# US 12,214,022 B2 · Sequence-specific antimicrobials by blocking DNA repair

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- Number: US 12,214,022 B2
- Document ID: US12214022B2
- Status: Granted
- Kind: B2
- Application number: 17/831,507
- Filed: 2022-06-03
- Published: 2022-11-03
- Granted: 2025-02-04
- Earliest claimed: 2015-07-13
- Assignees: Institut Pasteur; Eligo Bioscience
- Applicants: Institut Pasteur; Eligo Bioscience
- Inventors: David Bikard; Lun Cui; Xavier Duportet; Jesus Fernandez Rodriguez
- Web page: https://patentgrep.com/patent/US12214022B2

## CPC

- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61P 31/04: Antibacterial agents
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/10: 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)
- C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2795/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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).

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 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.

### Claim 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.

### Claim 11 (depends on 10)

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.

### Claim 12 (depends on 10)

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

### Claim 13 (depends on 10)

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.

### Claim 14 (depends on 10)

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.

### Claim 15 (depends on 14)

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.

### Claim 16 (depends on 1)

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