WO2016129656A1 - Agent for improving exogenous gene transfer efficiency to mammalian cells - Google Patents

Agent for improving exogenous gene transfer efficiency to mammalian cells Download PDF

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WO2016129656A1
WO2016129656A1 PCT/JP2016/054042 JP2016054042W WO2016129656A1 WO 2016129656 A1 WO2016129656 A1 WO 2016129656A1 JP 2016054042 W JP2016054042 W JP 2016054042W WO 2016129656 A1 WO2016129656 A1 WO 2016129656A1
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peptide
tyrosine kinase
kinase activity
foreign gene
improving
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浩史 冨田
江里子 菅野
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国立大学法人岩手大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

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  • the present invention relates to an agent for improving the efficiency of introducing a foreign gene into a mammalian cell.
  • AAV adeno-associated virus
  • the present inventors have energetically conducted research on therapeutic methods for visual impairment, and as a result, in gene therapy using AAV vectors, epithelium expressed in iris pigment epithelial cells as target cells.
  • EGFR growth factor receptor
  • tyrphostin which is a low molecular weight organic compound that inhibits kinase activity
  • Non-patent Document 1 tyrphostin is known to be cytotoxic at low concentrations (eg, 10 ⁇ M in in vitro experiments).
  • hydroxyurea used as an anticancer agent is also known to have an effect of improving the efficiency of introduction of a foreign gene when a foreign gene is introduced into a mammalian cell using an AAV vector. Its action is extremely weak.
  • an object of the present invention is to provide a novel agent for improving the efficiency of introducing a foreign gene, which is used when a foreign gene is introduced into a mammalian cell using an AAV vector.
  • a binding peptide of a peptide having membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR is applied to mammalian cells using an AAV vector. It has been found that when introducing a foreign gene, it is useful as an active ingredient of an agent for improving the efficiency of introducing a foreign gene.
  • An agent for improving the efficiency of introducing a foreign gene used when introducing a foreign gene into a mammalian cell using the AAV vector of the present invention made in view of the above points is, as described in claim 1, a membrane permeation agent A binding peptide of a peptide having sex and a peptide that inhibits the tyrosine kinase activity of EGFR is used as an active ingredient.
  • the foreign gene introduction efficiency improving agent according to claim 2 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the mammalian cell is a retinal cell.
  • the foreign gene introduction efficiency improving agent according to claim 3 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the foreign gene is a photosensitive cation selective channel gene.
  • the method for improving the efficiency of introducing a foreign gene into a mammalian cell comprises, as described in claim 4, a membrane permeability when the foreign gene is introduced into a mammalian cell using an AAV vector in vitro. And a peptide that inhibits the tyrosine kinase activity of EGFR.
  • the present invention also relates to a binding peptide comprising a peptide having membrane permeability and a peptide that inhibits tyrosine kinase activity of EGFR as described in claim 5.
  • the method for introducing a foreign gene into a mammalian cell using an AAV vector in vitro comprises a peptide having membrane permeability and a peptide inhibiting the tyrosine kinase activity of EGFR as described in claim 6. It is performed in the presence of a binding peptide.
  • a peptide having membrane permeability and an EGFR tyrosine kinase are used as active ingredients of an agent for improving the efficiency of introduction of a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Binding peptides of peptides that inhibit activity can be provided.
  • Example 2 is a graph showing the effect of various substances in Example 1 on the expression efficiency of the mCherry gene. It is a graph which shows the effect
  • FIG. 1 is a graph showing the effect of various substances in Example 1 on the expression efficiency of the mCherry gene. It is a graph which shows the effect
  • the agent for improving the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using the AAV vector of the present invention is a peptide having a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR.
  • the binding peptide is an active ingredient.
  • the peptide having membrane permeability in the present invention is a peptide having a permeability to the tyrosine kinase activity of EGFR, and the peptide having membrane permeability is a known amino acid sequence having an action of passing through the cell membrane into the cytoplasm.
  • YGRKKRRRRR SEQ ID NO: 1 derived from HIV-1 Tat
  • RRRRNRTRRRNRRRVR SEQ ID NO: 2
  • TRQARNRRRRRWRRQR SEQ ID NO: 3
  • RAMVRRAAARNRNRTAR SEQ ID NO: 4
  • TRRQRTRRRARNR SEQ ID NO: 5
  • HTLV-II Rex HTLV-II Rex, etc. Mentioned are (if necessary e.g., Shiro Niki, proteins nucleic acid enzymes, see Vol.47 No.11 (2002) 1415-1419). These amino acid sequences may have 1 to 5 amino acids deleted, substituted or added as long as they have membrane permeability.
  • the peptide that inhibits EGFR tyrosine kinase activity may have a known amino acid sequence, for example, DEYLI (SEQ ID NO: 6) derived from Y992 which is an autophosphorylation site of EGFR, VPEYINQ (SEQ ID NO: 7) derived from Y1068, DYQQD (SEQ ID NO: 8) derived from Y1148, ENAEYLR derived from Y1173 (SEQ ID NO: 9) (See, for example, Mineo Abe, et al., British Journal of Pharmacology, 2006; 147: 402-411) if necessary.
  • These amino acid sequences may be those in which 1 to 5 amino acids are deleted, substituted or added as long as they inhibit the EGFR tyrosine kinase activity.
  • the peptide having a membrane permeability and the peptide that inhibits EGFR tyrosine kinase activity may be bound to the C-terminus of the peptide having membrane permeability by binding a peptide that inhibits EGFR tyrosine kinase activity.
