阅读说明：本技术 细胞膜穿透肽 (Cell membrane penetrating peptides ) 是由 远藤英树 石坂幸人 石黑亮 高品智记 山本卓 佐久间哲史 于 2018-12-10 设计创作，主要内容包括：本发明的课题在于提供具有向细胞内的迁移性的细胞膜穿透肽。本发明人提供由序列号1所示的氨基酸序列构成的细胞膜穿透肽、由序列号2所示的氨基酸序列构成的细胞膜穿透肽和由序列号3所示的氨基酸序列构成的细胞膜穿透肽、以及包含该细胞膜穿透肽和功能性分子的复合物。(Disclosed is a cell membrane-penetrating peptide having a migration property into a cell. The present inventors provide a cell membrane-penetrating peptide comprising the amino acid sequence represented by sequence No. 1, a cell membrane-penetrating peptide comprising the amino acid sequence represented by sequence No. 2, and a cell membrane-penetrating peptide comprising the amino acid sequence represented by sequence No. 3, and a complex comprising the cell membrane-penetrating peptide and a functional molecule.)

1. A peptide selected from the group consisting of the following (1) to (3):

(1) a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) a peptide consisting of the amino acid sequence represented by SEQ ID No. 2; and

(3) a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

2. The peptide according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO. 1.

3. The peptide according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO. 2.

4. The peptide according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO. 3.

5. A complex comprising the peptide of claim 1 and a functional molecule.

6. A polynucleotide comprising a base sequence encoding the peptide of claim 1.

7. A polynucleotide comprising a base sequence encoding the complex of claim 5.

Technical Field

The present invention relates to cell membrane penetrating peptides.

Background

The cell membrane-penetrating peptide is a peptide having a function of penetrating a cell membrane and moving to the inside of the cell. As the cell membrane-penetrating peptide, various sequences such as Human Immunodeficiency Virus (HIV) -derived TAT (pennetrin), Oligoarginine (Oligoarginine), transporter (transporan), membrane transduction sequence (membrane transduction sequence) and the like are known (pharmacol. ther.,2015, vol.154, p.78-86). Further, a cell membrane penetrating peptide RIFIHFRIGC found from a peptide sequence contained in the viral protein R protein of HIV-1 has been reported (patent document 1).

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a novel cell membrane-penetrating peptide having migration into cells.

Means for solving the problems

The present inventors have made extensive studies in order to produce a cell membrane-penetrating peptide, and as a result, have produced a novel cell membrane-penetrating peptide (examples 1 and 2), and have found that the cell membrane-penetrating peptide has migration into cells (examples 3 and 4). As a result, the present inventors have provided the above cell membrane penetrating peptide and have completed the present invention.

That is, the present invention includes the following inventions as a medically or industrially useful substance or method.

[1] A peptide selected from the group consisting of the following (1) to (3):

(1) a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) a peptide consisting of the amino acid sequence represented by SEQ ID No. 2; and

(3) a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

[2] The peptide according to [1], which comprises an amino acid sequence represented by SEQ ID NO. 1.

[3] The peptide according to [1], which comprises an amino acid sequence represented by SEQ ID NO. 2.

[4] The peptide according to [1], which comprises an amino acid sequence represented by SEQ ID NO. 3.

[5] A complex comprising the peptide of [1] and a functional molecule.

[6] A polynucleotide comprising a base sequence encoding the peptide of [1 ].

[7] A polynucleotide comprising a base sequence encoding the complex of [5 ].

Effects of the invention

The cell membrane-penetrating peptide of the present invention can be used to penetrate any protein into a cell.

Drawings

FIG. 1 shows the intracellular uptake capacity of the CPP-EGFP protein. The vertical axis represents the ratio of the number of cells in which light emission by EGFP was observed among all the cells. Control represents the group of cells with solvent added only. Error bars represent the standard deviation of measurements from 3 replicate test samples.

FIG. 2 shows a comparison of the effect of increasing human TERT mRNA expression by CPP-TA L E-Activator the vertical axis shows the relative expression level of human TERT mRNA by each test sample assuming that the relative expression level of human TERT mRNA as a control is 1.

FIG. 3 shows a comparison of the effect of increasing human TERT mRNA expression by NTP-TA L E-VPR and TA L E-VPR, where the vertical axis represents the relative expression level of human TERT RNA for each test sample, assuming that the relative expression level of human TERT mRNA as a control is 1, and the error bars represent the standard deviation of the measured values for 3 replicate test samples.

