KR20080076622A - Oligomerizaed protein transduction domains and method for the intracellular transduction of viral vectors - Google Patents

Abstract

Oligomerized protein transduction domains are provided to increase transport and expression of a target material in a cell, and reduce the required amount of oligomers, so that they are useful for production of viral vaccine and proteins useful pharmaceutically and medically, and in gene therapy. The oligomerized protein transduction domains represented by the formula(1): (PTD)n-branch, wherein n is an integer of two or more, and the branch is a linker selected from multiple antigenic peptide(MAP), cysteine and carrier protein linked by a peptide linker, transduce a target material into a cell and are prepared by linking two or more of PTD(protein transduction domain) monomers, wherein the PTD monomer is HIV(human immunodeficiency virus) Tat(transactivator of transcription), Antp(Antennapedia) of drosophila, Mph-1(macrophage antigen 1) of mouse, HSV-1(herpes simplex virus type 1) VP22 and HP4(human protamine P4). A target material is intracellularly transduced by co-culturing and contacting the oligomerized protein transduction domains with the cell.

Description

Oligomerized Protein Transduction Domains and Methods for the Intracellular Transduction of Viral Vectors

1 is a cellular mass transfer capacity is the peptide (PTD) with the _{Tat 48 -60, Mph-1,} HP4 amino acid sequence, and they are the dimers, tetramers, illustration showing a structure in the form of arms oligomerization dimer schematically to be.

Figure 2 shows the maximum value (%) of adenovirus delivery efficiency of Tat _{48 -60} , Mph-1, HP4 and their oligomerized peptides in rat brain tumor cells C6Bu1, concentration of required peptide (uM), cells of 50% Table shows the concentration (uM) of peptides showing toxicity and the maximum concentration (uM) showing no cytotoxicity.

Figure 3 is a graph comparing the anticancer effect of the interleukin-12 adenovirus increased by the monomer PTD Tat _{48 -60} and tetramer PTD 4Tat _48-60 in mouse colon cancer animal experimental model.

The present invention relates to a new type of protein transporter oligomer oligomerized with protein transduction domain (PTD) and a method for delivering a viral vector into cells using the same.

In general, living cells are impermeable to large molecules such as proteins and nucleic acids. While only a few small substances can enter the cytoplasm or nucleus inside the cell at very low rates through the cell membrane, the fact that the macromolecule cannot enter the cell means that the macromolecule containing protein or nucleic acid It is a limiting factor for the treatment, prevention and diagnosis used. On the other hand, most of the materials produced for the purpose of treatment, prevention, and diagnosis have to be delivered intracellularly in a diagnostic, prophylactic and therapeutically effective amount thereof. Methods have been developed. As such, methods for delivering large molecules into cells in vitro include electroporation, membrane fusion using liposomes, high concentration projection using microprojectiles coated with DNA on the surface, and calcium-phosphorus. Cultures using DNA precipitates, transfection using DEAE-dextran, infection with modified viral nucleic acids, direct microinjection into single cells, and the like. However, these methods typically deliver only a fraction of the total cell number to be injected with the large molecules, and tend to have an undesirable effect on a large number of other cells. In addition, as a method of experimentally moving large molecules into cells in vivo, there are calcium-phosphorus precipitation and liposomes, but these techniques are extremely limited in their use in delivering substances into cells in vivo. There is a problem.

Proposed as a result of research to solve this problem is Protein Transduction Domain (PTD). These protein transduction domains (PTDs) are known to be short-charged peptides that can cross cell membranes, and PTDs can efficiently transfer not only proteins but also DNA, RNA, fat, carbohydrates, compounds, or viruses into cells. It is known that it can. The mechanism by which PTD penetrates the cell membrane is not yet known, but it is considered to be receptor-independent and independent of endocytosis or phagocytosis. The most well-known PTDs include HIV Tat, Drosophila Antp, Mph-1 (Korean Patent Publication No. 2004-0083429) of a mouse transcription factor, HSV-1 VP22, and recently identified HP4 (PCT Patent Application No. PCT / KOR2006 / 000759, PCT / KOR2006 / 000790). Among them, HIV Tat is the first PTD found (Schwarze. SR, Science, 285 (3): 1569-1572, 1999). Currently, many studies are being conducted on the delivery of DNA and the development of therapeutics through fusion with the target protein. have.