  • a peptide having membrane permeability may be bound to the C-terminus of the peptide that inhibits the tyrosine kinase activity of EGFR.
  • Specific examples include peptides in which VPYINQ derived from Y1068 and DYQQD derived from Y1148 are bound to the C-terminus of YGRKKRRQRRR derived from HIV-1 Tat, and RENEYLR derived from Y1173 at the C-terminus of RRRRNRTRRRNRRRVR derived from FHV coat. Examples thereof include a bound peptide and a peptide in which YGRKKRRQRRR derived from HIV-1 Tat is bound to the C-terminus of VPEYINQ derived from Y1068.
  • binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR can be chemically synthesized using a peptide synthesizer, but may be prepared by a genetic engineering technique.
  • the binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR maintains the stability when a foreign gene is introduced into a mammalian cell using an AAV vector, etc.
  • the N-terminus may be acetylated or the C-terminus may be amidated.
  • the binding peptide of the peptide having membrane permeability and the peptide inhibiting the tyrosine kinase activity of EGFR is, for example, a water-soluble powder. Therefore, when introducing a foreign gene into a mammalian cell in vitro using an AAV vector, the cell is infected with the virus after being dissolved in the cell culture medium and incubated at 37 ° C. for 1 to 24 hours, for example. Thus, the efficiency of introducing foreign genes can be improved.
  • a peptide-binding peptide of the present invention and a peptide that inhibits the tyrosine kinase activity of EGFR are dissolved in an AAV vector solution incorporating a foreign gene and then administered into the body.
  • the efficiency of introducing a foreign gene can be improved.
  • the binding amount of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR is dissolved in a cell culture solution or in a solution of an AAV vector incorporating a foreign gene, for example, 0. What is necessary is just to set suitably so that it may become the range of 1 micromol-10M.
  • the mammalian cells may be cells of various sites such as humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats and dogs.
  • the foreign gene may be a gene that exerts a therapeutic effect on a disease, for example, by being introduced into a target cell.
  • mammalian cells include mammalian retinal cells (such as neurons, glial cells, and pigment epithelial cells that constitute the retina).
  • foreign genes for photosensitive cation selective channels such as the photoreceptor channel rhodopsin that play an important role in visual function.
  • the binding peptide of the peptide possessing and the peptide that inhibits the tyrosine kinase activity of EGFR contributes to the restoration of more effective visual function by the AAV vector incorporating a smaller amount of the photosensitive cation selective channel gene.
  • Example 1 Cynomolgus monkey-derived fibroblast cell line CYNOM-K1 was seeded in a 96-well plate at 1 ⁇ 10 4 cells each, and incubated at 37 ° C. in MEM medium containing 10% FBS for 1 day.
  • the cells were washed twice with MEM medium containing 2% FBS, and then AAV-pmCherry (1 ⁇ 10 12 to 13 particles / particulate / 1 ⁇ 10 12-13 particle / mL) 47.5 ⁇ L of MEM medium containing 2.5 ⁇ L + 2% FBS was added as a virus solution and incubated for 2 hours at 37 ° C., and then 50 ⁇ L of MEM medium containing 18% FBS was added. After 1 day, the medium was changed and further cultured for 3 days.
  • AAV-pmCherry (1 ⁇ 10 12 to 13 particles / particulate / 1 ⁇ 10 12-13 particle / mL) 47.5 ⁇ L of MEM medium containing 2.5 ⁇ L + 2% FBS was added as a virus solution and incubated for 2 hours at 37 ° C., and then 50 ⁇ L of MEM medium containing 18% FBS was added. After 1 day, the medium was changed and further cultured for 3 days.
  • a Hoechst 33342 solution as a nuclear stain was added to 5 ⁇ g / mL, incubated at room temperature for 30 minutes, and a cell photograph was taken with a fluorescence microscope. From the photograph taken, the expression efficiency of the mCherry gene was calculated as the ratio of the number of cells stained red with mCherry to the total number of cells stained blue with Hoechst 33342. The results are shown in FIG. 1 (relative values using the untreated group as a control). As is apparent from FIG.
  • the binding peptide of the test substance which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, increases the expression efficiency by improving the efficiency of introducing the mCherry gene in a concentration-dependent manner. Improved and its action was comparable to tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 2 Similar to Example 1 except that various concentrations of Acetyl-YGRKKRRQRRRDYQQD-CONH 2 (SEQ ID NO: 11: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances.
  • SEQ ID NO: 11 water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8.
  • FIG. 2 the effect on the expression efficiency of the mCherry gene was evaluated. The results are shown in FIG. As apparent from FIG. 2, the effect of improving the expression efficiency of the mCherry gene of the test substance was comparable to that of tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 3 Similar to Example 1 except that various concentrations of Acetyl-VPEYINQYGRKKRRRRR-CONH 2 (SEQ ID NO: 12: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances. Then, when the effect on the expression efficiency of the mCherry gene was evaluated, the effect of improving the expression efficiency of the mCherry gene comparable to tyrphostin was shown. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 4 Tomita Hiroshi, et sl. Mol Ther. , 2014; 22 (8): 1434-1440, and the following experiment was conducted.
  • a modified channelrhodopsin (mVChR1) gene having a basic structure of channel rhodopsin (VChR1) derived from green algae Volbox was incorporated into an AAV vector, and an AAV vector solution (2.5 ⁇ 10 11 particles / mL) containing mVChR1 gene was 5 ⁇ L.
