JP7013626B2 - Methods for intracellularly transporting cell membrane penetrating peptides, constructs, and cargo molecules - Google Patents

Description

The present invention relates to a peptide having cell membrane permeability, and specifically to a peptide having cell membrane permeability capable of efficiently transporting a nucleic acid such as siRNA into a cell. It also relates to a construct consisting of the peptide and a cargo molecule. Further, the present invention relates to a method for transporting a cargo molecule into a cell.

Attention has been paid to a method of introducing a nucleic acid such as siRNA into a cell using a peptide having cell permeability (hereinafter, may be abbreviated as cell-penetrating Peptides (CPPs)). CPPs are chemically bound to a nucleic acid such as siRNA to be introduced into cells as an intracellular introduction vector, or a fusion protein of CPPs and a nucleic acid such as siRNA to be introduced is prepared by genetic engineering and used in a cell culture medium. By mixing, the target molecule is efficiently introduced into the cell.

Typical examples actually used as CPPs are (1) those rich in basic amino acids such as arginine, (2) amphipathic peptides having basic and hydrophobic moieties, and (3) hydrophobic sequences. Examples thereof include peptides containing a slight basic sequence, (4) hydrophobic peptides and the like.

Peptide vectors are effective for the introduction of various substances, but may not be suitable for the introduction of nucleic acids such as siRNA. It is difficult to realize a peptide vector that highly satisfies the two contradictory functions of stably retaining and protecting siRNA in the body, while rapidly releasing it once it reaches the target cell.

Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3 study CPPs that enable efficient intracellular introduction, and the importance of cationic functional groups in the membrane permeation of CPPs and oligoarginine. It is described that the peptide helical structure of the above contributes to the improvement of cell membrane permeability.

Non-Patent Document 4 describes the cell membrane penetrating peptide F-βAla- (Arg) _9- (Leu-Leu-Aib) 3 conjugated with nonaarginine (Arg) ₉ using a short-chain peptide (Leu-Leu-Aib) ₃ consisting of leucine and Aib as a helical promoter. Gly) _3- (L-Leu-L-Leu-Aib) ₃ -NH ₂ is described. However, since this cell membrane penetrating peptide is highly cytotoxic, the introduction of siRNA into target cells has not been confirmed.

M. Oba, Y. Demizu, H. Yamashita, M. Kurihara, M. Tanaka, Bioorg. Med. Chem. 2015, 23, 4911-4918. H. Yamashita, Y. Demizu, T. Shoda, Y. Sato, M. Oba, M. Tanaka, M. Kurihara, Bioorg. Med. Chem. 2014, 22, 2403-2408. H. Yamashita, M. Oba, T. Misawa, M. Tanaka, T. Hattori, M. Naito, M. Kurihara, Y. Demizu, ChemBioChem 2016, 17, 137-140. Development of Cell Membrane Highly Permeable Peptide for Intracellular Introduction of Hydrophilic Molecules Takashi Misawa Abstracts of the 136th Annual Meeting of the Pharmaceutical Society of Japan March 2016

An object of the present invention is to provide a cell membrane penetrating peptide that enables efficient intracellular introduction of nucleic acids such as siRNA.

The cell membrane penetrating peptide according to the present invention is a peptide having cell membrane permeability and represented by the following formula (I).
F- (L-Leu-L-Leu-Aib) _l- (Gly) _m- (Arg) _n -NH ₂・ ・ ・ (I)
In the equation, l is an integer of 3-5, m is an integer of 0-3, n is an integer of 8-10, and F is via a linker or It is a fluorosane compound as a fluorescent label attached to the N-terminal of the peptide without intervention.

According to the present invention, a cell membrane penetrating peptide that enables efficient intracellular introduction can be obtained.

It is the result of the CD spectrum measurement which shows the secondary structure under the physiological condition of the cell membrane penetrating peptide which concerns on this invention. It is an electrophoretic photograph showing the expression level of each protein when the modified siRNA or the standard type siRNA is introduced into the cell using the cell membrane penetrating peptide which concerns on this invention, and the bar graph which quantified it. It is an electrophoretic photograph showing the expression level of each protein to examine the site important for the siRNA introduction activity of a cell membrane penetrating peptide.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating the understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

The present inventors have a hydrophobic helical template sequence H- (L-Leu-L-Leu-Aib) _l -NH ₂ that induces the formation of a helical structure for oligoarginine (Arg) _n having cell membrane permeability. We designed a Block peptide in which By linking the hydrophobic helical template sequence to oligoarginine (Arg) _n , it is considered that the arginine sequence forms a helical structure and acquires amphipathicity.

The cell membrane penetrating peptide according to the present invention is represented by the following formula (I).