The most important concern of gene therapy is how efficiently the therapeutic gene can be delivered to the target cell. For this reason, in recent years, a lot of attempts have been made to transfer genes using viral vectors having high expression rates. For example, adenoviruses, retroviruses and vaccinia viruses are widely used for gene therapy. However, since these viral vectors are dependent on the type of cell and the degree of expression of the receptor, viral vector delivery to cancer cells or stem cells is limited. To overcome these limitations, strategies such as fusing viruses with receptors or using liposomes, cations and PTDs can be used. Among them, in the case of intracellular delivery of viral vectors using PTD, it has been reported that Antp and Tat can enhance intracellular delivery of adenovirus and retroviral vectors (Gratton. JP., Nature Medicine, 9 (3). ): 357-362, 2003). However, there is a limitation that the virus delivery method using the existing PTD is very impractical in that a large amount of PTD of 0.5mM is required to increase intracellular delivery efficiency by about 2 times.

Accordingly, the present inventors have synthesized a new type of peptide oligomerizing a variety of protein delivery sites (PTD), and found that the use of this oligomer PTD can effectively deliver viral vectors into cells, The present invention has been found to be able to significantly increase the intracellular delivery and expression of the gene of interest by using as a peptide for intracellular viral vector delivery.

One object of the present invention is to provide a protein carrier oligomer in which a monomeric protein transduction domain (PTD) is linked to two or more oligomers in a multimeric form.

Another object of the present invention is to provide the above protein carrier oligomers bound to a target substance to be delivered into the cytoplasm or nucleus of eukaryotic or prokaryotic cells.

Another object of the present invention is to provide a method for efficiently delivering a viral vector into eukaryotic or prokaryotic cells using the protein carrier oligomer.

In one embodiment, the present invention relates to a protein carrier oligomer in which a monomeric protein transduction domain (PTD) is oligomerized into a multimeric form.

As a specific embodiment, the present invention relates to a protein carrier oligomer represented by the following formula (1) oligomerized monomeric protein carrier (PTD) in the multimeric form.

(PTD) n-branch

Where

n is an integer of 2 or more,

A branch is a linker selected from the group consisting of multiple antigenic peptides (MAPs), cysteines and carrier proteins linked by a peptide linker.

In a preferred embodiment, Formula 1 is

n is an integer of 2, 4 or 8,

Branches are Multiple Antigenic Peptides (MAPs) or cysteines.

In a more preferred embodiment, the protein carrier oligomer of Formula 1 is a protein carrier oligomer selected from the group consisting of protein carrier oligomers represented by the following formulas (2) to (8).

In an even more preferred embodiment, the protein carrier oligomer is selected from the group consisting of protein carrier oligomers represented by the following formulas (3), (6) and (8).