  • Visual evoked potentials were measured 2 months after administration. Specifically, an LED light source is placed in front of the eyes of a rat that has undergone an implantation operation of a recording electrode in the brain visual cortex one week before the measurement, and 10 ms of light stimulation is performed 200 times at 1 second intervals. The associated changes in the potential of the visual cortex were recorded. The results are shown in FIG. As is clear from FIG. 3, the binding peptide of the test substance, which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, improves the expression efficiency by improving the efficiency of mVChR1 gene introduction into retinal cells. Improved and increased the amplitude of the visual evoked potential (the main cells to be transfected are ganglion cells).
  • the present invention has an industrial applicability in that it can provide a novel improver of the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Have.

Abstract

The problem of the present invention is to provide a novel agent for improving exogenous gene transfer efficiency to be used when transferring exogenous genes to mammalian cells using an adeno-associated viral vector. This agent for improving exogenous gene transfer and providing a solution to the aforementioned problem has an active ingredient in the form of a bound peptide comprising a peptide having membrane permeability and a peptide for inhibiting the tyrosine kinase activity of the epidermal growth factor receptors. The bound peptide comprising a peptide having membrane permeability and a peptide for inhibiting the tyrosine kinase activity of the epidermal growth factor receptors according to the present invention contributes, for example, to more effective recovery of visual function by an adeno-associated viral vector incorporating a smaller amount of light-sensitive cation-selective channel gene.

Description

哺乳動物細胞に対する外来遺伝子の導入効率の向上剤Agent for improving the efficiency of introduction of foreign genes into mammalian cells
　本発明は、哺乳動物細胞に対する外来遺伝子の導入効率の向上剤に関する。 The present invention relates to an agent for improving the efficiency of introducing a foreign gene into a mammalian cell.
　哺乳動物細胞に対して外来遺伝子を導入する方法には様々な方法が存在するが、その一つとしてウイルスベクターを利用する方法が知られている。この方法は、ゲノムに目的遺伝子を組み込んだウイルスを標的細胞に感染させることによって、目的遺伝子を標的細胞に送り込むものであり、ウイルスベクターのなかでもアデノ随伴ウイルス（ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄ　ｖｉｒｕｓ：以下「ＡＡＶ」と略称する）ベクターは、ヒトに対する安全性が高いウイルスベクターとして、すでに遺伝子治療において実用化されている。しかしながら、ＡＡＶの哺乳動物細胞への感染効率は、細胞の種類や由来によって様々であることから、感染効率が低い細胞が標的細胞である場合、標的細胞に対する目的遺伝子の導入効率が劣ることで、充分な遺伝子治療の効果が発揮されない。 There are various methods for introducing foreign genes into mammalian cells, and one of them is a method using a viral vector. In this method, the target gene is transferred to the target cell by infecting the target cell with a virus in which the target gene is incorporated into the genome. Among the viral vectors, adeno-associated virus (hereinafter referred to as “AAV”) is used. Vectors have already been put to practical use in gene therapy as viral vectors with high safety for humans. However, since the infection efficiency of AAV into mammalian cells varies depending on the cell type and origin, when a cell with low infection efficiency is a target cell, the efficiency of introducing the target gene into the target cell is poor. Insufficient gene therapy effect.
　本発明者らは、これまで視覚障害に対する治療方法についての研究を精力的に行ってきており、その成果として、ＡＡＶベクターを用いた遺伝子治療において、標的細胞である虹彩色素上皮細胞に発現する上皮成長因子受容体（ｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ：以下「ＥＧＦＲ」と略称する）のチロシンキナーゼ活性を阻害することで、目的遺伝子である脳由来神経栄養因子遺伝子の導入効率が向上することを、ＥＧＦＲのチロシンキナーゼ活性を阻害する低分子有機化合物であるチルホスチン（ｔｙｒｐｈｏｓｔｉｎ）を用いて確認している（非特許文献１）。しかしながら、チルホスチンは、低濃度（例えばインビトロの実験において１０μＭ）で細胞毒性を示すことが知られている。また、抗がん剤として用いられるヒドロキシ尿素も、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に外来遺伝子の導入効率を向上させる作用を有することが知られているが、その作用は極めて弱い。 The present inventors have energetically conducted research on therapeutic methods for visual impairment, and as a result, in gene therapy using AAV vectors, epithelium expressed in iris pigment epithelial cells as target cells. By inhibiting the tyrosine kinase activity of the growth factor receptor (hereinafter abbreviated as “EGFR”), the introduction efficiency of the brain-derived neurotrophic factor gene, which is the target gene, is improved. It has been confirmed using tyrphostin, which is a low molecular weight organic compound that inhibits kinase activity (Non-patent Document 1). However, tyrphostin is known to be cytotoxic at low concentrations (eg, 10 μM in in vitro experiments). Further, hydroxyurea used as an anticancer agent is also known to have an effect of improving the efficiency of introduction of a foreign gene when a foreign gene is introduced into a mammalian cell using an AAV vector. Its action is extremely weak.
　そこで本発明は、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の新規な向上剤を提供することを目的とする。 Therefore, an object of the present invention is to provide a novel agent for improving the efficiency of introducing a foreign gene, which is used when a foreign gene is introduced into a mammalian cell using an AAV vector.
　本発明者らは上記の点に鑑みて鋭意検討を行った結果、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドが、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際、外来遺伝子の導入効率の向上剤の有効成分として有用であることを見出した。 As a result of intensive studies in view of the above points, the present inventors have found that a binding peptide of a peptide having membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR is applied to mammalian cells using an AAV vector. It has been found that when introducing a foreign gene, it is useful as an active ingredient of an agent for improving the efficiency of introducing a foreign gene.