FIG. 4A shows the results of a Green Fluorescent protein fluorescence Detection Size Exclusion Chromatography (Green Fluorescent protein fluorescence-Detection Size-Exclusion Chromatography, GFP-FSEC) analysis. In the figure, the vertical axis represents the amount of GFP fusion protein measured by fluorescence intensity, and the horizontal axis represents the exclusion volume of the buffer. The results of the samples (1) NTP-GST-EGFP, (2) NTP (C10Q) -GST-EGFP, (3) NTP (I8Q) -GST-EGFP, and (4) NTP (ICQ2) -GST-EGFP are shown.

Fig. 4B is a view showing the results of the 4 samples shown in fig. 4A superimposed on each other.

Detailed Description

The present invention will be described in detail below. The following embodiments are illustrative of the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various ways without departing from the gist thereof. In addition, the present specification includes the contents described in the specification and drawings of japanese patent application (japanese patent application No. 2017-236660) filed on 11/12/2017 as a basis for the priority claims of the present application.

1.Cell membrane penetrating peptide of the present invention

The cell membrane-penetrating peptide of the present invention is a peptide selected from the group consisting of the following (1) to (3).

(1) A peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) a peptide consisting of the amino acid sequence represented by SEQ ID No. 2; and

(3) a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

The "cell membrane penetrating peptide" in the present specification means a peptide having a function of penetrating a cell membrane. Whether or not the cell membrane is penetrated can be confirmed using a well-known cell membrane penetration evaluation system. Examples of such an evaluation system include an intracellular marker protein detection system using a complex comprising a highly sensitive green fluorescent protein (EGFP) and a test peptide, and a gene expression evaluation system using a complex comprising a deoxyribonucleic acid (DNA) binding polypeptide, a transcription regulator, and a test peptide. When a complex containing EGFP and a test peptide is used, the cell membrane permeability of the test peptide can be evaluated using, for example, the luminescence of EGFP taken into cells as an index. In a gene expression evaluation system using a complex comprising a DNA-binding polypeptide, a transcription regulatory factor, and a peptide to be tested, the cell membrane permeability of the peptide to be tested can be evaluated using the expression level of a target gene as an index. As a specific evaluation method, for example, the method described in examples 3 and 4 described below can be used.

2.The composite of the invention

The complex of the present invention is a complex comprising the cell membrane penetrating peptide of the present invention and a functional molecule.

The functional molecule capable of combining with the cell membrane-penetrating peptide of the present invention is widely available, and the functional molecule contained in the complex of the present invention is not particularly limited as long as it shows the function possessed by the functional molecule, and examples thereof include physiologically active substances such as low molecular compounds, polynucleotides, polypeptides, lipids, saccharides and other high molecular compounds, magnetic particles, liposomes, and the like.

As the polynucleotide included in the complex of the present invention, for example, DNA or ribonucleic acid (including RNA, aptamer) of any length can be used. The DNA or ribonucleic acid used may be natural or artificial. The polynucleotide may be single-stranded or double-stranded. A variety of polynucleotides may also be used.

In the case where the polynucleotide is a DNA, a DNA encoding a polypeptide exhibiting physiological activity may be used. Examples of the polypeptide exhibiting physiological activity include hormones, growth factors, enzymes, cytokines, vaccine antigen peptides, receptors, antibodies, transcription factors, structural proteins, and fusion polypeptides.

When the polynucleotide is RNA, examples thereof include small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small temporal RNA (stRNA), small interfering RNA (siRNA), microRNA (miRNA), pre-miRNA (pre-miRNA), small hairpin RNA (shRNA), viral RNA (viral RNA), antisense RNA, and messenger RNA (messenger RNA, mRNA).

Examples of the polypeptide contained in the complex of the present invention include hormones, growth factors, enzymes, cytokines, vaccine antigen peptides, receptors, antibodies, transcription factors, structural proteins, and fusion polypeptides.

In one embodiment, as the fusion polypeptide contained in the complex of the present invention, there can be cited, for example, fusion polypeptides used as genome editing techniques (Proc. Natl. Acad. Sci. USA., 1996, Vol.93, p.1156-1160; Genetics, 2010, Vol.186, p.757-761; Science, 2013, Vol.339, p.819-823; Science, 2013, Vol.339, p.823-826; Methods mol.biol., 2016, Vol.1469, p.147-155; nat. Methods, 2013, Vol.10, p.977-979). In another embodiment, the complex of the invention also comprises a polynucleotide encoding the fusion polypeptide.