(GRKKRRQRRRPPQ) ₂ -K-βA-OH

(GRKKRRQRRRPPQ) ₄ -K ₂ -K-βA-OH

(GRKKRRQRRRPPQ) ₈ -K ₄ -K ₂ -K-βA-OH

(YARVRRRGPRR) ₂ -K-βA-OH

(YARVRRRGPRR) ₄ -K ₂ -K-βA-OH

(YARVRRRGPRR) ₈ -K ₄ -K ₂ -K-βA-OH

(RRRRPRRRTTRRRR) ₄ -K ₂ -K-βA-OH

Protein transduction domains (PTDs) of the present invention are directed to specific proteins, viruses, DNA, RNA, fats, carbohydrates, or chemical compounds that are directly or indirectly linked to them using chemical or physical covalent or non-covalent bonds. Refers to a substance peptide that is deliverable into the cytoplasm or nucleus of eukaryotic or prokaryotic cells. The protein carrier of the present invention is not limited thereto, but for example, Tat protein derived from human immunodeficiency virus type I (HIV-1), Antp (Antennapedia or penetratin), which is an antennapedia Homeodomain of Drosophila Peptides, Mph-1 of the mouse transcription factor, VP22 of HSV-1 and HP4 of herring protamine. In addition, protein transporters of the invention include cell-specific PTDs that have specificity for a particular cell type or condition. One example of such a cell-specific PTD is the Hn1 synthetic peptide described in US Patent Publication 2002-0102265. In addition, the protein transporters of the invention include synthetic peptides. One example of such a synthetic peptide is a peptide in which Ho et al. Modified 47-57 residues of Tat protein such that HIV's Tat protein optimizes protein delivery capacity (Ho et al. (2001) Cancer Res 61 (2): 474-). 7).

In a specific embodiment of the invention, the protein carrier is a protein carrier oligomer of Formula 2 to 4 comprises a Tat _48-60 (SEQ ID NO: 1) protein carrier of HIV, the protein carrier oligomer of Formula 5 to 7 is Mph- 1 (SEQ ID NO: 2) protein carrier, and the protein carrier oligomer of Formula 8 comprises the HP4 (SEQ ID NO: 3) protein transporter.

Protein carriers of the invention can be prepared by methods known in the art for synthesizing peptides. For example, it may be prepared by a method of synthesis in vitro through genetic recombination and protein expression systems or peptide synthesizers.

In the present invention, the protein carrier is used as a protein carrier oligomer in a multimeric form by linking two or more protein carriers in monomeric form by various known methods for linking peptides. In this application, the substance connecting the peptide is expressed as a branch.

The branch of the present invention includes, but is not limited to, Multiple Antigenic Peptide (MAP), Cysteine, Carrier protein.

The "Multiple Antigenic Peptide (MAP)" refers to a peptide that connects two or more protein carriers by using multiple lysine as a block, and the "cysteine" is a sulfur-containing neutral amino acid between cysteines. An amino acid that connects two or more protein carriers by disulfied bonds. The "carrier protein" is a membrane protein that binds two or more protein carriers and carries the protein carrier across the cell membrane. In order to connect the carrier protein and the protein carrier, MBS (m-maleimidobenzoyl-N-hydroxy) is used. Peptide linkers such as succinimide ester and glutaraldehyde are required. The carrier protein includes, but is not limited to, Keyhole limpet hemocyanin (KLH), Bovine Serum Albumin (BSA), and the like.

The "multimer" refers to a peptide formed by the aggregation of two or more units (or monomers).

In the present invention, "oligomerization" refers to connecting two or more peptides (or monomers) into a multimeric form. Oligomerization of the protein carrier (PTD) of the present invention is carried out by using two or more protein carriers using carrier proteins linked with multiple antigenic peptides (MAP), cysteine and peptide linker. (PTD) is linked to oligomerized to multimeric form. Specifically, by introducing two or more protein carriers in one molecule using the multi-antigen peptide, it is possible to oligomerize a protein carrier in monomeric form, and connect two or more protein carriers by using a carrier protein linked with a cysteine or peptide linker. As a result, the monomeric protein carrier can be oligomerized. More specifically, the carboxyl terminal of each peptide is linked to lysine blocks such as 2 to 4 to 8 of the multiple antigen complex to oligomerize in the form of dimers, tetramers, and tetramers, and cysteine. Protein carriers can be oligomerized into multimeric forms by linking to protein carrier ends to induce the formation of disulfied bonds between the two cysteines. In addition, by using a carrier protein and a peptide linker (peptide linker) it can be oligomerized in the form of a multimer by connecting multiple protein carriers.

