Sequence-specific antimicrobials by blocking DNA repair

Patent metadata

Field	Value
Publication number	US-12214022-B2
Application number	US-202217831507-A
Country	US
Kind code	B2
Filing date	Jun 3, 2022
Priority date	Jul 13, 2015
Publication date	Feb 4, 2025
Grant date	Feb 4, 2025

Abstract

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

First claim

The invention claimed is:

1. A method for killing a bacterium comprising contacting the bacterium with at least one recombinant phagemid(s) or plasmid(s);
wherein the recombinant phagemid(s) or plasmid(s) encodes an endonuclease that creates a double-stranded break (DSB) in the chromosomal or extrachromosomal DNA of the bacterium, and
an exogenous protein that inhibits DSB repair.

2. The method of claim 1 , wherein the exogenous protein is encoded by the same vector as the endonuclease or by a separate vector.

3. The method of claim 1 , wherein the endonuclease is one selected from a meganuclease, a Zinc Finger Nuclease and a Transcription Activator-Like Effector Nuclease (TALEN).

4. The method of claim 1 , wherein the endonuclease specifically cleaves the chromosomal or extrachromosomal DNA of the bacterium at less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sites.

5. The method of claim 1 , wherein the at least one recombinant phagemid(s) or plasmid(s) encodes an exogenous protein that inhibits DNA repair selected from the group consisting of Mu phage Gain protein, a lambda phage Gain protein and a phage T7 gp5.9 protein.

6. The method of claim 1 , wherein the at least one recombinant phagemid(s) is selected from the group consisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1 Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qß, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage, Pseudomonas phage ϕ6, ϕ29 phage and 186 phage.

7. The method of claim 1 , wherein the bacterium comprises a recBCD homologous repair pathway or addAB system.

8. The method of claim 1 , wherein the bacterium is selected from the group consisting of Enterobacter , Streptococci, Staphylococci, Enterococci, Salmonella, Pseudomonas , and Mycobacterium.

9. A method for making a bacterium more susceptible to an antibiotic comprising contacting the bacterium with at least one recombinant phagemid(s) or plasmid(s) and the antibiotic;
wherein the recombinant phagemid(s) or plasmid(s) encode(s) an endonuclease that creates a double-stranded break (DSB) in an antibiotic resistance gene encoded by the bacterium, and
an exogenous protein that inhibits DSB repair.

10. A phagemid or plasmid vector encoding an endonuclease selected from a meganuclease, a Zinc Finger Nuclease, and a TALEN, and an exogenous protein inhibiting DSB repair selected from the group consisting of Mu phage Gam protein, a lambda phage Gam protein, a phage T7 gp5.9 protein.

11. The phagemid or plasmid vector of claim 10 , wherein the recombinant phagemid(s) is selected from the group consisting of comprising M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage, Pseudomonas phage ϕ6, ϕ29 phage and 186 phage.

12. The phagemid or plasmid vector of claim 10 , wherein the phagemid vector is a λ bacteriophage.

13. A host cell comprising:
(i) the phagemid or plasmid vector of claim 10 , or
(ii) a phagemid or plasmid vector encoding an endonuclease and a vector encoding a protein inhibiting DSB repair.

14. A pharmaceutical composition comprising:
(i) the vector of claim 10 , or
(ii) a phagemid or plasmid vector encoding an endonuclease and a vector encoding a protein inhibiting DSB repair, or
(iii) a phagemid or plasmid vector encoding an endonuclease and a protein inhibiting DSB repair,
and a pharmaceutical acceptable vehicle.

15. The pharmaceutical composition of claim 14 further comprising an antibiotic suitable for treating infection due to a bacterium selected from the group of Enterobacter , Streptococci, Staphylococci, Enterococci, Salmonella, Pseudomonas , and Mycobacterium.

16. The method of claim 1 , wherein the endonuclease specifically cleaves the chromosomal or extrachromosomal DNA of the bacterium at a single site.

Assignees

• Pasteur Institut
• Eligo Bioscience

Inventors

• Bikard David
• Cui Lun
• Duportet Xavier
• Fernandez Rodriguez Jesus

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title