The cell membrane penetrating peptide and the functional molecule contained in the complex of the present invention may be directly bound or may be indirectly bound via a linker.

The linker for binding the cell membrane-penetrating peptide and the functional molecule is not limited as long as the complex of the present invention penetrates the cell membrane and shows the function of the functional molecule.

The size (diameter, length, etc.) of the complex of the present invention is not limited as long as it is a size capable of penetrating cell membranes, and is, for example, in the range of about 0.1nm to about 500 nm. The length of the functional molecule included in the complex of the present invention is, for example, about 5000 nucleotides or less in the case where the functional molecule is RNA, about 2 ten thousand base pairs (hereinafter abbreviated as bp) or less in the case where the functional molecule is DNA, and about 3000 amino acids or less in the case where the functional molecule is polypeptide, but is not limited thereto.

3.Polynucleotides of the invention

The polynucleotides of the invention include: a polynucleotide comprising a base sequence encoding the cell membrane-penetrating peptide of the present invention, and a polynucleotide comprising a base sequence encoding the complex of the present invention.

In one embodiment, the polynucleotide of the present invention is a polynucleotide selected from the group consisting of:

(1) a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 2; and

(3) a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

In one embodiment, the polynucleotide of the present invention is a polynucleotide selected from the group consisting of:

(1) a polynucleotide comprising the base sequence of SEQ ID NO. 33 having base numbers 274 to 303;

(2) a polynucleotide comprising the base sequence of SEQ ID NO. 34 in base numbers 274 to 303; and

(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO. 35 having nucleotide numbers 274 to 303.

The polynucleotide of the present invention can be synthesized by a gene synthesis method known in the art (e.g., J.biol.chem., 1982, Vol.257, p.9226-9229) based on a base sequence designed based on the amino acid sequence of the cell membrane-penetrating peptide of the present invention or the complex of the present invention.

4.Expression vectors of the invention

The expression vector of the present invention comprises: an expression vector comprising a polynucleotide comprising a base sequence encoding the cell membrane-penetrating peptide of the present invention, and an expression vector comprising a polynucleotide comprising a base sequence encoding the complex of the present invention.

In one embodiment, the expression vector of the present invention is an expression vector comprising the polynucleotide of the present invention selected from the group consisting of:

(1) an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 2; and

(3) an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

The expression vector for expressing the polynucleotide of the present invention is not particularly limited as long as it can express a polynucleotide comprising a base sequence encoding the cell membrane-penetrating peptide of the present invention or the complex of the present invention in various host cells of eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and/or prokaryotic cells (e.g., Escherichia bacteria), or a cell extract used for cell-free protein synthesis (hereinafter, referred to as a cell-free protein synthesis solution, and examples thereof include a wheat germ extract, an Escherichia coli extract, a rabbit reticulocyte extract, and an insect cell extract), and can produce a polypeptide encoded by these polynucleotides. Examples of such expression vectors include plasmid vectors and viral vectors (e.g., adenovirus, adeno-associated virus, retrovirus, and Sendai virus), and pEU-E01-MCS (CellFreeSesciences), pET20b (+) (Novagen), and pCold vector-I (Takara Bio) can be preferably used.

The expression vector of the present invention may contain a promoter functionally linked to the polynucleotide of the present invention, examples of promoters for expressing the polynucleotide of the present invention in animal cells include promoters derived from viruses such as Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), Simian virus 40(Simian virus 40, SV40), actin promoters, Elongation Factor (EF) 1 α promoters, heat shock promoters, and the like, examples of promoters for expressing in bacteria such as escherichia bacteria include trp promoters, lac promoters, λ P L promoters, tac promoters, T3 promoters, T7 promoters, SP6 promoters, and the like, examples of promoters for expressing in yeast include GA L promoters, GA L promoters, PH 5 promoters, adhk promoters, GAP promoters, RNA promoters for expressing in yeast, RNA promoters for reacting with RNA polymerase, SP 5835, and the like.