F- (L-Leu-L-Leu-Aib) _l- (Gly) _m- (Arg) _n -NH ₂・ ・ ・ (I)
In the formula, l is an integer of 3 to 5, m is an integer of 0 to 3, and n is an integer of 8 to 10. F is a fluorosane compound as a fluorescent label attached to the N-terminus of the peptide with or without a linker. The cell-penetrating peptide represented by the formula (I) shows almost no cytotoxicity, and has the property of stably retaining siRNA in the cell culture medium and rapidly releasing it when it reaches the target cell. Efficient intracellular introduction of siRNA and suppression of gene expression are possible.

F is preferably a fluorescent label mediated by a linker, and examples of the linker include β-Ala and the like. The fluorescent label is not particularly limited as long as it is a fluorescein compound, and examples thereof include the compounds shown below. It is preferably 5-FAM or 6-FAM or a mixture thereof.

The structural formula of the cell membrane penetrating peptide according to this embodiment is shown below. In the following equation, R is, for example, OH.

In the formula (I), l is preferably 3, m is preferably 3, and n is preferably 9. The structural formula of the cell membrane penetrating peptide according to this most preferable embodiment is shown.

The construct according to the present embodiment includes the cell membrane penetrating peptide according to the present embodiment and the cargo molecule to be transported into the cell. The cell membrane penetrating peptide and the cargo molecule bind covalently or non-covalently.

The cargo molecule is not particularly limited, but is, for example, a nucleic acid, a protein, a drug, or nanoparticles, and is preferably a nucleic acid.

The nucleic acid, which is a cargo molecule, may be a polynucleotide or an oligonucleotide, or may be a DNA or an RNA molecule. In the case of DNA, it may be plasmid DNA, cDNA, genomic DNA or synthetic DNA. DNA and RNA may be double-stranded or single-stranded. The cargo molecule is particularly preferably siRNA, which is a double-stranded RNA.

The method for transporting the cargo molecule according to the present embodiment into the cell is a step of binding the cargo molecule to be transported into the cell and the peptide according to the present embodiment to obtain a structure, and introducing the structure into the cell. It has a process.

The method for administering the construct according to the present embodiment to a living body (animals including humans, particularly mammals including humans) may be oral, injection, instillation, nasal instillation, transpulmonary, or absorption through the skin. Injection is preferred. For example, the construct according to this embodiment can be systemically administered by intravenous injection or locally administered by injecting into the affected area.

According to the construct according to this embodiment, it is possible to investigate the intracellular function into which a gene or protein is introduced. For example, it is possible to investigate the function of a gene by introducing a siRNA nucleic acid that suppresses the expression of the gene whose function is desired into cells and suppressing the expression of the gene.

Further, according to the construct according to this embodiment, the properties of cells can be changed. For example, if siRNA capable of degrading a specific mRNA is administered to a cell as a cargo molecule, a cell having a reduced expression level of a functional protein encoded by the mRNA can be obtained.

(1) Peptide synthesis Peptides of (Arg) ₉ , A6K, and Block 3 to Block 9 were synthesized by the Fmoc solid phase method using microwaves. The peptides of Block 3 to Block 9 are represented by the following formula (I'), and F, l, m, n are represented by Table 1 below. 5,6-FAM is an isomer mixture of 5-FAM and 6-FAM. 5,6-TAMRA is an isomer mixture of 5-TAMRA and 6-TAMRA. Blocks 3, 4, 5, 9 are peptides according to this example. (Arg) ₉ , A6K and Block6,7,8 are peptides for comparative examples.

F-β-Ala-(L-Leu-L-Leu-Aib) _l- (Gly) _m- (Arg) _n -NH ₂・ ・ ・ (I')

(Arg) ₉ is a peptide represented by the following formula (II).

5,6-FAM-βAla- (Arg) ₉ -NH ₂・ ・ ・ (II)
Arg is shown below.

The A6K peptide is represented by the following formula (III). A is alanine and K is lysine.

5,6-FAM-β-Ala-AAAAAAK-NH ₂・ ・ ・ (III)
(2) Secondary structure analysis of peptide The obtained peptide was purified by reverse phase HPLC and identified by MALDI-MS and LC-TOFMS. The secondary structure of the peptide in solution was analyzed by CD spectrum measurement (190-260 nm) using 20 mM PBS buffer solution (pH = 7.4): methanol = 1: 1 solvent. As a result, it was shown that the block peptide according to the present invention forms a helical structure (Fig. 1).

(3) Introduction of siRNA
Regarding the siRNA introduction effect, 3 types of siRNA (Stealth RNAiTM siRNA (Thermo Fisher Scientific); 25-mer) chemically modified for estrogen receptor ERα and XIAP, which inhibits apoptosis, are used in Opti-MEM (registered trademark) (Thermo Fisher). Scientific) was suspended with various peptides and added to cells 10 minutes later. Modified siRNA (25-mer) with high target specificity and serum stability was used as siRNA for XIAP and ERα. In addition, three or two unmodified standard siRNAs (21-mer) for the aromatic hydrocarbon receptor (AhR) and the transcription factor Notch1 are available on Opti-MEM® (Thermo Fisher Scientific). It was suspended with the peptide and added to the cells 10 minutes later.

