WO2007102631A1

WO2007102631A1 - Novel intracellular transduction peptides

Info

Publication number: WO2007102631A1
Application number: PCT/KR2006/000790
Authority: WO
Inventors: Young Chul Sung; Je-In Youn; Sang Hoon Park
Original assignee: Postech Foundation; Lee, Ok Hee
Priority date: 2006-03-07
Filing date: 2006-03-07
Publication date: 2007-09-13

Abstract

The present invention relates to a novel peptide having a protein transduction domain (PTD), and to a method for delivering a target material into a cell using the same. The peptide can efficiently deliver a viral vector, a DNA, an RNA, a protein, a lipid, a carbohydrate, or a chemical compound into a cell. The peptide can be also used for a virus vaccine, a DNA vaccine, production of a pharmaceutically or medically effective protein, or gene therapy using the same.

Description

NOVEL INTRACELLULAR TRANSDUCTION PEPTIDES

Technical Field

The present invention relates to a peptide having a protein transduction domain (PTD), and to a method for delivering a target material into a cell using the same. [Background Art]

A substance having useful information such as DNA, peptide, protein cannot pass through lipid layers of a cell membrane. Therefore, it has been known that such substances alone are rarely transferred into a cell. To overcome this difficulty, methods, such as microinjection, electroporation using an electric shock, and cationic liposome-mediated delivery, have been developed. However, the methods have various problems including low transduction efficiency, cytotoxicity, and induction of immunity, thereby limited in application. Recently, a peptide capable of passing through a cell membrane has been studied as a new method for delivering a DNA or a protein. It was discovered in 1980s that a HIV TAT protein has ability to pass through a cell membrane, and in 1999 that a specific region of the HIV TAT protein has ability to transfer a protein into a cell directly. Subsequently, many studies have beenmade on a PTD, a protein transduction domain inducing intracellular transduction (Schwarze. S. R., Science, 285(3) : 1569-1572, 1999) .^' The internalization of PTD is a new type of translocation, which is different from the general mechanism for receptor mediated endocytosis or phagocytosis, but its mechanism is not clearly identified yet. The known PTDs include HIV Tat, drosophila Antp, a mouse transcription factor, Mph-1 (Korean patent publication No. 2004-0083429), and HSV-I VP22.

Accordingly, the present inventors found that a part of the peptide sequence of the herring protamine can translocate a cell membrane, and that a target protein, viral vector, nucleic acid, chemical compound, or the like can be effectively delivered using the peptide as an intracellular transduction peptide, thereby completing the present invention.

Technical Solution

It is an object of the present invention to provide a peptide, which is a novel protein transduction domain (PTD) having an amino acid sequence of Rn-X-R-R-R-Y-Y-Rn (wherein n is an integer of 3 to 5, R is an arginine, and X and Y are each an amino acid other than aspartic acid and glutamic acid) .

It is another object of the present invention to provide a method for delivering a bioactive material into the cytoplasm or nucleus of eukaryotic or prokaryotic cells in vivo and in vitro using the peptide. It is still another object of the present invention to provide a therapy using a viral vector vaccine, a DNA or RNA vaccine, a protein, and a small molecule including the peptide . [Description of Drawings] Fig.1 is a graph showing their transduction efficiencies of adenoviral vector, when each of Mph-1, Tat4₉_₅7, Tat₄8-60_f K9, R9, protamine sulfate from herring, HPl, HP2, HP3, HP4, HP5, HPβ, HP4-1 and HP4-2 is co-treated into rat glioma CβBul cell line and monkey kidney Cos7 cell line. Fig. 2 is a graph showing the internalization activity of HP4 in rat glioma CβBul cell line and monkey kidney Cos7 cell line, compared with those of Mph-1, Tat₄₉-₅₇, Antp and HP4-D, which are the known PTD.