The “protein carrier oligomer” of the present invention is a protein carrier (PTD) formed of a multimeric form by oligomerizing a protein carrier (PTD) in monomeric form by the above method. Refers to a protein carrier that delivers into a cell. The "target material" is a therapeutic or prophylactic material for delivery into a cell for the treatment or prevention of a disease of an individual including a human, for example, a virus, DNA, RNA, protein, fat, carbohydrate or compound.

In specific embodiments of the present invention, a lesser amount of a multimeric form of the protein carrier oligomer than the monomeric form of the protein carrier has been delivered more effectively into the cells of the individual, eg, a viral vector. From these results, the protein carrier oligomer of the present invention can effectively deliver a target substance, such as a virus, DNA, RNA, protein, fat, carbohydrate or compound, etc. into the cell.

In another embodiment, the present invention provides a method of mixing and contacting a protein carrier oligomer of the present invention fused or bound with a target substance to be delivered into a cell by chemical or physical methods, with the eukaryotic or prokaryotic cell to which the target substance is to be delivered. It relates to a method of delivering a target substance into a cell, comprising the step.

In the present invention, the target substance to be delivered into the cell is a virus, DNA, RNA, protein, fat, carbohydrate or compound, preferably a viral vector. The viral vector is a virus that delivers a gene for synthesizing a functional modulator having a biological activity that regulates all physiological phenomena in a living body, but is not limited thereto, and examples thereof include adenovirus, retrovirus, and vaccinia virus. .

As a method for preparing a complex in a form in which the target substance and the protein carrier oligomer are fused or bound, various known methods may be used, for example, the method disclosed in US Pat. No. 7,166,692. Specifically, crosslinking of glutathione and glutathione-S-transferase or the like using a chemical crosslinker such as a homobifunctional cross-linker or a heterobifunctional cross-linker Pairs of bridging molecules can be used to prepare the complex.

As used herein, the term "contact" means that the protein carrier oligomers fused or bound with a target substance are intramuscular, intraperitoneal, intravenous, oral, nasal, It means contact with eukaryotic or prokaryotic cells by subcutaneous, intradermal, mucosal or inhale routes, whereby protein carrier oligos fused or bound with the target substance are eukaryotic or Deliver the desired substance into the cytoplasm or nucleus of prokaryotic cells.

In a specific embodiment of the present invention, when the adenovirus vector is transferred into the C6Bul cell line using the protein carrier oligomer represented by Formulas 2 to 8 of the present invention, the monomeric protein carrier (PTD) Tat _{48 -60} (SEQ ID NO: 1), Mph-1 (SEQ ID NO: 2), HP4 When (SEQ ID NO: 3) was treated, it was delivered into cells at a better rate, and as the number of protein carriers (PTDs) included in the protein carrier oligomer increased, intracellular delivery efficiency of the adenovirus vector was increased. In addition, in terms of quantity, as the number of protein carriers (PTDs) included in the protein carrier oligomers increased, the amount of protein carriers required to exhibit the maximum intracellular delivery efficiency decreased. In addition to these results, animal experiments using the tetrameric form of the protein carrier oligomer 4Tat _48-60 (Formula 3) also induced a better anticancer effect than the monomeric protein carrier (PTD) (Fig. 3). From the above results, the protein carrier oligomer of the present invention can solve the problem that a large amount is required when using the protein carrier in monomer form, which has been a problem in the past.

In another aspect, the invention provides a method for treating a disease comprising a protein carrier oligomer of the invention, which is fused or bound by chemical or physical methods with a target substance to be delivered into a cell, formulated together with a pharmaceutically acceptable carrier. Or to a pharmaceutical composition for prevention.

As used herein, the term “pharmaceutically acceptable carrier” includes any or all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and physiologically compatible analogs. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, epithelial administration (eg, injection or infusion). Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of acids or other natural environments that may inactivate the substance.

