CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to JP 2019-069134, filed Mar. 29, 2019.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 24, 2020, is named sequence.txt and is 1,659 bytes.

FIELD OF THE INVENTION

The present invention relates to a novel polypeptide comprising a cell-penetrating sequence.

The present invention also relates to a cell-invasive composition comprising the polypeptide and a substance of interest to be introduced into a cell.

The present invention further relates to a method for transporting a substance of interest into a cell using the polypeptide.

BACKGROUND OF THE INVENTION

A cell-penetrating peptide is known as a tool that is extensively used for transporting a substance, such as a certain peptide, protein, or oligonucleotide (also referred to as a “cargo molecule”), into a cell. While such cellular uptake was deduced to occur by directly passing through cell membrane in the beginning, at present, it is known that endocytosis significantly contributes to the cellular uptake (M. Fotin-Mleczek et al, Curr. Pharm. Design., 2005, 11: 3613-3628).

A cell-penetrating peptide is applicable to fields including cellular biology, medicine, and agriculture. For example, a cell-penetrating peptide can be used for intracellular drug delivery, plant variety improvement by genome editing, and cell modification. Although numerous cell-penetrating peptides have been proposed, it is said that desired peptides are (poly)peptides that have properties including the capacity of efficient delivery of a wide variety of cargo molecules into cells without damaging cell membranes. Triggered by the finding in 1988 that the Tat protein, which is an AIDS virus transcription factor, penetrates a cell membrane, the research and development of cell-penetrating peptides, including the Tat peptide, became active.

Examples of cell-penetrating peptides reported include (poly)peptides comprising cationic functional groups including naturally-occurring or non-naturally-occurring amino acids, such as lysine (Lys) or arginine (Arg) (e.g., JP 2016-190813 A), amphipathic (poly)peptides comprising polycationic sequences and polyanionic sequences (e.g., JP 2017-527300 A), and telomerase-derived (poly)peptides (e.g., JP 2018-172396 A).

SUMMARY OF THE INVENTION

While many cell-penetrating peptides have been reported, drawbacks thereof are known. Examples of such peptides include: peptides that disrupt cell membranes to leak the cytoplasm or interrupt normal functions of membrane proteins; peptides that gives rise to a cytotoxic or immunogenic effect; peptides that are rapidly degraded in the cytoplasm; peptides that are captured in the endosome and degraded in that state in the lysosome; peptides that cannot make a cargo molecule free; and peptides that are applicable to a limited range of cargo molecules (Naoki Kajiwara, Futoshi Shibasaki, Journal of the Japanese Pharmacological Society, 2013, 141: 220-221).

Under the above circumstances, there are needs for: cell-penetrating peptides with no or little the above-mentioned drawbacks; or cell-penetrating peptides that are capable of highly efficient cellular uptake of various types of cargo molecules and have low toxicity. In the present invention, we have now found polypeptides having at least properties that they exhibit a higher ability of cell invasion (internalization) over a long period of time compared with conventional cell-penetrating Tat peptide or R9 peptide (polyarginine), that they enable introduction of a cargo molecule into a cell over a long period of time because of high resistance to enzymatic degradation; and that they have almost no cytotoxicity. This has led to the completion of the present invention.

The present invention includes the following features.

[1] A polypeptide represented by Formula (I):


wherein n is a number of 2 to 20; R and R may independently represent a C1-C4alkyl group or may bind to each other to form a ring; R3represents a C1-C10primary aminoalkyl group; R4represents a hydrogen atom or a C1-C10hydrocarbon group; the “n” number of R1, R2, R3, and R4may independently be the same with or different from each other; and the N-terminus and the C-terminus may or may not be modified independently.
[2] The polypeptide according to [1], wherein R1and R2are both methyl groups.
[3] The polypeptide according to [1] or [2], wherein R4is a hydrogen atom or a C1-C3alkyl group.
[4] The polypeptide according to any of [1] to [3], wherein the C-terminus is modified.
[5] The polypeptide according to any of [I] to [4], wherein the N-terminus is modified.
[6] The polypeptide according to any of [1] to [5], which comprises 2-20 tripeptide units represented by Formula (II):
—Y1-Y2-Y3-  (II)
wherein Y1 is a cationic amino acid selected from the group consisting of Lys and Orn; Y3 is a non-cationic amino acid selected from the group consisting of Gly, Ala, Phe, Leu, Val, and Ile; and Y2 is a non-naturally-occurring amino acid comprising at least one quaternary carbon, each of Y1, Y2, and Y3 is present in a number of 2 to 20 and may independently be the same with or different from each other, and the N-terminus and the C-terminus of the polypeptide may or may not be modified independently.
[7] The polypeptide according to any of [1] to [6], which comprises a reactive functional group on at least one of the N-terminus and the C-terminus.
[8] A cell-invasive composition comprising a polypeptide, as a first polypeptide, according to any of [1] to [7] and a substance of interest, wherein the substance of interest is united with the polypeptide or is present independently from the polypeptide.
[9] The composition according to [8], wherein the substance of interest is selected from the group consisting of a protein, a peptide, a glycoprotein, a naturally-occurring or non-naturally-occurring nucleic acid, DNA, RNA, a DNA/RNA hybrid, an oligonucleotide, a polynucleotide, an anti-sense molecule, miRNA, siRNA, a plasmid, a low-molecular-weight compound, a sugar, a lipid, a glycolipid, a contrast substance, a drug, nanoparticles, and a quantum dot.
[10] The composition according to [8] or [9], wherein one amino acid or a combination of two or more amino acids selected from among Lys, His, and Arg further comprises/comprise a second polypeptide comprising two or more continuous cationic sequences.
[11] The composition according to [10], wherein at least part of the first polypeptide is bound to at least part of the second polypeptide.
[12] A kit for intracellular delivery comprising a polypeptide according to any of [1] to [7].
[13] A method for transporting at least one substance of interest into a cell, comprising bringing a polypeptide according to any of [1] to [7] into contact with a cell and thereby introducing at least one substance of interest into a cell, wherein the substance is united with the polypeptide and/or is present independently from the polypeptide.

According to the present invention, a polypeptide that has almost no cytotoxicity, that has high resistance to enzymatic degradation, and that exhibits a high ability of cell invasion (internalization) over a long period of time, is provided.

