# US 2020/0216858 A1 · Polynucleotide for modifying target sequence and use thereof

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- Number: US 2020/0216858 A1
- Document ID: US20200216858A1
- Status: Published application
- Kind: A1
- Application number: 16/631,407
- Filed: 2018-07-19
- Published: 2020-07-09
- Earliest claimed: 2017-07-21
- Assignees: I'rom Group Co., Ltd.
- Applicants: I'rom Group Co., Ltd.
- Inventors: Kohji KUSANO; Takayuki KITOGO; Makoto INOUE; Tsugumine SHU; Toyotaka MORI
- Web page: https://patentgrep.com/patent/US20200216858A1

## CPC

- C12N 15/85: for animal cells
- C12N 15/907: {in mammalian cells}
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/86: Viral vectors
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 2320/00: Applications; Uses
- C12N 2710/16643: viral genome or elements thereof as genetic vector
- C12N 2760/18011: Paramyxoviridae
- C12N 2760/18842: virus or viral particle as vehicle, e.g. encapsulating small organic molecule
- C12N 2760/18843: viral genome or elements thereof as genetic vector
- C12N 2800/10: Plasmid DNA
- C12N 2810/6072: negative strand RNA viruses
- 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)

## Abstract

The present invention provides novel donor polynucleotides formed by linking the two ends of a genomic fragment containing a cleavable site by a polynucleotide carrying a positive selection marker gene and a negative selection marker gene. Use of the donor polynucleotide makes it possible to modify only a target gene with avoiding the possibility of introducing mutations to sequences, called “off-target”, which are other than the target sequence, by introducing cleavage in a homologous site of the donor polynucleotide without introducing cleavage in a target gene locus.

## Claims

### Claim 1

A donor polynucleotide for modifying a genomic sequence, wherein the donor polynucleotide comprises a genomic fragment comprising one or more modifications, wherein both ends of the genomic fragment are linked by a polynucleotide, wherein the linker polynucleotide comprises within it both a positive selection marker gene and a negative selection marker gene, wherein the genomic fragment is cleavable, and wherein the donor polynucleotide may be linear when the cleavable site is severed to become the two ends of the donor polynucleotide chain, or the donor polynucleotide may be circular when the site is connected in the donor polynucleotide.

### Claim 2 (depends on 1)

The donor polynucleotide of claim 1, wherein a cleavage sequence is added to the cleavable site.

### Claim 3 (depends on 2)

The donor polynucleotide of claim 2, wherein the cleavage sequence added to the site is not contained in a sequence of a target cell genomic fragment corresponding to the genomic fragment comprised in the donor polynucleotide.

### Claim 4 (depends on 1, 2, 3)

The donor polynucleotide of any one of claims 1 to 3, wherein in the genomic fragment, the one or more modifications are included only on one side of the site.

### Claim 5 (depends on 1, 2, 3, 4)

The donor polynucleotide of any one of claims 1 to 4, wherein the linker polynucleotide is a polynucleotide of a plasmid.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The donor polynucleotide of any one of claims 1 to 5, which does not comprise a target cell genomic sequence in between the positive selection marker gene and the negative selection marker gene.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The donor polynucleotide of any one of claims 1 to 6, wherein the positive selection marker gene and the negative selection marker gene are fused, and the positive selection marker and the negative selection marker are expressed as a fusion protein.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

A method for modifying a genomic sequence, which comprises the steps of: (a) introducing the donor polynucleotide of any one of claims 1 to 7 into cells; (b) selecting, using a positive selection marker, cells into which the donor polynucleotide has been introduced; and (c) selecting, using a negative selection marker, cells in which the linker polynucleotide has been removed.

### Claim 9 (depends on 8)

The method of claim 8, wherein step (a) comprises a step of introducing into cells a linear donor polynucleotide formed by severing the site of the donor polynucleotide

### Claim 10 (depends on 8)

The method of claim 8, wherein step (a) comprises a step of introducing into cells a circular donor polynucleotide, in which the site is connected, and a cleavage enzyme that cleaves the site or a vector that expresses the enzyme.

### Claim 11 (depends on 10)

The method of claim 10, which comprises introducing the circular donor polynucleotide simultaneously with the enzyme or the vector.

### Claim 12 (depends on 10, 11)

The method of claim 10 or 11, wherein the vector that expresses the enzyme is a minus strand RNA virus vector that expresses the enzyme.

### Claim 13 (depends on 8, 9, 10, 11, 12)

The method of any one of claims 8 to 12, which further comprises a step of selecting a cell comprising a modification of interest in its genome.

### Claim 14 (depends on 8, 9, 10, 11, 12, 13)

The method of any one of claims 8 to 13, which is used to convert a disease-causing sequence to a normal sequence in a causative gene of a hereditary disease.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7)

A cell having a structure in which the donor polynucleotide of any one of claims 1 to 7 has been incorporated into its genome, wherein the modified genomic fragment included in the donor polynucleotide and a fragment corresponding thereto derived from the genome of the cell have a structure in which they are arranged in tandem in no particular order via the linker polynucleotide, and wherein the linker polynucleotide comprises within it a positive selection marker gene and a negative selection marker gene.

### Claim 16 (depends on 15)

A method for producing a cell with a modified genome, which comprises a step of subjecting the cell of claim 15 to selection using a negative selection marker to select cells in which the linker polynucleotide has been removed.

### Claim 17 (depends on 12)

A minus strand RNA virus vector for use in the method of claim 12, which encodes an endonuclease that cleaves the cleavage site of the donor polynucleotide.

### Claim 18 (depends on 17)

The vector of claim 17, which is a Sendai virus vector.

### Claim 19 (depends on 17, 18)

The vector of claim 17 or 18, wherein the endonuclease is I-SceI.

### Claim 20 (depends on 12, 17, 18, 19)

A composition for use in the method of claim 12, which comprises the vector of any one of claims 17 to 19.