　上記の点に鑑みてなされた本発明のＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の向上剤は、請求項１記載の通り、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを有効成分とする。
　また、請求項２記載の外来遺伝子の導入効率の向上剤は、請求項１記載の外来遺伝子の導入効率の向上剤において、哺乳動物細胞が網膜細胞である。
　また、請求項３記載の外来遺伝子の導入効率の向上剤は、請求項１記載の外来遺伝子の導入効率の向上剤において、外来遺伝子が光感受性陽イオン選択的チャネル遺伝子である。
　また、本発明の哺乳動物細胞に対する外来遺伝子の導入効率を向上させる方法は、請求項４記載の通り、インビトロにおいてＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを存在させることによる。
　また、本発明は、請求項５記載の通り、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドに関する。
　また、本発明のインビトロにおいてＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する方法は、請求項６記載の通り、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドの存在下で行うものである。 An agent for improving the efficiency of introducing a foreign gene used when introducing a foreign gene into a mammalian cell using the AAV vector of the present invention made in view of the above points is, as described in claim 1, a membrane permeation agent A binding peptide of a peptide having sex and a peptide that inhibits the tyrosine kinase activity of EGFR is used as an active ingredient.
The foreign gene introduction efficiency improving agent according to claim 2 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the mammalian cell is a retinal cell.
The foreign gene introduction efficiency improving agent according to claim 3 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the foreign gene is a photosensitive cation selective channel gene.
The method for improving the efficiency of introducing a foreign gene into a mammalian cell according to the present invention comprises, as described in claim 4, a membrane permeability when the foreign gene is introduced into a mammalian cell using an AAV vector in vitro. And a peptide that inhibits the tyrosine kinase activity of EGFR.
The present invention also relates to a binding peptide comprising a peptide having membrane permeability and a peptide that inhibits tyrosine kinase activity of EGFR as described in claim 5.
The method for introducing a foreign gene into a mammalian cell using an AAV vector in vitro according to the present invention comprises a peptide having membrane permeability and a peptide inhibiting the tyrosine kinase activity of EGFR as described in claim 6. It is performed in the presence of a binding peptide.
　本発明によれば、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の向上剤の有効成分として、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを提供することができる。 According to the present invention, a peptide having membrane permeability and an EGFR tyrosine kinase are used as active ingredients of an agent for improving the efficiency of introduction of a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Binding peptides of peptides that inhibit activity can be provided.
実施例１における各種の物質のｍＣｈｅｒｒｙ遺伝子の発現効率に対する作用を示すグラフである。2 is a graph showing the effect of various substances in Example 1 on the expression efficiency of the mCherry gene. 実施例２における各種の物質のｍＣｈｅｒｒｙ遺伝子の発現効率に対する作用を示すグラフである。It is a graph which shows the effect | action with respect to the expression efficiency of the mCherry gene of the various substances in Example 2. 実施例４においてｍＶＣｈＲ１遺伝子の発現によって発生する視覚誘発電位を被験物質が増大させる効果を有することを示すグラフである。It is a graph which shows that a test substance has an effect which increases the visual evoked potential generate | occur | produced by expression of mVChR1 gene in Example 4. FIG.
　本発明のＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の向上剤は、膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを有効成分とする。 The agent for improving the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using the AAV vector of the present invention is a peptide having a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR. The binding peptide is an active ingredient.
　本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドにおいて、膜透過性を有するペプチドは、細胞膜を通過して細胞質内へ移行する作用を持つ自体公知のアミノ酸配列を有するもの（Ｃｅｌｌ　Ｐｅｎｅｔｒａｔｉｎｇ　Ｐｅｐｔｉｄｅ：ＣＰＰ）であってよく、例えば、ＨＩＶ－１　Ｔａｔに由来するＹＧＲＫＫＲＲＱＲＲＲ（配列番号１）、ＦＨＶ　ｃｏａｔに由来するＲＲＲＲＮＲＴＲＲＮＲＲＲＶＲ（配列番号２）、ＨＩＶ－１　Ｒｅｖに由来するＴＲＱＡＲＲＮＲＲＲＲＷＲＥＲＱＲ（配列番号３）、ＢＭＶ　Ｇａｇに由来するＲＡＱＲＲＡＡＡＲＲＮＲＷＴＡＲ（配列番号４）、ＨＴＬＶ－ＩＩ　Ｒｅｘに由来するＴＲＲＱＲＴＲＲＡＲＲＮＲ（配列番号５）などが挙げられる（必要であれば例えば、二木史朗、蛋白質核酸酵素、Ｖｏｌ．４７　Ｎｏ．１１（２００２）１４１５－１４１９を参照のこと）。これらのアミノ酸配列は、膜透過性を有する限りにおいて１～５個のアミノ酸が欠失、置換または付加したものであってもよい。 The peptide having membrane permeability in the present invention is a peptide having a permeability to the tyrosine kinase activity of EGFR, and the peptide having membrane permeability is a known amino acid sequence having an action of passing through the cell membrane into the cytoplasm. For example, YGRKKRRRRR (SEQ ID NO: 1) derived from HIV-1 Tat, RRRRNRTRRRNRRRVR (SEQ ID NO: 2) derived from FHV coat, and HIV-1 Rev. TRQARNRRRRRWRRQR (SEQ ID NO: 3), RAMVRRAAARNRNRTAR (SEQ ID NO: 4) derived from BMV Gag, TRRQRTRRRARNR (SEQ ID NO: 5) derived from HTLV-II Rex, etc. Mentioned are (if necessary e.g., Shiro Niki, proteins nucleic acid enzymes, see Vol.47 No.11 (2002) 1415-1419). These amino acid sequences may have 1 to 5 amino acids deleted, substituted or added as long as they have membrane permeability.
