JP2020191872A - Agents for improving exogenous gene transfer efficiency to mammalian cells - Google Patents
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- C12N15/09—Recombinant DNA-technology
Abstract
Description
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 mammalian cells.
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 a foreign gene into mammalian cells, and one of them is a method using a viral vector. In this method, a target gene is sent to the target cell by infecting the target cell with a virus in which the target gene is integrated into the genome. Among the viral vectors, adeno-associated virus (hereinafter referred to as “AAV”” is used. The vector (abbreviated as) has already been put into practical use in gene therapy as a viral vector having high safety to humans. However, since the efficiency of AAV infecting 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 inferior. Sufficient gene therapy effect is not exhibited.
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 been energetically conducting research on treatment methods for visual impairment, and as a result, epidermal growth factor expressed in iris pigment epidermal cells, which are target cells, in gene therapy using AAV vector. By inhibiting the tyrosine kinase activity of the growth factor receptor (epidermal growth factor receptor: hereinafter abbreviated as "EGFR"), the efficiency of introduction of the brain-derived neurotrophic factor gene, which is the target gene, is improved. It has been confirmed by 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). Hydroxyurea, which is used as an anticancer agent, is also known to have an effect of improving the efficiency of introducing a foreign gene when introducing a foreign gene into mammalian cells 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 introducing a foreign gene 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 diligent studies in view of the above points, the present inventors have obtained a binding peptide of a peptide having membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR against mammalian cells using an AAV vector. When introducing a foreign gene, it was found to be 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、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た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、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た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、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た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、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た結合ペプチドの存在下で行うものである。
As described in claim 1, the agent for improving the efficiency of introducing a foreign gene, which is 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 EGFR. The active ingredient is a binding peptide in which a peptide having membrane permeability is bound to the C-terminal of the peptide that inhibits tyrosine kinase activity.
Further, in the agent for improving the transfer efficiency of a foreign gene according to claim 2, the mammalian cell is a retinal cell in the agent for improving the transfer efficiency of a foreign gene according to claim 1.
Further, in the agent for improving the transfer efficiency of a foreign gene according to
Further, as described in claim 4, the method for improving the efficiency of introducing a foreign gene into a mammalian cell of the present invention is that when a foreign gene is introduced into a mammalian cell using an AAV vector in vitro, EGFR tyrosine is used. This is due to the presence of a binding peptide to which a peptide having membrane permeability is bound to the C-terminal of the peptide that inhibits kinase activity.
Further, as described in claim 5, the present invention relates to a binding peptide in which a peptide having membrane permeability is bound to the C-terminal of a peptide that inhibits the tyrosine kinase activity of EGFR.
In addition, the method of introducing a foreign gene into a mammalian cell using an AAV vector in vitro of the present invention provides membrane permeability to the C-terminal of a peptide that inhibits the tyrosine kinase activity of EGFR, as described in claim 6. This is performed in the presence of a binding peptide to which the peptide to be possessed is bound.
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 a tyrosine kinase of EGFR are used as an active component of an agent for improving the efficiency of introducing a foreign gene, which is used when introducing a foreign gene into a mammalian cell using an AAV vector. Binding peptides of peptides that inhibit activity can be provided.
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, which is used when introducing a foreign gene into a mammalian cell using the AAV vector of the present invention, is a peptide having membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR. The binding peptide is the 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。 Among the binding peptides of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention, the peptide having membrane permeability is a known amino acid sequence having an action of translocating through the cell membrane into the cytoplasm. (Cell Penetrating Peptide: CPP), for example, YGRKKRRQRRR (SEQ ID NO: 1) derived from HIV-1 Tat, RRRRRNRTRRNRRRRVR (SEQ ID NO: 2) derived from FHV coat, and HIV-1 Rev. Examples thereof include TRQARRNRRRRWRERRQR (SEQ ID NO: 3), RAQRRARAARRNRWTAR derived from BMV Gag (SEQ ID NO: 4), TRRQRRTRARRNR derived from HTLV-II Rex (SEQ ID NO: 5), and the like (for example, Shiro Futaki, protein nucleic acid enzyme). , Vol. 47 No. 11 (2002) 1415-1419). These amino acid sequences may be those in which 1 to 5 amino acids have been 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。 Among the binding peptides of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention, the peptide that inhibits the tyrosine kinase activity of EGFR may have an amino acid sequence known per se, for example. DEYLI (SEQ ID NO: 6) derived from Y992, which is the self-phosphorylating site of EGFR, VPEYINQ (SEQ ID NO: 7) derived from Y1068, DYQQD (SEQ ID NO: 8) derived from Y1148, and ENAEYLR (SEQ ID NO: 9) derived from Y1173. ) Etc. (see, for example, Mineo Abe, et al., British Journal of Pharmacology, 2006; 147: 402-411). These amino acid sequences may be deleted, substituted or added with 1 to 5 amino acids as long as they inhibit the tyrosine kinase activity of EGFR.