The target substance to be delivered into the cell is a virus, DNA, RNA, protein, fat, carbohydrate or compound, preferably a viral vector. The viral vector is a virus that delivers a gene for synthesizing a functional modulator having a biological activity that regulates all physiological phenomena in a living body, but is not limited thereto, and examples thereof include adenovirus, retrovirus, and vaccinia virus. .

By delivering the target substance into cells, the disease can be treated or prevented by the action of the target substance. The disease is determined in relation to the target substance for delivery into the cell. For example, when the target substance is an attenuated virus for preventing or treating hepatitis, it can be delivered intracellularly in combination with the protein carrier oligomer of the present invention to treat or prevent hepatitis.

The pharmaceutical composition may further comprise a pharmaceutically acceptable salt. “Pharmaceutically acceptable salts” maintain the desired biological activity of the original substance and note any undesirable toxic effects (see Berge, SM, et al. (1977) J. Pharm . Sci . 66: 1-19). Refers to salts that do not.

The pharmaceutical composition may be in various forms. These include, for example, liquid, semisolid and solid formulations such as liquid solutions (eg, injectable or injectable solutions), dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. The formulation depends on the method of administration desired and the therapeutic application.

Conventional compositions are in the form of injectable or injectable solutions, such as compositions similar to those used to administer antibodies to the human body. Typical methods of administration include parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular) administration. Preferred methods of administration are administration by intramuscular or subcutaneous injection. Exemplary routes of administration include oral administration, epidermal tissue (eg skin) or mucosal (eg eye) administration, or inhalation. Other routes of administration may also be used.

As used herein, the phrase “parenteral administration” or “administered parenterally” refers to methods by injection and intravenous, intramuscular, intraarterial, intrastratum (brain, spinal), intradermal, intraorbital ), Including, but not limited to, intracardiac, intradermal, intraperitoneal, respiratory, subcutaneous, subepithelial, intraarticular, subcapsular, subarachnoid, spinal, epidural and intrasternal injections and infusions It doesn't work.

The dosage of the pharmaceutical composition may vary depending on the type and severity of the disease to be treated or prevented. Specific dosing regimens for any particular subject should be adjusted with sufficient time depending on the needs of the individual and the professional judgment of the person administering or administering the composition.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example 1: Preparation of PTD Peptides

To prepare the peptide used in the present invention, it was prepared by the organic synthesis method by ordering to Peptron Co., Ltd. (385-19, Doyong-dong, Yuseong-gu, Daejeon), and the prepared peptide was confirmed to have a purity of 70% or more through RP-HPLC. The synthesized peptide is as follows.

Tat _48-60 : GRKKRRQRRRPPQ (SEQ ID NO: 1)

2Tat _{48 -60} : (GRKKRRQRRRPPQ) ₂ -K-βA-OH (Formula 2)

4Tat _{48 -60} : (GRKKRRQRRRPPQ) ₄ -K ₂ -K-βA-OH (Formula 3)

8Tat _{48 -60} : (GRKKRRQRRRPPQ) ₈ -K ₄ -K ₂ -K-βA-OH (Formula 4)

Mph-1: YARVRRRGPRR (SEQ ID NO: 2)

2Mph-1: (YARVRRRGPRR) _2- K-βA-OH (Formula 5)

4Mph-1: (YARVRRRGPRR) ₄ -K ₂ -K-βA-OH (Formula 6)

8Mph-1: (YARVRRRGPRR) ₈ -K ₄ -K ₂ -K-βA-OH (Formula 7)

HP4: RRRRPRRRTTRRRR (SEQ ID NO: 3)

4HP4: (RRRRPRRRTTRRRR) ₄ -K ₂ -K-βA-OH (Formula 8)

Example 2: Effect of Enhancing Delivery Efficiency of Adenovirus Vectors of Oligomerized PTD