In the case of using animal cells, insect cells or yeast as host cells, or in the case of using cell-free protein synthesis solutions, the expression vector of the present invention may contain an initiation codon and a stop codon. In this case, the expression vector of the present invention may include an enhancer sequence, untranslated regions on the 5 'and 3' sides of the gene encoding the fusion polypeptide of the present invention, a secretion signal sequence, a splice, a polyadenylation site, a replicable unit, and the like. In the case of using an Escherichia bacterium as a host cell, the expression vector of the present invention may contain an initiation codon, a stop codon, a terminator region and a replicable unit. In this case, the expression vector of the present invention may contain a selection marker (e.g., tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) that is generally used according to the purpose.

5.The transformed host cell of the present invention

Transformed host cells of the invention include: a host cell transformed with an expression vector comprising a base sequence encoding the cell membrane-penetrating peptide of the present invention, and a host cell transformed with an expression vector comprising a base sequence encoding the complex of the present invention.

In one embodiment, the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (1) to (3):

(1) a host cell transformed with an expression vector containing a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1;

(2) a host cell transformed with an expression vector containing a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 2; and

(3) a host cell transformed with an expression vector containing a polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO. 3.

The host cell to be transformed is not particularly limited as long as it is suitable for the expression vector to be used and can be transformed with the expression vector to express a protein. Examples of the host cell to be transformed include various cells (for example, animal cells (for example, CHO cells), insect cells (for example, Sf9), bacteria (for example, Escherichia bacteria), yeasts (for example, Saccharomyces and Pichia bacteria), and the like) such as natural cells or artificially established cells which are generally used in the technical field of the present invention, and preferably animal cells such as CHO cells, HEK293 cells and NS0 cells and Escherichia bacteria can be used.

The method for transforming the host cell is not particularly limited, and for example, a calcium phosphate method, an electroporation method, and the like can be used.

6.Method for producing cell membrane-penetrating peptide of the present invention

The cell membrane-penetrating peptide of the present invention can be produced by a peptide synthesis method known in the art or a known genetic engineering method. Examples of the peptide synthesis method include solid phase synthesis methods (Nature, 2011, Vol.480, p.471-479). As the genetic engineering method, for example, Methods shown in Methods in enzymol, 1987, vol.154, p.221-533, and philis. trans. a math. phys. eng.sci, 2009, vol.367, p.1705-1726, and the like can be used.

The method for producing the cell membrane penetrating peptide of the present invention may comprise: culturing the host cell of the present invention to express the cell membrane-penetrating peptide; or a step of reacting mRNA synthesized using the expression vector of the present invention with a cell-free protein synthesis solution to express a cell membrane-penetrating peptide. The method for producing the cell membrane-penetrating peptide of the present invention may include a step of culturing the transformed host cell of the present invention to express the cell membrane-penetrating peptide, a step of reacting the mRNA synthesized by the expression vector of the present invention with a cell-free protein synthesis solution to express the cell membrane-penetrating peptide, and a step of recovering, preferably isolating and purifying the expressed cell membrane-penetrating peptide. Examples of the separation or purification method include a method utilizing solubility such as salting-out and solvent precipitation, a method utilizing a difference in molecular weight such as dialysis, ultrafiltration and gel filtration, a method utilizing charge such as ion exchange chromatography and hydroxyapatite chromatography, a method utilizing specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, and a method utilizing a difference in isoelectric point such as isoelectric point electrophoresis. Preferably, the cell membrane-penetrating peptide accumulated in the culture supernatant can be purified by various chromatographies.

The transformed host cell can be cultured by a known method. The culture conditions, such as temperature, pH of the medium and culture time, are appropriately selected. The cell membrane penetrating peptide of the present invention can be produced by culturing host cells.

The cell membrane penetrating peptide of the present invention also includes a cell membrane penetrating peptide produced using the method for producing the cell membrane penetrating peptide of the present invention.

7.Method for producing the composite of the invention

The complex of the present invention can be easily produced by a person skilled in the art by binding the cell membrane-penetrating peptide of the present invention to a functional molecule using a binding method well known in the art (Nucleic Acids res., 2009, vol.37, p.2574-2583).

The method for producing the complex of the present invention includes a step of binding to a cell membrane-penetrating peptide directly or indirectly via a linker by a chemical binding method using a functional group present at the end or inside of a functional molecule. Examples of the chemical bonding method in this case include covalent bonds such as amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, and S — S bonds, and non-covalent bonds such as ionic bonds, electrostatic bonds, intermolecular forces, and hydrogen bonds.

