# US 10,765,761 B2 · Nucleic acid condensing peptide, nucleic acid condensing peptide set, nucleic acid delivery carrier, nucleic acid delivery method, cell production method, cell detection method and kit

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- Number: US 10,765,761 B2
- Document ID: US10765761B2
- Status: Granted
- Kind: B2
- Application number: 16/119,738
- Filed: 2018-08-31
- Published: 2019-06-06
- Granted: 2020-09-08
- Earliest claimed: 2016-11-22
- Assignees: Kabushiki Kaisha Toshiba
- Applicants: Kabushiki Kaisha Toshiba
- Inventors: Mitsuko Ishihara; Eiichi Akahoshi; Emi Nozaki
- Web page: https://patentgrep.com/patent/US10765761B2

## CPC

- A61K 48/0033: {the non-active part being non-polymeric}
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 41/0028: {Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 35/00: Antineoplastic agents
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/001: {by chemical synthesis}
- C07K 14/003: {Peptide-nucleic acids (PNAs)}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- A61K 31/4184: condensed with carbocyclic rings, e.g. benzimidazoles
- A61K 31/575: substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
- A61K 33/243: Platinum; Compounds thereof
- A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)}
- C07K 2319/00: Fusion polypeptide
- C12Q 1/66: involving luciferase
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

According to one embodiment, a nucleic acid condensing peptide includes one terminal having a first sequence RRRRRR and another terminal having a second sequence RQRQR. Between the first sequence and the second sequence, none or one or more intermediate sequences each consisting of RRRRRR or RQRQR are included. Further, of the first sequence, the second sequence and the intermediate sequences, two or more neutral amino acids are included between any two sequences adjacent to each other.

## Claims

### Claim 1

A nucleic acid condensing peptide comprising:
a first sequence RRRRRR at one terminal, a second sequence RQRQR at another terminal,
none or one or more intermediate sequences comprising RRRRRR or RQRQR between the first sequence and the second sequence, and
two or more neutral amino acids between two adjacent sequences of the first sequence, the second sequence and the intermediate sequence.

### Claim 2 (depends on 1)

The peptide of claim 1, wherein the neutral amino acids contain G or Y.

### Claim 3 (depends on 1)

The peptide of claim 1, wherein a full length of the peptide is 55 or less amino acids.

### Claim 4 (depends on 1)

The peptide of claim 1, wherein the R is contained at a rate of 45% or higher to the entire sequence.

### Claim 5 (depends on 1)

The peptide of claim 1, wherein the peptide is composed of salts containing sulfate ion.

### Claim 6 (depends on 1)

The peptide of claim 1, wherein a sequence of the peptide is RQRQRYYRQRQRGGRRRRRR or RQRQRGGRRRRRR.

### Claim 7 (depends on 1)

A peptide set for condensing nucleic acid, comprising:
the nucleic acid condensing peptide of claim 1, as a first nucleic acid condensing peptide; and a second nucleic acid condensing peptide and/or a third nucleic acid condensing peptide,
wherein the second nucleic acid condensing peptide comprises: a third sequence RRRRRR at one terminal, a fourth sequence RRRRRR at another terminal, none or one or more intermediate sequences comprising RRRRRR or RQRQR between the third sequence and the fourth sequence, and two or more neutral amino acids between two adjacent sequences of the third sequence, the fourth sequence and the intermediate sequence; and
the third nucleic acid condensing peptide comprises an amino acid sequence GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.

### Claim 8 (depends on 7)

The peptide set of claim 7, wherein an amino acid sequence of the second nucleic acid condensing peptide is RRRRRRYYRQRQRGGRRRRRR.

### Claim 9 (depends on 7)

The peptide set of claim 7, wherein an arbitrary amino acid sequence is linked to one end of the third nucleic acid condensing peptide.

### Claim 10 (depends on 9)

The peptide set of claim 9, wherein the arbitrary amino acid sequence is an amino acid sequence which binds to the nucleic acid.

### Claim 11 (depends on 7)

The peptide set of claim 7, wherein
when the peptide set comprises comprising the third nucleic acid condensing peptide, the first nucleic acid condensing peptide and/or the second nucleic acid condensing peptide are linked to the third nucleic acid condensing peptide.

### Claim 12 (depends on 1)

A nucleic acid delivery carrier comprising:
a complex comprising the nucleic acid condensing peptide of claim 1 and a nucleic acid; and a liposome,
wherein the complex is encapsulated in the liposome.

### Claim 13 (depends on 7)

A nucleic acid delivery carrier comprising:
a complex comprising the nucleic acid condensing peptide set of claim 7 and a nucleic acid; and a liposome,
wherein the complex is encapsulated in the liposome.

### Claim 14 (depends on 12)

The nucleic acid delivery carrier of claim 12, further comprising: a function improving agent, wherein the function improving agent is encapsulated in the liposome, and wherein the function improving agent is at least one selected from the group consisting of:
a reagent for detecting a signal from a reporter gene;
a drug; and
a transcription promoter.

