US-12617817-B2 - Carrier peptide fragment and use thereof

US12617817B2US 12617817 B2US12617817 B2US 12617817B2US-12617817-B2

Abstract

A technology for efficiently introducing a foreign substance into at least cytoplasm of eukaryotic cells from outside the cells is provided. The method for introducing a foreign substance of interest in vitro or in vivo into at least cytoplasm of eukaryotic cells from outside the cells includes (1) a step of preparing a foreign substance introduction construct including a carrier fragment composed of (SEQ ID NO: 1) KKRTLRKSNRKKR and the foreign substance of interest linked to the N-terminal and/or C-terminal side of the carrier peptide fragment; (2) a step of supplying the foreign substance introduction construct to a sample containing a target eukaryotic cell; and (3) a step of incubating the sample, to which the foreign substance introduction construct has been supplied, to thereby introduce the construct into the eukaryotic cell in the sample.

Inventors

Nahoko BAILEYKOBAYASHI
Tetsuhiko Yoshida

Assignees

TOAGOSEI CO., LTD.

Dates

Publication Date: 20260505
Application Date: 20210910
Priority Date: 20200914

Claims (9)

1 . A method of introducing a foreign substance of interest into at least cytoplasm of eukaryotic cells from outside the cells, the method comprising: (1) a step of preparing a foreign substance introduction construct including a carrier peptide fragment composed of the amino acid sequence below (SEQ ID NO: 1) KKRTLRKSNRKKR and the foreign substance of interest linked to the N-terminal and/or C-terminal side of the carrier peptide fragment; (2) a step of supplying the foreign substance introduction construct to a sample containing a target eukaryotic cell; and (3) a step of incubating the sample, to which the foreign substance introduction construct has been supplied, to thereby introduce the foreign substance introduction construct into the eukaryotic cell in the sample.
2 . The method according to claim 1 , wherein the foreign substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes, and drugs.
3 . The method according to claim 2 , wherein the foreign substance is a mature polypeptide derived from any species of organism, or a precursor polypeptide thereof, and the foreign substance introduction construct is a synthetic polypeptide comprising an amino acid sequence corresponding to a mature polypeptide or precursor polypeptide thereof as the foreign substance, together with the amino acid sequence of the carrier peptide fragment.
4 . The method according to claim 3 , wherein the amino acid sequence corresponding to the mature polypeptide or precursor polypeptide thereof as the foreign substance is linked to the N-terminal side of the carrier peptide fragment.
5 . The method according to claim 1 , wherein the target eukaryotic cell into which the foreign substance introduction construct is introduced is a human or non-human mammalian cell.
6 . A foreign substance introduction construct prepared for introducing a foreign substance of interest into at least cytoplasm of eukaryotic cells from outside the cells, the foreign substance introduction construct comprising: a carrier peptide fragment composed of KKRTLRKSNRKKR (SEQ ID NO: 1), and the foreign substance of interest linked to the N-terminal and/or C-terminal side of the carrier peptide fragment.
7 . The construct according to claim 6 , wherein the foreign substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes, and drugs.
8 . The construct according to claim 7 , wherein the foreign substance is a mature polypeptide delivered from any species of organism, or a precursor polypeptide thereof, and the construct is a synthetic polypeptide comprising an amino acid sequence corresponding to the mature polypeptide or precursor polypeptide thereof as the foreign substance, together with the amino acid sequence of the carrier peptide fragment.
9 . The construct according to claim 8 , wherein the amino acid sequence corresponding to the mature polypeptide or precursor polypeptide thereof as the foreign substance is linked to the N-terminal side of the carrier peptide fragment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority on the basis of Japanese Patent Application No. 2020-153492 filed in Japan on Sep. 14, 2020, the entire contents of which are incorporated herein by reference. REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “TOAGOSEI CO., LTD.” created on Sep. 6, 2021 and is 1,000 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety. TECHNICAL FIELD The disclosure relates to a method of introducing (transporting) a foreign substance into a eukaryotic cell from outside the cell, and to a carrier peptide fragment for use in this method. BACKGROUND Conventionally, foreign substances, such as peptides and particularly physiologically active substances, are introduced into the cells of humans and other mammals and the like (eukaryotic cells) to alter the traits of those cells (and moreover tissues and organs composed of such cells) or to improve or enhance the functions of those cells. For example, Japanese Patent No. 3854995 discloses a cell-penetrating carrier peptide for introducing foreign substances such as polypeptides and DNA into cells. This patent literature describes introducing highly effectively physiologically active substances, such as polypeptides and DNA, into cells by using a carrier peptide conjugate having a cell-penetrating carrier peptide linked to a heterologous polypeptide, DNA or the like. Methods are also being sought that alter the traits of target cells or improving (or enhancing) their functions by easily introducing whole polypeptides with relatively large molecular weights, as foreign substances (physiologically active substances), into the target cells without using any specialized equipment. Focusing on specific functions of polypeptides, methods are also being sought that efficiently introduce amino acid sequence parts that are the smallest parts capable of expressing such functions, or in other words, amino acid sequence (foreign substances) constituting peptide motifs (peptide fragments), into cells, instead of introducing whole polypeptides or proteins. SUMMARY Meanwhile, WO 2011/013700 describes a foreign substance introduction construct containing a foreign substance of interest together with the amino acid sequence (carrier peptide fragment) of SEQ ID NO:2 (described in Journal of Biological Chemistry, 281(35), 2006, pp. 25223-25230), which is known as a nucleolar localization signal (hereunder called a “NoLS”). This construct can pass highly efficiently through the cell membranes of a eukaryotic cell, whereby the foreign substance of interest can be effectively introduced into the eukaryotic cell from outside the cell. Cell-penetrating peptides such as the above NoLS have attracted increased interest in recent years from a medical perspective and the like, and there is demand for the development of technologies for more efficiently introducing foreign substances into target cells. It is an object of the present disclosure, created to meet such demands, to provide a method capable of efficiently introducing a foreign substance of interest into at least the cytoplasm of eukaryotic cells from outside the cells. Another object of the disclosure is to provide a construct including a carrier peptide fragment and a foreign substance of interest and capable of efficiently introducing that foreign substance into the cytoplasm of eukaryotic cells from outside the cells. To more efficiently introduce the foreign substance introduction construct disclosed in WO 2011/013700 above into the cytoplasm of eukaryotic cells from outside the cells, the inventors substituted various amino acid residues in the amino acid sequence of SEQ ID NO:2 to prepare a construct with greater cell penetrating ability. As a result, the inventors found that cell penetrating ability was improved in a mutant having serine substituted for the 8th position asparagine and asparagine substituted for the 9th position aspartic acid counting from the N-terminal of the amino acid sequence of SEQ ID NO:2. Surprisingly, substitution of the 9th position amino acid residue was not a so-called conservative substitution of amino acid residues (such as substitution of a basic amino acid for another basic amino acid). That is, the amino acid sequence of SEQ ID NO:1 disclosed here was created through extensive trial and error on the part of the inventors. The method disclosed here is a method of introducing (transporting) a foreign substance of interest into at least the cytoplasm (preferably also into the nucleus) of eukaryotic cells (especially various animal cells such as human and other mammal cells lacking cell walls) from outside those cells (that is, from outside the cell membrane). That is, the foreign substance intr