# US 12,760,282 B2 · Method for producing a modified bacteriophage without genome modification

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- Number: US 12,760,282 B2
- Document ID: US12760282B2
- Status: Granted
- Kind: B2
- Application number: 17/627,861
- Filed: 2020-07-16
- Published: 2022-08-18
- Granted: 2026-10-13
- Earliest claimed: 2019-07-18
- Assignees: Technische Universität München
- Applicants: Technische UniversitäT MüNchen
- Inventors: Kilian Vogele; Friedrich Simmel; Quirin Emslander
- Web page: https://patentgrep.com/patent/US12760282B2

## CPC

- C12N 7/02: Recovery or purification
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61P 31/04: Antibacterial agents
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The invention relates to a method for producing a modified bacteriophage in a cell-free expression system wherein the expression of at least one gene of interest is suppressed by a molecule specifically inhibiting its expression. The invention further relates to a composition and a kit for producing a modified bacteriophage. Moreover, the invention relates to a bacteriophage which is not modified on the genomic level but on the proteomic level and its use for therapy, for diagnostic and detection assays.

## Claims

### Claim 1

A method for producing a modified bacteriophage in a cell-free expression system comprising the steps of:
contacting a cell lysate of a microorganism with a genome of the bacteriophage; and
suppressing expression of at least one gene of interest encoded by the genome of the bacteriophage by adding a molecule specifically inhibiting the expression of the endogenous version of the at least one gene of interest; and
expressing a modified version of the at least one gene of interest by adding a molecule encoding a modified version of the gene of interest, and wherein
the molecule inhibiting the expression of the endogenous version of the at least one gene of interest is a DNAi molecule binding to the ribosome binding side of the gene of interest, and wherein the DNAi molecule does not bind to the ribosome binding site of the modified version of the gene of interest.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the gene of interest is an essential gene or a non-essential gene.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the genome of the bacteriophage is not modified.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the gene of interest encodes a capsid protein or a tail fiber protein of the bacteriophage.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the modified bacteriophage protein comprises a modification selected from the group consisting of an affinity tag, a detection marker, a protein for the improvement of the bacteriophage or mutation or combinations thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the bacteriophage is a bacteriophage of the Myoviridae family.

### Claim 7 (depends on 1)

A composition for carrying out the method of claim 1 comprising:
a cell lysate of a microorganism; and
a genome of the bacteriophage; and
a molecule specifically inhibiting the expression of the endogenous version of the gene of interest; and
a molecule encoding a modified version of the gene of interest, and wherein the molecule inhibiting the expression of the endogenous version of the at least one gene of interest is a DNAi molecule binding to the ribosome binding side of the gene of interest, and wherein the DNAi molecule does not bind to the ribosome binding site of the modified version of the gene of interest.

### Claim 8 (depends on 1)

A kit for carrying out the method of claim 1 comprising:
a cell lysate of a microorganism; and
a genome of the bacteriophage; and
a molecule specifically inhibiting the expression of the endogenous version of the gene of interest; and
a molecule encoding a modified version of the gene of interest, and wherein the molecule inhibiting the expression of the endogenous version of the at least one gene of interest is a DNAi molecule binding to the ribosome binding side of the gene of interest, and wherein the DNAi molecule does not bind to the ribosome binding site of the modified version of the gene of interest.

### Claim 9 (depends on 4)

The method of claim 4, wherein the gene of interest encodes a highly immunogeneic outer capsid protein (HOC).

### Claim 10 (depends on 6)

The method of claim 6, wherein the bacteriophage is of the Tevenvirinae subfamily.

### Claim 11 (depends on 10)

The method of claim 10, wherein the bacteriophage is Escherichia virus T4.