　本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドにおいて、ＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドは、自体公知のアミノ酸配列を有するものであってよく、例えば、ＥＧＦＲの自己リン酸化部位であるＹ９９２に由来するＤＥＹＬＩ（配列番号６）、Ｙ１０６８に由来するＶＰＥＹＩＮＱ（配列番号７）、Ｙ１１４８に由来するＤＹＱＱＤ（配列番号８）、Ｙ１１７３に由来するＥＮＡＥＹＬＲ（配列番号９）などが挙げられる（必要であれば例えば、Ｍｉｎｅｏ　Ａｂｅ，ｅｔ　ａｌ．，Ｂｒｉｔｉｓｈ　Ｊｏｕｒｎａｌ　ｏｆ　Ｐｈａｒｍａｃｏｌｏｇｙ，２００６；１４７：４０２－４１１を参照のこと）。これらのアミノ酸配列は、ＥＧＦＲのチロシンキナーゼ活性を阻害する限りにおいて１～５個のアミノ酸が欠失、置換または付加したものであってもよい。 In the binding peptide of a peptide having membrane permeability in the present invention and a peptide that inhibits EGFR tyrosine kinase activity, the peptide that inhibits EGFR tyrosine kinase activity may have a known amino acid sequence, for example, DEYLI (SEQ ID NO: 6) derived from Y992 which is an autophosphorylation site of EGFR, VPEYINQ (SEQ ID NO: 7) derived from Y1068, DYQQD (SEQ ID NO: 8) derived from Y1148, ENAEYLR derived from Y1173 (SEQ ID NO: 9) (See, for example, Mineo Abe, et al., British Journal of Pharmacology, 2006; 147: 402-411) if necessary. These amino acid sequences may be those in which 1 to 5 amino acids are deleted, substituted or added as long as they inhibit the EGFR tyrosine kinase activity.
　本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、膜透過性を有するペプチドのＣ末端にＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドを結合させてもよいし、ＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドのＣ末端に膜透過性を有するペプチドを結合させてもよい。具体例としては、ＨＩＶ－１　Ｔａｔに由来するＹＧＲＫＫＲＲＱＲＲＲのＣ末端にＹ１０６８に由来するＶＰＥＹＩＮＱやＹ１１４８に由来するＤＹＱＱＤを結合させたペプチド、ＦＨＶ　ｃｏａｔに由来するＲＲＲＲＮＲＴＲＲＮＲＲＲＶＲのＣ末端にＹ１１７３に由来するＥＮＡＥＹＬＲを結合させたペプチド、Ｙ１０６８に由来するＶＰＥＹＩＮＱのＣ末端にＨＩＶ－１　Ｔａｔに由来するＹＧＲＫＫＲＲＱＲＲＲを結合させたペプチドなどが挙げられる。 In the present invention, the peptide having a membrane permeability and the peptide that inhibits EGFR tyrosine kinase activity may be bound to the C-terminus of the peptide having membrane permeability by binding a peptide that inhibits EGFR tyrosine kinase activity. A peptide having membrane permeability may be bound to the C-terminus of the peptide that inhibits the tyrosine kinase activity of EGFR. Specific examples include peptides in which VPYINQ derived from Y1068 and DYQQD derived from Y1148 are bound to the C-terminus of YGRKKRRQRRR derived from HIV-1 Tat, and RENEYLR derived from Y1173 at the C-terminus of RRRRNRTRRRNRRRVR derived from FHV coat. Examples thereof include a bound peptide and a peptide in which YGRKKRRQRRR derived from HIV-1 Tat is bound to the C-terminus of VPEYINQ derived from Y1068.
　本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、ペプチド合成機を用いて化学合成することができるが、遺伝子工学的手法によって調製してもよい。 The binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR can be chemically synthesized using a peptide synthesizer, but may be prepared by a genetic engineering technique.
　なお、本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際の安定性を保つことなどを目的として、そのＮ末端をアセチル化したりＣ末端をアミド化したりしてもよい。 In addition, the binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR maintains the stability when a foreign gene is introduced into a mammalian cell using an AAV vector, etc. For this purpose, the N-terminus may be acetylated or the C-terminus may be amidated.