The base sequence of the above-mentioned siRNA is shown below. The terminal tt is an overhang and is a thymidine 2 base in DNA.
XIAP siRNA # 1: 5'-ACACUGGCACGAGCAGGGUUUCUUU-3' (SEQ ID NO: 1)
XIAP siRNA # 2: 5'-GAAGGAGAUACCGUGCGGUGCUUUA-3' (SEQ ID NO: 2)
XIAP siRNA # 3: 5'-CCAGAAUGGUCAGUACAAAGUUGAA-3' (SEQ ID NO: 3)
ERalpha siRNA # 1: 5'-CGACAUGCUGCUGGCUACAUCAUCU-3' (SEQ ID NO: 4)
ERalpha siRNA # 2: 5'-UCACAGACACUUUGAUCCACCUGAU-3' (SEQ ID NO: 5)
ERalpha siRNA # 3: 5'-GACCGAAGGAGGGAGAAUGUUGA-3' (SEQ ID NO: 6)
AhR siRNA # 1: 5'-GCUCUGAAUGGCUUUGUAUtt-3' (SEQ ID NO: 7)
AhR siRNA # 2: 5'-GCUACCACAUCCACUCUAAtt-3' (SEQ ID NO: 8)
AhR siRNA # 3: 5'-CCUGUAAUCAGCCUGUAUUtt-3' (SEQ ID NO: 9)
NOTCH1 siRNA # 1: 5'-GCAACAGCUCCUUCCACUUtt-3' (SEQ ID NO: 10)
NOTCH1 siRNA # 2: 5'-GCAUGGUGCCGAACCAAUAtt-3' (SEQ ID NO: 11)
The intracellular expression level of each target protein was detected by Western blotting and quantified to evaluate the transport efficiency.

(4) Block 3 experimental results
Block3 was found to reduce the expression level of target proteins (XIAP, ERα) in a siRNA sequence-dependent manner when treated with modified siRNA in MCF-7 cells (Fig. 2 (a)).

Block 3 can also be treated with MCF-7 cells together with standard siRNA (chemically modified modified siRNA may be referred to as standard siRNA, but unmodified siRNA may be referred to as standard siRNA). It was revealed that the expression level of the target protein (AhR, NOTCH1)) was reduced in an siRNA sequence-dependent manner (Fig. 2 (b)).

It was also revealed that Block 3 reduces the expression level of the target protein (XIAP) in a siRNA sequence-dependent manner even when treated with modified siRNA into HeLa cells or HepG2 cells (Fig. 2 (c)).

(5) Experimental results of other peptides
The activity was compared with other peptides to determine which structure of Block 3 is important for siRNA transduction activity (Fig. 3).

With reference to Lane 8,10,12,14 in Fig. 3 (a), it was clarified that Block4 without a glycine linker has a slightly diminished siRNA introduction effect as compared with Block3.

With reference to Lanes 2, 4, and 6 in Fig. 3 (a), it was clarified that Block 5 in which the fluorescent label was changed to FITC had a slightly diminished siRNA introduction effect as compared with Block 3. With reference to Lane 8 in Fig. 3 (c), it was clarified that Block 9 in which the fluorescent label was changed to TAMRA had a slightly diminished siRNA introduction effect as compared with Block 3. From these results, it was found that the fluorosane compound is preferably 5-FAM or 6-FAM for the siRNA introduction efficiency.

Referring to Lane 2 in FIG. 3 (c), (Arg) ₉ could not suppress the expression level of XIAP. With reference to Lane 4 in FIG. 3 (c), A6K was unable to suppress the expression level of XIAP. With reference to FIG. 3 (b), Blocks 6, 7 and 8 could not suppress the expression level of XIAP. From these results, (Arg) ₉ and the helical promoter sequence alone did not show the siRNA introduction effect, and the ligation of the helical promoter sequence of appropriate length (l = 3-5) for (Arg) ₉ was the siRNA introduction. It turned out to be essential for activity.

It can be used for investigating gene functions in target cells, modifying target cells, and the like.

SEQ ID NOs: 1-4: Primers

Claims (5)

The following formula (I) with cell membrane permeability
F- (L-Leu-L-Leu-Aib) _l- (Gly) _m- (Arg) _n -NH ₂・ ・ ・ (I)
[In the formula,
l is an integer of 3 to 5,
m is an integer of 0 to 3 and is
n is an integer of 8 to 10.
F is a fluorosane compound as a fluorescent label attached to the N-terminus of the peptide with or without a linker. ] Peptide represented by. The peptide according to claim 1, wherein the fluorescein compound is 5-FAM or 6-FAM. A construct comprising the peptide according to claim 1 or 2 and a cargo molecule to be transported into a cell. The construct according to claim 3, wherein the cargo molecule is a nucleic acid. The construct according to claim 4, wherein the nucleic acid is siRNA.

Images