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。 The binding peptide of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention may bind a peptide that inhibits tyrosine kinase activity of EGFR to the C-terminal of the peptide having membrane permeability. , A peptide having membrane permeability may be bound to the C-terminal of the peptide that inhibits the tyrosine kinase activity of EGFR. As a specific example, a peptide in which VPEYINQ derived from Y1068 or DYQQD derived from Y1148 is bound to the C-terminal of YGRKKRRQRRRR derived from HIV-1 Tat, and ENAEYLR derived from Y1173 at the C-terminal of RRRRNRTRRNRRRRVR derived from FHV cot. Examples thereof include a bound peptide, a peptide in which YGRKKRRQRRR derived from HIV-1 Tat is bound to the C-terminal 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 and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention 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。 The binding peptide of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention maintains stability when introducing a foreign gene into a mammalian cell using an AAV vector. The N-terminal may be acetylated or the C-terminal may be amidated for the purpose of.
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。 The binding peptide of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention is, for example, a water-soluble powder. Therefore, when introducing a foreign gene into a mammalian cell using an AAV vector in vitro, the cell is infected with the virus after being dissolved in a cell culture solution and incubated at 37 ° C. for, for example, 1 to 24 hours. Therefore, the efficiency of introducing foreign genes can be improved. In vivo, the binding peptide of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR in the present invention is dissolved in a solution of an AAV vector incorporating a foreign gene and then administered into the body. By infecting cells with a virus, the efficiency of introducing foreign genes can be improved. In the present invention, the amount of the peptide binding peptide of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR dissolved in the cell culture solution or the amount of the AAV vector incorporating the foreign gene incorporated in the solution is, for example, 0. It may be appropriately set so as to be in the range of 1 μM to 10 M. 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, for example, one that exerts a therapeutic effect on a disease by being introduced into a target cell. Specifically, examples of mammalian cells include mammalian retinal cells (nerve cells, glial cells, pigment epithelial cells, etc. that constitute the retina). Foreign genes include genes for photosensitive cation-selective channels such as the photoreceptive channel rhodopsin, which plays an important role in visual function. Integration of the photosensitive cation-selective channel gene into the AAV vector and introduction of the AAV vector incorporating the light-sensitive cation-selective channel gene into the retinal cells are described, for example, in Tomita Hiroshi, et sl. , Mol Ther. , 2014; 22 (8): 1434-1440. The present inventors have reported in this document that the visual function is restored by introducing an AAV vector incorporating a light-sensitive cation-selective channel gene into retinal cells, but the membrane permeability in the present invention is described. The binding peptide between the peptide and the peptide that inhibits the tyrosine kinase activity of EGFR contributes to more effective restoration of 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:
CYNOM-K1 which is a fibroblast cell line derived from cynomolgus monkey was seeded in 96-well plates in an amount of 1 × 10 4 each, and incubated in MEM medium containing 10% FBS at 37 ° C. for 1 day. After washing twice with 1 × DPBS, 40 mM hydroxyurea (HU) (including 1 mM sodium butyrate), 10 μM tyrphostin (Tyr), various concentrations of Acetyl-YGRKKRRQRRRBPEYINQ-CONH 2 as a test substance (SEQ ID NO: 10: Water-soluble white powder by chemical synthesis using a peptide synthesizer PSSM-8 manufactured by Shimadzu) was added, and the mixture was incubated at 37 ° C. for 5 hours. After completion of the incubation, wash twice in MEM medium containing 2% FBS, and then AAV-pmCherry (1 × 10 12-13 virus /) prepared according to the manufacturer's manual using commercially available AAV and pmCherry-N1 Virus. mL) 47.5 μL of MEM medium containing 2.5 μL + 2% FBS was added as a virus solution, and after incubating at 37 ° C. for 2 hours, 50 μL of MEM medium containing 18% FBS was added. The medium was changed after 1 day, and the cells were cultured for another 3 days. Then, a solution of Hoechst33342, a nuclear stain, was added to 5 μg / mL, incubated for 30 minutes at room temperature, and the cells were photographed with a fluorescence microscope. From the photographs taken, the expression efficiency of the mCherry gene was calculated as the ratio of the number of cells whose nuclei were stained red by mCherry to the total number of cells whose nuclei were stained blue by Hoechst33342. The results are shown in FIG. 1 (relative values with the unprocessed group as the control). As is clear from FIG. 1, the binding peptide of the peptide having membrane permeability, which is the test substance, and the peptide that inhibits the tyrosine kinase activity of EGFR increases the expression efficiency by improving the introduction efficiency of the mCherry gene in a concentration-dependent manner. It was improved and its action was comparable to that of tylhostin. This test substance was less toxic than tyrphostin (for example, even 1 mM did 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:
Same as Example 1 except that Acetyl-YGRKKRRQRRDYQQD-CONH 2 (SEQ ID NO: 11: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) is used as a test substance. The effect of the mCherry gene on the expression efficiency was evaluated. The results are shown in FIG. As is clear from FIG. 2, the effect of improving the expression efficiency of the mCherry gene of the test substance was comparable to that of tylhostin. This test substance was less toxic than tyrphostin (for example, even 1 mM did 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:
Same as Example 1 except that Acetyl-VPEYINQYGRKKRRQRR-CONH 2 (SEQ ID NO: 12: water-soluble white powder by chemical synthesis using the peptide synthesizer PSSM-8 of Shimadzu) is used as a test substance. 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 that of tylhostin was shown. This test substance was less toxic than tyrphostin (for example, even 1 mM did 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, the following experiments were performed. A modified channelrhodopsin (mVChR1) gene based on the green algae Volvox-derived channelrhodopsin (VChR1) was incorporated into an AAV vector, and an AAV vector solution (2.5 × 10 11 parts / mL) containing the mVChR1 gene was incorporated into the AAV vector. Was administered under anesthesia into one of the intravitreals of hereditary retinal degeneration (RCS, rdy / rdy) rats (purchased from Claire Japan) 6 months or older who led to blindness using a 32-gauge Hamilton microsyringe. did. In the other intravitreal, 5 μL of a solution containing the AAV vector incorporating the mVChR1 gene and the test substance (Acetyl-YGRKKRRQRRVPEYINQ-CONH 2 : Tat-Y1068) according to Example 1 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). The visual evoked potential was measured 2 months after administration. Specifically, an LED light source was placed in front of the eyes of a rat who underwent an operation to implant a recording electrode in the visual cortex of the brain one week before the measurement, and light stimulation of 10 ms was performed 200 times at 1-second intervals for light stimulation. The accompanying potential changes in the visual cortex were recorded. The results are shown in FIG. As is clear from FIG. 3, the binding peptide of the peptide having membrane permeability, which is the test substance, and the peptide that inhibits the tyrosine kinase activity of EGFR improves the expression efficiency by improving the transfer efficiency of the MVChR1 gene into retinal cells. Improves and increases the amplitude of the visual evoked potential (the main cell into which the gene is introduced is the ganglion cell).
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 provides industrial applicability in that it can provide a novel agent for improving the efficiency of introducing a foreign gene, which is used when introducing a foreign gene into a mammalian cell using an AAV vector. Have.
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た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た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た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た結合ペプチドの存在下で行うものである。
As described in claim 1, the agent for improving the efficiency of introducing a foreign gene, which is 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 membrane permeation. The active ingredient is a binding peptide in which a peptide that inhibits the tyrosine kinase activity of EGFR is bound to the C-terminal of the peptide having sex .
Further, in the agent for improving the transfer efficiency of a foreign gene according to claim 2, the mammalian cell is a retinal cell in the agent for improving the transfer efficiency of a foreign gene according to claim 1.
Further, in the agent for improving the transfer efficiency of a foreign gene according to
Further, as described in claim 4, the method for improving the efficiency of introducing a foreign gene into a mammalian cell of the present invention is membrane permeability when introducing a foreign gene into a mammalian cell using an AAV vector in vitro. This is due to the presence of a binding peptide in which a peptide that inhibits the tyrosine kinase activity of EGFR is bound to the C-terminal of the peptide having .
Further, as described in claim 5, the present invention relates to a binding peptide in which a peptide that inhibits the tyrosine kinase activity of EGFR is bound to the C-terminal of a peptide having membrane permeability .
In addition, the method of introducing a foreign gene into a mammalian cell using an AAV vector in vitro of the present invention inhibits the tyrosine kinase activity of EGFR at the C-terminal of a peptide having membrane permeability, as described in claim 6. It is carried out in the presence of a binding peptide to which the peptide to be bound is bound.
Claims (6)
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