In order to confirm that the oligomerized PTD is effective in improving the delivery efficiency of adenovirus vectors, rAd / EGFP, an adenovirus vector expressing GFP (Green Fluorescent Protein), which is a self-produced fluorescent label, was used. First, rAd / EGFP was prepared using 0.1 to 300 uM of monomers (SEQ ID NOs: 1, 2 and 3) and dimer PTDs (Formulas 2 and 5), tetramers of 0.003 to 3 uM (Formulas 3, 6 and 8) and octahedral PTDs 4 and 7) were mixed for 30 minutes at room temperature, and then infected with mouse brain tumor cells C6Bu1 for 2 hours. The use of PTDs above the concentrations used in this experiment was virtually meaningless because of their very low practicality. The amount of adenovirus used at infection was 100 multiplicity of infection. After 2 hours, remove the virus mixture, and DMEM for after standard transfer to the culture medium or two CO ₂ incubator (CO ₂ water Jacketed Incubator, Forma Scientific ^TM) in the culture, and were removed to infected cells with trypsin (trypsin) intracellular GFP levels Was measured using FACS (Fluorescence Activated Cell Sorter, BD).

The resulting maximum transfer efficiency and optimum concentration are shown in FIG. 2. In the case of the monomer and dimer PTD, the maximum transfer efficiency and the concentration (the optimal concentration) could not be determined because the transfer efficiency increased in a concentration-dependent manner up to a concentration of 300 uM or less. The tetramer and octahedral PTD showed similar maximum transfer efficiency at concentrations of 0.1-0.3 uM. On the other hand, under certain concentrations, the transfer efficiency of tetramer and octahedron PTD was lower than that of monomer and dimer PTD. In the case of HP4, tetramer PTD (4HP4) showed much higher transfer efficiency even at lower concentration than monomer PTD (HP4).

Example 3: Comparison of Cytotoxicity of Monomer PTD and Oligomeric PTD

After PTD the oligomerization as cytotoxic how monomer of 0.01 ~ 300uM in brain tumor cells, C6Bu1 cells of the rat to see if change and dimer PTD, process the tetramer and eight tetramer PTD of 0.01 ~ 100uM for 24 hours, CO ₂ Cultured in the incubator. After 24 hours, PTD solution was removed, followed by MTT solution for 4 hours, and 0.04N hydrochloric acid / isopropanol solution was treated for 15 hours in a dark place at room temperature to lyse the cells. After measuring the OD570 value using an ELISA Reader, the cell survival rate was calculated by dividing this value by the value of a sample that was not treated with anything.

The resulting 'concentration of PTD showing 50% cytotoxicity' and 'maximum concentration showing no cytotoxicity' are shown in FIG. 2. As a result, only octahedral PTD (8Tat, 8Mph-1) and one tetrameric PTD (4HP4) showed 50% cytotoxicity within the available concentration range. However, monomeric PTDs, dimer PTDs, and some tetrameric PTDs (4Tat _{48 -60} , 4Mph-1) did not show cytotoxicity below 50% within the concentration range of the experiment, thus showing no maximum cytotoxicity. Is indicated. In addition, other concentrations other than those shown in FIG. 2 generally showed high cytotoxicity in the order of octahedron, tetramer, dimer, and monomer.

When all three components of maximum transfer efficiency, optimal concentration, and cytotoxicity shown in FIG. 2 were combined, superiority of tetramer, octahedron, dimer, and monomer was shown. As a result, the tetramer PTD was found to be the most optimal form of PTD.

Example  4: Oligomer PTD Anticancer effect of antiviral vaccine

Based on the result that the tetramer PTD of Example 3 was the most optimal PTD, an animal experiment was performed using the representative tetramer PTD 4Tat _{48 -60 and the} monomer PTD Tat _{48 -60} . Mouse colon cancer cells CT26 were first infected with adenovirus 300MOI expressing interleukin-12 for 2 hours. At this time, the adenovirus was mixed with the two types of PTD (300 uM of Tat _{48 -60} to 0.3 uM of 4Tat _{48 -60} ) and left at room temperature for 30 minutes. After infection for 2 hours, the virus mixture was removed, changed into DMEM culture medium, incubated in a CO ₂ incubator for 4 hours, and cells were removed with trypsin and washed three times with PBS. The CT26 cells thus transferred were subcutaneously injected 5 × 10 ⁵ in BALB / c mice. Thereafter, tumor size was measured at 3 day intervals for about 30 days.