When the binding is performed by a chemical binding method, the linker is not particularly limited as long as it has a structure that can link two molecules by having a reactive group at both ends, for example. Examples of the reactive group include a maleimide group, an N-succinimide ester group, an epoxy group, and an avidin group.

In one embodiment, in the case where the functional molecule is DNA or RNA, the cell membrane penetrating peptide of the present invention may be bound to DNA or RNA using, for example, disulfide bonds (FEBS L etters, 2004, Vol.558, p.63-68). further, in one embodiment, the cell membrane penetrating peptide of the present invention may be bound to DNA or RNA via a nucleic acid binding peptide such as protamine (Theranostics, 2017, Vol.7, p.2495-2508).

In one embodiment, in the case where the functional molecule is an antibody, the cell membrane penetrating peptide of the present invention can be bound to the antibody using a known method such as maleimidobenzoate succinimide ester (MBS) method (NanoBiotechnology Protocols, 2005, Vol.2, p.88), a crosslinking method using carbodiimide (Methods in enzymol., 2012, Vol.502, p.102), or a Conjugation method (Wong S, Chemistry of Protein Conjugation and Cross-L addition, CRC Press Inc., Boca Raton, 1993).

In addition, regarding the complex of the present invention, those skilled in the art can easily produce from a polynucleotide comprising a base sequence encoding the cell membrane penetrating peptide of the present invention and a functional molecule using genetic engineering methods well known in the art (appl. microbiol.biotechnol., 2006, vol.72, p.211, appl. microbiol.biotechnol., 2016, vol.100, p.5719-5728).

In the case of using a genetic engineering method, the method for producing the complex of the present invention comprises: a step of culturing the transformed host cell of the present invention to express the complex, or a step of reacting mRNA synthesized by the expression vector of the present invention with a cell-free protein synthesis solution to express the complex. The method for producing the complex of the present invention may include a step of culturing the transformed host cell of the present invention to express the complex, a step of reacting the mRNA synthesized by the expression vector of the present invention with a cell-free protein synthesis solution to express the complex, and a step of recovering, preferably isolating and purifying the expressed complex. As a method for isolation or purification, for example, the method described in <6 > method for producing a cell membrane-penetrating peptide of the present invention > can be used.

The composites of the invention also include composites produced using the methods of producing the composites of the invention.

8.Pharmaceutical compositions of the invention

The pharmaceutical compositions of the invention include pharmaceutical compositions comprising a complex of the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a commonly used method using a pharmaceutical excipient, a pharmaceutical carrier, and the like, which are commonly used excipients in this field. Examples of the dosage form of these pharmaceutical compositions include parenteral preparations such as injections and intravenous drip preparations, and administration can be performed by intravenous administration or subcutaneous administration. When formulated, excipients, carriers, additives and the like corresponding to these dosage forms may be used within a pharmaceutically acceptable range.

The amount of the complex of the present invention to be added in the preparation varies depending on the degree and age of the symptoms of the patient, the dosage form of the preparation to be used, or the functional molecule, and may be, for example, about 0.001mg/kg to about 100 mg/kg.

The pharmaceutical composition of the present invention can be used for the prevention and/or treatment of various diseases according to the function of the functional molecule contained in the complex of the present invention. For example, in the case where the functional molecule included in the complex is a compound, polynucleotide or polypeptide for the treatment of the disease, the pharmaceutical composition of the present invention can be used for the treatment or prevention of the disease.

In the present invention, the disease to be treated or prevented is not particularly limited, and may be selected according to the function of the functional molecule contained in the complex of the present invention, and examples thereof include cancer, immune diseases, nervous system diseases, endocrine system diseases, cardiovascular system diseases, and the like.

The present invention includes a pharmaceutical composition for prevention or treatment of a disease comprising the complex of the present invention. In addition, the invention includes a method of treating or preventing a disease comprising the step of administering to a patient a therapeutically effective amount of a complex of the invention. In addition, the invention includes complexes of the invention for use in the prevention or treatment of a disease. In addition, the present invention includes the use of the complex of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease.

In the present invention, the patient is not limited as long as it is a mammal, and examples thereof include a mouse, a rat, a dog, a pig, a monkey, and a human.

While the present invention has been described in full, specific embodiments referenced herein are provided for further understanding. These examples are for illustrative purposes and are not intended to limit the present invention.