherein the C-terminal region amino sequence identity is less than 80%.
16. The method according to claim 12 , wherein the N-terminal region has at least 95% amino acid sequence identity with the N-terminal region of bacteriophage Phi33.
17. The method according to claim 16 , wherein the N-terminal region is from any one of bacteriophage Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH-4, PTP47, C36, PTP92 and PTP93.
18. The method according to claim 12 , wherein the tail fibre protein has more than 80% amino acid sequence identity with the tail fibre amino acid sequence of bacteriophage Phi33.
19. The method according to claim 18 , wherein the tail fibre protein is from a bacteriophage selected from Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH-4, PTP47, C36, PTP92 and PTP93.
20. The method according to claim 18 , wherein the amino acid sequence identity is more than 95%.
21. The method according to claim 12 , wherein the second host cell is a Pseudomonas host cell.