Tumor growth with time obtained as a result is shown in FIG. 3. 4Tat _{48 -60} more Tat _{48 -60} showed better anti-cancer effects of interleukin -12 Atheros furnace virus. In the case of the tetramer PTD, the concentration of the tetramer PTD is about 250 times lower than that of the monomer PTD. Therefore, oligomerized PTD can be effectively used for developing therapeutics using adenoviruses such as adenovirus anticancer vaccines.

The protein carrier oligomers of the present invention can deliver viral vaccines, such as adenovirus anticancer vaccines, into cells in smaller amounts than protein carriers in monomer form, thereby effectively developing gene therapy and vaccine therapeutics.

Claims (11)

A protein transport oligomer represented by the following formula (1), wherein two or more monomeric protein transduction domains (PTDs) are linked and oligomerized into a multimeric form. Formula 1 (PTD) n-branch In Chemical Formula 1, n is an integer of 2 or more, A branch is a linker selected from the group consisting of multiple antigenic peptides (MAPs), cysteines and carrier proteins linked by a peptide linker. The method of claim 1, n is an integer of 2, 4 or 8, A branch is a protein carrier oligomer that is a multiple antigenic peptide (MAP) or cysteine. The protein carrier oligomer of claim 2, wherein the protein carrier oligomer is selected from the group consisting of protein carrier oligomers represented by Formulas 2-8. Formula 2 (GRKKRRQRRRPPQ) ₂ -K-βA-OH Formula 3 (GRKKRRQRRRPPQ) ₄ -K ₂ -K-βA-OH Formula 4 (GRKKRRQRRRPPQ) ₈ -K ₄ -K ₂ -K-βA-OH Formula 5 (YARVRRRGPRR) ₂ -K-βA-OH Formula 6 (YARVRRRGPRR) ₄ -K ₂ -K-βA-OH Formula 7 (YARVRRRGPRR) ₈ -K ₄ -K ₂ -K-βA-OH Formula 8 (RRRRPRRRTTRRRR) ₄ -K ₂ -K-βA-OH The protein carrier oligomer of claim 3, wherein the protein carrier oligomer is selected from the group consisting of protein carrier oligomers represented by formulas (3), (6) and (8). Formula 3 (GRKKRRQRRRPPQ) ₄ -K ₂ -K-βA-OH Formula 6 (YARVRRRGPRR) ₄ -K ₂ -K-βA-OH Formula 8 (RRRRPRRRTTRRRR) ₄ -K ₂ -K-βA-OH 5. The protein carrier oligomer of claim 1, wherein said monomeric protein carrier is selected from the group consisting of Tat, Antp, Mph-1, VP22 and HP4. 6. The protein carrier oligomer of any one of claims 1 to 4 combined with a substance of interest to be delivered into the cytoplasm or nucleus of a eukaryotic or prokaryotic cell. The protein delivery oligomer of claim 6, wherein the target substance is selected from the group consisting of viral vectors, DNA, RNA, proteins, fats, carbohydrates and compounds. 8. The protein delivery oligomer of claim 7, wherein said viral vector is a virus selected from the group consisting of adenovirus, retrovirus and vaccinia virus. A method of delivering a substance of interest to a cell comprising the step of mixing and contacting the protein carrier oligomer of any one of claims 1 to 4 with the cell to which the substance of interest is to be delivered. The method of claim 9, wherein the target material is selected from the group consisting of viral vectors, DNA, RNA, proteins, fats, carbohydrates, and compounds. The method of claim 10, wherein said viral vector is a virus selected from the group consisting of adenovirus, retrovirus and vaccinia virus.

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