Fig.3 is a graph showing improvement in a DNA transduction efficiency by 4HP4 in monkey kidney Cos7 cell line. [Best Mode]

According to one aspect, the present invention relates to a peptide having an amino acid sequence of Formula 1. [Form-¹- ¹¹

Rn-X-R-R-R-Y-Y-Rn

wherein n is an integer of 3 to 5, R is an arginine, and

X and Y are each an amino acid other than aspartic acid and glutamic acid. The term "peptide" as used herein means a polymer consisting of amino acids connected by amide bonds (or peptide bonds) , and specifically a peptide delivering a target material into a cell . The intracellular transduction peptide of the invention is derived from a peptide consisting of amino acid sequence of SEQ ID NO: 1 of herring {Clupea pallasi) protamine, and functions to transfer a viral vector, a DNA, an RNA, a protein, a lipid, a carbohydrate, or a chemical compound into a cell. The above intracellular transduction peptide has a sequence of Rn-X-R-R-R-Y-Y-Rn. n is an integer of 3 to 5, preferably an integer of 3 or 4. R is an arginine. X is an amino acid other than aspartic acid and glutamic acid, preferably an amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptopahn and valine, and more preferably proline. Y is an amino acid other than aspartic acid and glutamic acid, preferably an amino acid selected from the group consisting of asparagine, cysteine, glutamine, glycine, serine, threonine and tyrosine, and more preferably threonine.

In a specific embodiment of the invention, the transduction efficiency of intracellular transduction peptide consisting of an amino acid sequence of SEQ ID NO: 5 was better than that of the conventional PTD (protein transduction domain) . Further, in the case of intracellular transduction peptide consisting of the amino acid sequence of SEQ ID NO: 5, in which first and second amino acid are removed, or in which two threonines at position 9 and 10 are substituted with aspartic acid, its transduction efficiency was better than that of the conventional PTD (protein transduction domain) , but lower than that of the intracellular transduction peptide consisting of amino acid sequence of Formula 1 of the invention. The peptide of the invention can be prepared by- conventional methods well known in the art, including in vitro synthetic methods using genetic recombination, and a protein expression system or peptide synthesizer. In a specific embodiment, the peptide of the invention was prepared by an organic synthetic method. It was found that the transduction efficiency of the intracellular transduction peptide consisting the amino acid sequence of SEQ ID NO: 5 was most excellent, as compared with each other including the amino acid sequences of SEQ ID NOs: 2 to 7, which were prepared by random cutting of the amino acid sequence of SEQ ID NO: 1 with 14-mer.

In another aspect, the present invention relates to a peptide consisting of amino acid sequence of Formula 1, which is connected with a target material to be transferred into the cytoplasm or nucleus of eukaryotic or prokaryotic cells. The term "target material" as used herein means a functional material having a bioactivity to regulate all physiological phenomena in vivo. Examples of the target material include a viral vector, a DNA, an RNA, a protein, a lipid, a carbohydrate, and a chemical compound. The viral vector is used for gene therapy. Examples of the viral vector include, but not limited thereto, an adenovirus, and a retrovirus. The chemical compound is a chemical compound capable of regulating cellular functions. Examples of the chemical compound include, but not limited thereto, an anti-cancer drug, a therapeutic agent for immune diseases, an antiviral therapeutic agent, and a mammalian growth, development or differentiation factor.

The target material is fused or connected with the peptide of the invention by a chemical or physical method, so as to be transferred into the cytoplasm or nucleus of eukaryotic or prokaryotic cells with high efficiency. In a specific embodiment, when an adenoviral vector was transferred into a cell line, CβBul or Cos7 using the peptide of the invention, its transduction efficiency was better than those of Tat₄₉_₅₇

(SEQ ID NO: 10), Tat₄₈-60 (SEQ ID NO: 11), Mph-1 (SEQ ID NO:

13), K₉ (SEQ ID NO: 14), R₉ (SEQ ID NO: 15) (Fig. 1), which are other conventional PTDs . When the cell lines were treated with HP4 labeled with FITC (Fluorescein isothiocyanate) , which is a fluorescent material having a lowmolecular weight, FITC was more effectively transferred into the cell lines, compared to the cell lines treated with Tat₄₉-₅₇ (SEQ ID NO: 10) of HIV (human immunodeficiency virus) , Antp (SEQ ID NO: 12) of drosophila, Mph-1 (SEQ IDNO: 13) , amouse transcription factor, labeled with FITC (Fig. 2) . In the case of treating with 4HP4, a tetramer of HP4, and a DNA expressing a GFP (Green Fluorescent Protein) , its DNA transduction efficiency was improved, while the GFP was not expressed at all when treated with the DNA alone (Fig. 3) . In another aspect, the present invention relates to a method for delivering a target material into a cell, comprising the steps of co-culturing and contacting the peptide of Formula 1, which is fused with a target material to be transferred into the cytoplasm or nucleus of eukaryotic or prokaryotic cells, or connected with the target material by a chemical or physical method, with the cell to be transduced.