　本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、例えば水溶性の粉末である。従って、インビトロにおいてＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際、細胞の培養液に溶解して例えば１～２４時間、３７℃でインキュベートした後、ウイルスを細胞に感染させることで、外来遺伝子の導入効率を向上させることができる。また、インビボにおいては、本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを、外来遺伝子を組み込んだＡＡＶベクターの溶液に溶解してから、体内に投与してウイルスを細胞に感染させることで、外来遺伝子の導入効率を向上させることができる。本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドの、細胞の培養液への溶解量や外来遺伝子を組み込んだＡＡＶベクターの溶液への溶解量は、例えば０．１μＭ～１０Ｍの範囲になるように適宜設定すればよい。なお、本発明において、哺乳動物細胞は、ヒト、サル、マウス、ラット、ハムスター、モルモット、ウシ 、ブタ、ウマ、ウサギ、ヒツジ、ヤギ、ネコ、イヌなどの各種の部位の細胞であってよい。外来遺伝子は、標的細胞に対して導入されることで、例えば疾患の治療効果を発揮するものなどであってよい。具体的には、哺乳動物細胞としては、哺乳動物の網膜細胞（網膜を構成する神経細胞やグリア細胞や色素上皮細胞など）が挙げられる。外来遺伝子としては、視覚機能に重要な役割を果たす光受容チャネルロドプシンなどの光感受性陽イオン選択的チャネルの遺伝子が挙げられる。ＡＡＶベクターへの光感受性陽イオン選択的チャネル遺伝子の組み込みや、光感受性陽イオン選択的チャネル遺伝子を組み込んだＡＡＶベクターの網膜細胞への導入は、例えばＴｏｍｉｔａ　Ｈｉｒｏｓｈｉ，ｅｔ　ｓｌ．，Ｍｏｌ　Ｔｈｅｒ．，２０１４；２２（８）：１４３４－１４４０に記載の方法に従って行うことができる。本発明者らは、この文献において、光感受性陽イオン選択的チャネル遺伝子を組み込んだＡＡＶベクターを網膜細胞に導入にすることによって視覚機能が回復することを報告したが、本発明における膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、より少量の光感受性陽イオン選択的チャネル遺伝子を組み込んだＡＡＶベクターによる、より効果的な視覚機能の回復に寄与する。 In the present invention, the binding peptide of the peptide having membrane permeability and the peptide inhibiting the tyrosine kinase activity of EGFR is, for example, a water-soluble powder. Therefore, when introducing a foreign gene into a mammalian cell in vitro using an AAV vector, the cell is infected with the virus after being dissolved in the cell culture medium and incubated at 37 ° C. for 1 to 24 hours, for example. Thus, the efficiency of introducing foreign genes can be improved. Further, in vivo, a peptide-binding peptide of the present invention and a peptide that inhibits the tyrosine kinase activity of EGFR are dissolved in an AAV vector solution incorporating a foreign gene and then administered into the body. By infecting cells with a virus, the efficiency of introducing a foreign gene can be improved. In the present invention, the binding amount of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR is dissolved in a cell culture solution or in a solution of an AAV vector incorporating a foreign gene, for example, 0. What is necessary is just to set suitably so that it may become the range of 1 micromol-10M. In the present invention, the mammalian cells may be cells of various sites such as humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats and dogs. The foreign gene may be a gene that exerts a therapeutic effect on a disease, for example, by being introduced into a target cell. Specific examples of mammalian cells include mammalian retinal cells (such as neurons, glial cells, and pigment epithelial cells that constitute the retina). Examples of foreign genes include genes for photosensitive cation selective channels such as the photoreceptor channel rhodopsin that play an important role in visual function. Incorporation of a photosensitive cation selective channel gene into an AAV vector and introduction of an AAV vector incorporating a photosensitive cation selective channel gene into retinal cells are described in, for example, Tomita Hiroshi, et sl. , Mol Ther. , 2014; 22 (8): 1434-1440. In this document, the present inventors have reported that visual function is restored by introducing an AAV vector incorporating a photosensitive cation-selective channel gene into retinal cells. The binding peptide of the peptide possessing and the peptide that inhibits the tyrosine kinase activity of EGFR contributes to the restoration of more effective visual function by the AAV vector incorporating a smaller amount of the photosensitive cation selective channel gene.
　以下、本発明を実施例によって詳細に説明するが、本発明は以下の記載に限定して解釈されるものではない。 Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not construed as being limited to the following description.
実施例１：
　カニクイザル由来の繊維芽細胞株であるＣＹＮＯＭ－Ｋ１を９６ウェルプレートにそれぞれ１×１０４個ずつ播種し、１０％ＦＢＳを含むＭＥＭ培地で、１日、３７℃でインキュベートした。１×ＤＰＢＳで２回ｗａｓｈした後、４０ｍＭのヒドロキシ尿素（ＨＵ）（１ｍＭの酪酸ナトリウムを含む）、１０μＭのチルホスチン（Ｔｙｒ）、被験物質としての各種の濃度のＡｃｅｔｙｌ－ＹＧＲＫＫＲＲＱＲＲＲＶＰＥＹＩＮＱ－ＣＯＮＨ２（配列番号１０：Ｓｈｉｍａｄｚｕ社のペプチド合成機ＰＳＳＭ－８を用いた化学合成による水溶性の白色粉末）をそれぞれ加え、５時間、３７℃でインキュベートした。インキュベートを終了した後、２％ＦＢＳを含むＭＥＭ培地で２回ｗａｓｈしてから、市販のＡＡＶとｐｍＣｈｅｒｒｙ－Ｎ１　Ｖｅｃｔｏｒを用いてメーカのマニュアルに従って作製したＡＡＶ－ｐｍＣｈｅｒｒｙ（１×１０１２～１３ｐａｒｔｉｃｌｅ／ｍＬ）２．５μＬ＋２％ＦＢＳを含むＭＥＭ培地４７．５μＬをウイルス溶液として加え、２時間、３７℃でインキュベートした後、１８％ＦＢＳを含むＭＥＭ培地を５０μＬ加えた。１日後に培地交換を行い、さらに３日間培養した。その後、核染色剤であるＨｏｅｃｈｓｔ３３３４２溶液を５μｇ／ｍＬになるように加え、３０分間、室温でインキュベートし、蛍光顕微鏡で細胞の写真を撮影した。撮影した写真から、ｍＣｈｅｒｒｙ遺伝子の発現効率を、Ｈｏｅｃｈｓｔ３３３４２によって核が青色に染色された全細胞数に対する、ｍＣｈｅｒｒｙによって核が赤色に染色された細胞数の比率として計算した。結果を図１に示す（無処理群をコントロールとする相対値）。図１から明らかなように、被験物質である膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、濃度依存的にｍＣｈｅｒｒｙ遺伝子の導入効率を向上させることで発現効率を向上させ、その作用はチルホスチンに匹敵するものであった。なお、この被験物質は、チルホスチンよりも低毒性であった（例えばインビトロの実験において１ｍＭでも細胞毒性を示さない）。 Example 1:
Cynomolgus monkey-derived fibroblast cell line CYNOM-K1 was seeded in a 96-well plate at 1 × 10 4 cells each, and incubated at 37 ° C. in MEM medium containing 10% FBS for 1 day. After washing twice with 1 × DPBS, 40 mM hydroxyurea (HU) (containing 1 mM sodium butyrate), 10 μM tyrphostin (Tyr), various concentrations of Acetyl-YGRKKRRQRRRRVPEYINQ-CONH 2 (SEQ ID NO: 10: water-soluble white powder obtained by chemical synthesis using a peptide synthesizer PSSM-8 manufactured by Shimadzu Co., Ltd.) was added, respectively, and incubated at 37 ° C. for 5 hours. After the incubation was completed, the cells were washed twice with MEM medium containing 2% FBS, and then AAV-pmCherry (1 × 10 12 to 13 particles / particulate / 1 × 10 12-13 particle / mL) 47.5 μL of MEM medium containing 2.5 μL + 2% FBS was added as a virus solution and incubated for 2 hours at 37 ° C., and then 50 μL of MEM medium containing 18% FBS was added. After 1 day, the medium was changed and further cultured for 3 days. Thereafter, a Hoechst 33342 solution as a nuclear stain was added to 5 μg / mL, incubated at room temperature for 30 minutes, and a cell photograph was taken with a fluorescence microscope. From the photograph taken, the expression efficiency of the mCherry gene was calculated as the ratio of the number of cells stained red with mCherry to the total number of cells stained blue with Hoechst 33342. The results are shown in FIG. 1 (relative values using the untreated group as a control). As is apparent from FIG. 1, the binding peptide of the test substance, which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, increases the expression efficiency by improving the efficiency of introducing the mCherry gene in a concentration-dependent manner. Improved and its action was comparable to tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
実施例２：
　被験物質としての各種の濃度のＡｃｅｔｙｌ－ＹＧＲＫＫＲＲＱＲＲＲＤＹＱＱＤ－ＣＯＮＨ２（配列番号１１：Ｓｈｉｍａｄｚｕ社のペプチド合成機ＰＳＳＭ－８を用いた化学合成による水溶性の白色粉末）を用いること以外は実施例１と同様にしてそのｍＣｈｅｒｒｙ遺伝子の発現効率に対する作用を評価した。結果を図２に示す。図２から明らかなように、被験物質のｍＣｈｅｒｒｙ遺伝子の発現効率の向上作用はチルホスチンに匹敵するものであった。なお、この被験物質は、チルホスチンよりも低毒性であった（例えばインビトロの実験において１ｍＭでも細胞毒性を示さない）。 Example 2:
Similar to Example 1 except that various concentrations of Acetyl-YGRKKRRQRRRDYQQD-CONH 2 (SEQ ID NO: 11: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances. Thus, the effect on the expression efficiency of the mCherry gene was evaluated. The results are shown in FIG. As apparent from FIG. 2, the effect of improving the expression efficiency of the mCherry gene of the test substance was comparable to that of tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
実施例３：
　被験物質としての各種の濃度のＡｃｅｔｙｌ－ＶＰＥＹＩＮＱＹＧＲＫＫＲＲＱＲＲＲ－ＣＯＮＨ２（配列番号１２：Ｓｈｉｍａｄｚｕ社のペプチド合成機ＰＳＳＭ－８を用いた化学合成による水溶性の白色粉末）を用いること以外は実施例１と同様にしてそのｍＣｈｅｒｒｙ遺伝子の発現効率に対する作用を評価したところ、チルホスチンに匹敵するｍＣｈｅｒｒｙ遺伝子の発現効率の向上作用を示した。なお、この被験物質は、チルホスチンよりも低毒性であった（例えばインビトロの実験において１ｍＭでも細胞毒性を示さない）。 Example 3:
Similar to Example 1 except that various concentrations of Acetyl-VPEYINQYGRKKRRRRR-CONH 2 (SEQ ID NO: 12: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances. Then, when the effect on the expression efficiency of the mCherry gene was evaluated, the effect of improving the expression efficiency of the mCherry gene comparable to tyrphostin was shown. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
実施例４：
　Ｔｏｍｉｔａ　Ｈｉｒｏｓｈｉ，ｅｔ　ｓｌ．，Ｍｏｌ　Ｔｈｅｒ．，２０１４；２２（８）：１４３４－１４４０に記載の方法に従って以下の実験を行った。緑藻類ボルボックス由来のチャネルロドプシン（ＶＣｈＲ１）を基本構造とした改変型チャネルロドプシン（ｍＶＣｈＲ１）の遺伝子をＡＡＶベクターに組み込み、ｍＶＣｈＲ１遺伝子を組み込んだＡＡＶベクターの溶液（２．５×１０１１ｐａｒｔｉｃｌｅｓ／ｍＬ）５μＬを、３２ゲージハミルトンマイクロシリンジを用いて、失明に至った６ヶ月齢以上の遺伝性網膜変性症（ＲＣＳ，ｒｄｙ／ｒｄｙ）ラット（日本クレア社より購入）の一方の硝子体内に麻酔下で投与した。他方の硝子体内には、ｍＶＣｈＲ１遺伝子を組み込んだＡＡＶベクターと、実施例１に記載の被験物質（Ａｃｅｔｙｌ－ＹＧＲＫＫＲＲＱＲＲＲＶＰＥＹＩＮＱ－ＣＯＮＨ２：Ｔａｔ－Ｙ１０６８）を含む溶液５μＬを投与した（ｍＶＣｈＲ１遺伝子を組み込んだＡＡＶベクターの最終濃度は対側眼と同じで被験物質の最終濃度は４０μＭ）。投与から２ヶ月後に視覚誘発電位を測定した。具体的には、測定の１週間前に脳視覚野に記録用電極の埋め込み手術を行ったラットの眼前にＬＥＤ光源を配置し、１秒間隔で１０ｍｓの光刺激を２００回行い、光刺激に伴って誘発される視覚野の電位変化を記録した。結果を図３に示す。図３から明らかなように、被験物質である膜透過性を有するペプチドとＥＧＦＲのチロシンキナーゼ活性を阻害するペプチドの結合ペプチドは、網膜細胞へのｍＶＣｈＲ１遺伝子の導入効率を向上させることで発現効率を向上させ、視覚誘発電位の振幅を増大させた（遺伝子導入される主たる細胞は神経節細胞である）。 Example 4:
Tomita Hiroshi, et sl. Mol Ther. , 2014; 22 (8): 1434-1440, and the following experiment was conducted. A modified channelrhodopsin (mVChR1) gene having a basic structure of channel rhodopsin (VChR1) derived from green algae Volbox was incorporated into an AAV vector, and an AAV vector solution (2.5 × 10 11 particles / mL) containing mVChR1 gene was 5 μL. Was administered under anesthesia into one vitreous of a 6-month-old or older hereditary retinal degeneration (RCS, rdy / rdy) rat (purchased from CLEA Japan), which led to blindness, using a 32-gauge Hamilton microsyringe. did. In the other vitreous, 5 μL of a solution containing the AAV vector incorporating the mVChR1 gene and the test substance described in Example 1 (Acetyl-YGRKKRRQRRRRVPEYINQ-CONH 2 : Tat-Y1068) was administered (AAV incorporating the mVChR1 gene) The final concentration of the vector is the same as that of the contralateral eye and the final concentration of the test substance is 40 μM). Visual evoked potentials were measured 2 months after administration. Specifically, an LED light source is placed in front of the eyes of a rat that has undergone an implantation operation of a recording electrode in the brain visual cortex one week before the measurement, and 10 ms of light stimulation is performed 200 times at 1 second intervals. The associated changes in the potential of the visual cortex were recorded. The results are shown in FIG. As is clear from FIG. 3, the binding peptide of the test substance, which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, improves the expression efficiency by improving the efficiency of mVChR1 gene introduction into retinal cells. Improved and increased the amplitude of the visual evoked potential (the main cells to be transfected are ganglion cells).
　本発明は、ＡＡＶベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の新規な向上剤を提供することができる点において、産業上の利用可能性を有する。 The present invention has an industrial applicability in that it can provide a novel improver of the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Have.

Claims (6)

  1. 　膜透過性を有するペプチドと上皮成長因子受容体のチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを有効成分とする、アデノ随伴ウイルスベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際に用いる、外来遺伝子の導入効率の向上剤。 Used to introduce foreign genes into mammalian cells using adeno-associated virus vectors, which contain as an active ingredient a binding peptide consisting of a membrane-permeable peptide and a peptide that inhibits tyrosine kinase activity of epidermal growth factor receptor. Enhancer of foreign gene transfer efficiency.
  2. 　哺乳動物細胞が網膜細胞である請求項１記載の外来遺伝子の導入効率の向上剤。 The agent for improving the efficiency of introducing a foreign gene according to claim 1, wherein the mammalian cell is a retinal cell.
  3. 　外来遺伝子が光感受性陽イオン選択的チャネル遺伝子である請求項１記載の外来遺伝子の導入効率の向上剤。 The agent for improving the efficiency of introducing a foreign gene according to claim 1, wherein the foreign gene is a photosensitive cation selective channel gene.
  4. 　インビトロにおいてアデノ随伴ウイルスベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する際、膜透過性を有するペプチドと上皮成長因子受容体のチロシンキナーゼ活性を阻害するペプチドの結合ペプチドを存在させることによる、哺乳動物細胞に対する外来遺伝子の導入効率を向上させる方法。 When a foreign gene is introduced into mammalian cells in vitro using adeno-associated virus vectors, a peptide having membrane permeability and a peptide that inhibits tyrosine kinase activity of epidermal growth factor receptor are present. A method for improving the efficiency of introducing a foreign gene into a mammalian cell.
  5. 　膜透過性を有するペプチドと上皮成長因子受容体のチロシンキナーゼ活性を阻害するペプチドの結合ペプチド。 A binding peptide of a peptide having membrane permeability and a peptide that inhibits tyrosine kinase activity of epidermal growth factor receptor.
  6. 　インビトロにおいてアデノ随伴ウイルスベクターを用いて哺乳動物細胞に対して外来遺伝子を導入する方法であって、膜透過性を有するペプチドと上皮成長因子受容体のチロシンキナーゼ活性を阻害するペプチドの結合ペプチドの存在下で行う方法。 A method for introducing a foreign gene into a mammalian cell using an adeno-associated virus vector in vitro, the presence of a peptide having a membrane permeability and a peptide that inhibits the tyrosine kinase activity of epidermal growth factor receptor How to do below.
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