### Claim 15 (depends on 12)

A nucleic acid delivery method comprising:
preparing the nucleic acid delivery carrier of claim 12; and
bringing the nucleic acid delivery carrier into contact with a cell, thereby delivering the nucleic acid into the cell.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cell is a cell in a living body.

### Claim 17 (depends on 15)

The method of claim 15, further comprising: opening a pore in a cell membrane of the cell before or after bringing the nucleic acid delivery carrier and the cell into contact with each other.

### Claim 18 (depends on 17)

The method of claim 17, wherein the opening the pore is carried out by applying voltage or ultrasonic waves to the cell.

### Claim 19 (depends on 15)

A method of transforming a cell, comprising:
executing the nucleic acid delivery method of claim 15;
culturing the cell obtained; and
selecting a cell into which the nucleic acid is transfected into of the cells.

### Claim 20 (depends on 15)

A cell detection method comprising:
executing the nucleic acid delivery method of claim 15, wherein the nucleic acid comprises a reporter gene expression cassette containing a reporter gene; and
detecting a signal from the reporter gene.

### Claim 21 (depends on 12)

A kit comprising: the nucleic acid delivery carrier of claim 12; and a function improving agent, wherein the function improving agent is at least one selected from the group consisting of:
a reagent for detecting a signal from a reporter gene;
a drug; and
a transcription promoter.

### Claim 22 (depends on 21)

The kit of claim 21, wherein
the kit is a kit for detecting a cell, the nucleic acid contained in the nucleic acid delivery carrier contains a reporter gene expression cassette for expressing a reporter gene, and the function improving agent contains the reagent for detecting a signal from the reporter gene.

### Claim 23 (depends on 21)

The kit of claim 21, wherein the kit is a kit for modifying a function of a cell taken out of an organism. the nucleic acid contained in the nucleic acid delivery carrier contains a gene for modifying the function of the cell, and the function improving agent is a drug that promotes differentiation.

### Claim 24 (depends on 21)

The kit of claim 21, wherein
the kit is a kit for killing a cell, the nucleic acid contained in the nucleic acid delivery carrier contains a gene which leads the cell to apoptosis, and the function improving agent contains actinomycin-D, cisplatin, bendamustine hydrochloride, betulic acid or a combination of any of these.

### Claim 25 (depends on 21)

The kit of claim 21, wherein
the kit is a kit for producing a substance in a cell, the nucleic acid contained in the nucleic acid delivery carrier contains a gene for producing a desired substance in the cell, and the function improving agent contains a transcription promoter.

### Claim 26 (depends on 1)

The peptide of claim 1, wherein the neutral amino acids contain two to five amino acids.

### Claim 27 (depends on 7)

The peptide of claim 7, wherein the neutral amino acids contain G or Y.

### Claim 28 (depends on 7)

The peptide of claim 7, wherein a full length of the peptide is 55 or less amino acids.

### Claim 29 (depends on 7)

The peptide of claim 7, wherein the R is contained at a rate of 45% or higher to the entire sequence.

### Claim 30 (depends on 7)

The peptide of claim 7, wherein the peptide is composed of salts containing sulfate ion.

### Claim 31 (depends on 7)

The peptide of claim 7, wherein a sequence of the peptide is RQRQRYYRQRQRGGRRRRRR or RQRQRGGRRRRRR.

### Claim 32 (depends on 7)

The peptide of claim 7, wherein the neutral amino acids contain two to five amino acids.

### Claim 33 (depends on 12)

The peptide of claim 12, wherein the neutral amino acids contain G or Y.

### Claim 34 (depends on 12)

The peptide of claim 12, wherein a full length of the peptide is 55 or less amino acids.

### Claim 35 (depends on 12)

The peptide of claim 12, wherein the R is contained at a rate of 45% or higher to the entire sequence.

### Claim 36 (depends on 12)

The peptide of claim 12, wherein the peptide is composed of salts containing sulfate ion.

### Claim 37 (depends on 12)

The peptide of claim 12, wherein a sequence of the peptide is RQRQRYYRQRQRGGRRRRRR or RQRQRGGRRRRRR.

### Claim 38 (depends on 12)

The peptide of claim 12, wherein the neutral amino acids contain two to five amino acids.

### Claim 39 (depends on 13)

The peptide of claim 13, wherein the neutral amino acids contain G or Y.

### Claim 40 (depends on 13)

The peptide of claim 13, wherein a full length of the peptide is 55 or less amino acids.

### Claim 41 (depends on 13)

The peptide of claim 13, wherein the R is contained at a rate of 45% or higher to the entire sequence.

### Claim 42 (depends on 13)

The peptide of claim 13, wherein the peptide is composed of salts containing sulfate ion.

### Claim 43 (depends on 13)

The peptide of claim 13, wherein a sequence of the peptide is RQRQRYYRQRQRGGRRRRRR or RQRQRGGRRRRRR.

### Claim 44 (depends on 13)

The peptide of claim 13, wherein the neutral amino acids contain two to five amino acids.