The term "contacting" as used herein means that the peptide fused or connected with a target material is contacted with eukaryotic or prokaryotic cells via numerous routes including intramuscular, intraperitoneal, intravein, oral, nasal, subcutaneous, intradermal, mucosal and inhale. Accordingly, the peptide fused or connected with a target material can be delivered into the cytoplasm or nucleus of eukaryotic or prokaryotic cells. Hereinafter, the present invention will be described

— 1 — in more detail with reference to Examples. However, it will be apparent to those skilled in the art that these Examples are for the illustrative purpose only and the invention is not intended to be limited by these Examples. [Mode for Invention]

Example 1 : Preparation of PTD peptide In order to prepare the peptides used in the present invention, the peptides were ordered and prepared using an organic synthetic method by Peptron, Inc. (385-19 Doryong-dong, Yusung-gu, Daejeon, Korea). It was confirmed by RP-HPLC that the prepared peptides had purity of 85% or more. The synthesized peptides are as following.

HP: ARRRRSSSRPIRRRRPRRRTTRRRRAGRRRR (SEQ ID NO: 1) HPl: ARRRRSSSRPIRRR (SEQ ID NO: 2)

HP2: RSSSRPIRRRRPRR (SEQ ID NO: 3)

HP3: RPIRRRRPRRRTTR (SEQ ID NO: 4)

HP4: RRRRPRRRTTRRRR (SEQ ID NO: 5)

HP5: RPRRRTTRRRRAGR (SEQ ID NO: 6) HP6: RRTTRRRRAGRRRR (SEQ ID NO: 7)

HP4-1: RRPRRRTTRRRR (SEQ ID NO: 8)

HP4-2: RRRTTRRRR (SEQ ID NO: 9)

Tat₄₉-₅₇: RKKRRQRRR (SEQ ID NO: 10)

Tat₄₈-6o: GRKKRRQRRRPPQ (SEQ ID NO: 11) Antp: RQIKIWFQNRRMKWKK (SEQ ID NO: 12) Mph-1: YARVRRRGPRR (SEQ ID NO: 13)

K₉: KKKKKKKKK (SEQ ID NO: 14)

R₉: RRRRRRRRR (SEQ ID NO: 15)

HP4-D: RRRRPRRRDDRRRR (SEQ ID NO: 16)

Further, a HP4-FITC, a Tat-FITC, a Mph-1-FITC, and an

Antp-FITC, in which FITC, a fluorescent compound, is labeled at their Carboxyl-terminals, were ordered and prepared by

Peptron, Inc. It was confirmed that the prepared peptides had purity of 85% or more.

Example 2 : Effect of HP4 for improving transduction efficiency of adenoviral vector

To test which peptide is effective in improving the transduction efficiency of an adenoviral vector among HPl to 6 (SEQ ID NOs : 2 to 7 ) , which are partial peptides of herring, the conventional PTDs (SEQ ID NOs : 10 to 15) , and the peptides

(SEQ ID NOs: 8 and 9) , in which partial sequences of HP4 were substituted, an rAd/EGFP, an adenoviral vector expressing a GFP (Green Fluorescent Protein) , was used. First, the rAd/EGFP was mixed with 25 uM of HPl to 6, Tat₄₉-57, Tat₄₈-60,

Mph-1, K₉, R₉, HP4-1 and HP4-2, respectively. After the mixtures were incubated at room temperature for 30 minutes, rat glioma CβBul cell lines and monkey kidney Cos7 cell lines were infected with the mixture for 2 hours. The amount of adenoviral vector, used upon infection, is 100 MOI (multiplicity of infection) in C6Bul cell lines, and 50 MOI in Cos7 cell lines. After 2 hours, the virus mixtures were removed, and the cells were cultured in DMEM culture media in a CO₂ incubator (CO₂ water Jacketed Incubator, Forma Scientific™) for 2 days. After the infected cells were treated with trypsin and collected, GFP expression levels of the cells weremeasuredusing a FACS (Fluorescence Activated Cell Sorter, BD) . The results are shown in Fig.1. The GFP genes were barely transferred, in the case where CβBul cells were treated with the adenoviral vector alone, or in the case where the cells were even treated with each mixture of adenoviral vector and Tat49-5₇, Tat48-60_f Mph-1, or K₉. It was found that while about 10% of the cells expressed GFP when treated with the mixture of adenoviral vector and protamine sulfate of herring, which is Rg or cationic, about 50% of the cells- expressed GFP when treated with the mixture of adenoviral vector and HP4. In the case where Cos7 cells were treated with each mixture of adenoviral vector and Tat49-5₇, Tat48-6o^ Mph-1, K₉, or protamine sulfate of herring, which is R₉ or cationic, their transduction efficiencies were also improved, as compared with the cells treated with the adenoviral vector alone. However, in the case of treating with the mixture of adenoviral vector and HP4, the highest transduction efficiency was observed. It was found that four arginines (R) at the start of

