# US 12,767,944 B2 · Method for noninvasively acquiring micro-RNA from cells

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- Number: US 12,767,944 B2
- Document ID: US12767944B2
- Status: Granted
- Kind: B2
- Application number: 18/576,490
- Filed: 2022-06-15
- Published: 2024-09-26
- Granted: 2026-10-20
- Earliest claimed: 2021-07-08
- Assignees: National Institute of Advanced Industrial Science and Technology
- Applicants: National Institute of Advanced Industrial Science and Technology
- Inventors: Fumio Maeda; Shungo Adachi; Tohru Natsume
- Web page: https://patentgrep.com/patent/US12767944B2

## CPC

- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 15/1017: {by filtration, e.g. using filters, frits, membranes}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for noninvasively acquiring a microRNA from a cell, the method comprising (1) introducing a nucleic acid coding a microRNA-binding protein and a nucleic acid coding a vesicle-forming protein into the cell, wherein the microRNA-binding protein comprises a first portion consisting of an MID domain and a PIWI domain of an Argonaute protein and a second portion consisting of a virus protein R, and the vesicle-forming protein comprises: a palmitoylation or myristoylation signal or a PH domain; a self-assembling domain; an ESCRT or ESCRT-related factor-binding domain; and a Gag p6 domain; thereby producing an exosome-like vesicle comprising a micro-RNA, (2) collecting an extracellular fluid of the cell, and (3) extracting the micro-RNA from the extracellular fluid.

## Claims

### Claim 1

A method for noninvasively acquiring a microRNA from an in vitro cell, the method comprising:
(1) introducing a nucleic acid coding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein into the cell, wherein the microRNA-binding protein comprises a first portion consisting of an MID domain and a PIWI domain of an Argonaute protein and a second portion consisting of a virus protein R, and the vesicle-forming protein comprises: a palmitoylation or myristoylation signal or a pleckstrin homology domain; a self-assembling domain; an ESCRT or ESCRT-related factor-binding domain; and a Gag p6 domain; thereby producing an exosome-like vesicle comprising a microRNA,
(2) collecting an extracellular fluid of the cell, and
(3) extracting the micro-RNA from the extracellular fluid.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the microRNA-binding protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the vesicle-forming protein comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the extracellular fluid is a culture supernatant.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the vesicle-forming protein comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the extracellular fluid is a culture supernatant.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the extracellular fluid is a culture supernatant.

### Claim 8

An exosome-like vesicle comprising:
(a) a microRNA,
(b) a microRNA-binding protein comprising a first portion consisting of an MID domain and a PIWI domain of an Argonaute protein and a second portion consisting of a virus protein R, and
(c) a nanocage composed of a vesicle-forming protein comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

### Claim 9 (depends on 8)

The exosome-like vesicle according to claim 8, further comprising (d) a membrane fusion protein.