HP4 sequence play a crucial role in PTD effect of HP4, as compared with HP4-1 (SEQ ID NO: 8) and HP4-2 (SEQ ID NO: 9)

(Fig. 1) , in which several arginines (R) at the start of HP4 sequence (SEQ ID NO: 5) were removed.

Example 3 : Effect of HP4 for internalization efficiency of low molecular weight compound

To compare the internalization efficiency of a low molecular weight compound between HP4 and Tat and Antp, which are the known PTD, a Mph-1-FITC, a Tat₄₉-₅₇-FITC, anAntp-FITC, a HP4-FITC, and a HP4-D-FITC, in which FITC was labeled at their Carboxyl-terminals, were ordered and prepared by Peptron, Inc. Rat glioma CβBul cell lines and monkey kidney Cos7 cell lines were treated with each prepared peptide in the concentration of 10 uM and 100 uM at 37⁰C for 30 minutes. The intracellular presence of FITC was measured using a FACS. Thus, the internalization efficiency of each peptide was confirmed. The results are shown in Fig. 2. It was found that the effect of HP4 for internalization efficiency of a low molecular weight compound was much higher in both cell lines of CβBul and Cos7, as compared to Tat₄₉_₅₇, Mph-1, or Antp, which are the known PTD. In the case where the cells were treated with HP4-D (SEQ ID NO: 16), in which the neural amino acid, TT (threonine) were substituted with the negative amino acid, DD (aspartic acid) in the sequence of HP4, the internalization efficiency decreased dramatically, as compared to HP4(Fig. 2) . Accordingly, it can be assumed that TT (threonine) of HP4 plays an important role in intracellular transduction.

Example 4 : Effect of HP4 for improving DNA transduction efficiency

To confirm whether HP4 of the invention improves DNA transduction efficiency or not, a pCIN/EGFP, a DNA vector expressing a GFP (Green Fluorescent Protein) , was used. First, the pCIN/EGFP was mixed with 5 uM, 10 uM, 20 uM of 4HP4, a tetramer of HP4, respectively. After the mixtures were incubated at room temperature for 30 minutes, and Cos7 cell lines were infected with the mixture. After 4 hours, the

DNA mixtures were removed, and the cells were cultured in culture media in a CO₂ incubator for 2 days . After the infected cells were treated with trypsin and collected, GFP expression levels of the cells were measured using a FACS. From the results, it was found that the GFP expressions were increased in the cells treated with 4HP4 in proportion to its concentration (Fig. 3) .

Claims

CLAIMS [Claim 1]

A peptide having an amino acid sequence of Formula 1, [Formula 1] Rn-X-R-R-R-Y-Y-Rn wherein n is an integer of 3 to 5,

R is an arginine, and

X and Y are each an amino acid other than aspartic acid and glutamic acid. [Claim 2]

The peptide according to claim 1, which consists of the amino acid sequence of SEQ ID NO: 5. [Claim 3] The peptide according to claim 1, which is connected with a target material to be transferred into the cytoplasm or nucleus of eukaryotic or prokaryotic cells. [Claim 4]

The peptide according to claim 3, wherein the target material is at least one of a viral vector, a DNA, an RNA, a protein, a lipid, a carbohydrate, and a chemical compound. [Claim 5]

A method for delivering a target material into a cell, comprising the steps of co-culturing and contacting the peptide of claim 1 with the cell to be transduced with the target molecule. [Claim 6]

The method according to claim 5, wherein the target material is at least one of a viral vector, a DNA, an RNA, a protein, a lipid, a carbohydrate, and a chemical compound.