JP2013102747A - Vascular endothelial cell-specific promotor - Google Patents
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Abstract
Description
本発明は、血管内皮細胞特異的プロモーターに関する。 The present invention relates to a vascular endothelial cell specific promoter.
現在、疾患部位特異的に様々な遺伝子やshRNA、miRNA等を導入し疾患の治療を行う試みがなされており、細種胞特異的な転写活性をもつプロモーター配列が求められている。基礎研究においても、臓器や細胞特異的に遺伝子を発現させることによってその臓器、細胞における遺伝子の機能解析を行うことは重要であり、細胞種特異的な転写活性を持つプロモーター配列は重要である。 At present, attempts have been made to treat diseases by introducing various genes, shRNAs, miRNAs, etc. in a disease site-specific manner, and a promoter sequence having transcriptional activity specific to a cell type is required. In basic research, it is important to analyze the function of a gene in the organ or cell by expressing the gene in the organ or cell specifically, and a promoter sequence having transcriptional activity specific to the cell type is important.
これまでに、血管内皮細胞特異的プロモーターとしていくつかのもの（Tie-2, VE-cadherin等）が報告されており（特許文献１等）、既に基礎研究において使用されている。しかしながら、これら公知のプロモーターは血管内皮細胞だけでなく平滑筋細胞や血球系細胞でも活性があり、さらに組織においても様々な組織で活性がみられ、必ずしも血管内皮細胞特異的とはいえなかった。 So far, several vascular endothelial cell-specific promoters (Tie-2, VE-cadherin, etc.) have been reported (Patent Document 1, etc.) and have already been used in basic research. However, these known promoters are active not only in vascular endothelial cells but also in smooth muscle cells and blood cells, and also in various tissues, and are not necessarily specific for vascular endothelial cells.
一方、特許文献２には、血管形成の調節に有用な数種類の遺伝子が開示されている。例えばBAZFについては、その発現が心臓と肺に追いやられていること、及び活性化リンパ球で前初期遺伝子として誘導されること、血管形態形成時に高発現することが記載されている。しかしながら、該遺伝子が血管内皮細胞に特異的に高発現するといった記載や示唆は全くない。 On the other hand, Patent Document 2 discloses several types of genes useful for regulating angiogenesis. For example, it is described that BAZF is driven to the heart and lungs, is induced as an immediate early gene in activated lymphocytes, and is highly expressed during blood vessel morphogenesis. However, there is no description or suggestion that the gene is highly expressed specifically in vascular endothelial cells.
従って、本発明は、従来の血管内皮細胞特異的プロモーターよりも特異性及び転写活性に優れた新規な血管内皮細胞特異的プロモーターを提供し、血管内皮特異的に所望の遺伝子やshRNA、miRNA等を高発現させることができる手段を提供することにある。 Accordingly, the present invention provides a novel vascular endothelial cell-specific promoter that is superior in specificity and transcriptional activity to conventional vascular endothelial cell-specific promoters, and provides a desired gene, shRNA, miRNA, etc. specifically in the vascular endothelium. The object is to provide means capable of high expression.
本願発明者らは、オリジナルのジーンチップを用いて内皮細胞増殖因子VEGF-Aで刺激したHUVECにおける遺伝子発現を解析し、発現が増幅する遺伝子を鋭意スクリーニングすることにより、BAZF(B-cell lymphoma 6 associated zinc finger protein)を同定した。そして、BAZFのプロモーター領域を鋭意同定し、このプロモーター領域を用いたレポーターアッセイにより、BAZFプロモーターの血管内皮細胞への特異性が極めて高いこと、さらにその転写活性についても、公知の強発現用プロモーターであるCMVプロモーターの転写活性よりも遥かに強い転写活性を有することを見出し、本願発明を完成した。 The inventors of the present application analyzed the gene expression in HUVEC stimulated with endothelial cell growth factor VEGF-A using the original gene chip, and eagerly screened for the gene whose expression is amplified, whereby BAZF (B-cell lymphoma 6 associated zinc finger protein) was identified. Then, the BAZF promoter region was earnestly identified, and the reporter assay using this promoter region showed that the BAZF promoter has a very high specificity for vascular endothelial cells. It has been found that it has a transcription activity far stronger than that of a certain CMV promoter, and the present invention has been completed.
すなわち、本発明は、下記いずれかのDNAからなり、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有する、血管内皮細胞特異的プロモーターを提供する。
(a) 配列番号１に示す塩基配列中の連続する709塩基以上の領域からなり、4312nt〜5020ntの領域を含むDNA。
(b) (a)と９０％以上の同一性を有する塩基配列からなり、かつ、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有するDNA。
また、本発明は、上記本発明のプロモーターを含む、血管内皮細胞特異的に所望のRNAを産生させるための発現ベクターを提供する。さらに、本発明は、上記本発明の発現ベクターを含む細胞を提供する。さらに、本発明は、上記本発明の発現ベクターを含む、血管内皮細胞特異的に所望のRNAを産生させるための組成物を提供する。さらに、本発明は、上記本発明の発現ベクターを含む、血管内皮細胞特異的に所望のRNAを産生させるためのキットを提供する。さらに、本発明は、上記本発明のプロモーターと、該プロモーターに機能的に連結された所望のRNAをコードするDNAとがゲノム中に組み込まれ、該所望のRNAを血管内皮細胞特異的に産生するトランスジェニック非ヒト動物を提供する。さらに、本発明は、上記本発明のプロモーターと、該プロモーターに機能的に連結された所望のRNAをコードするDNAとを非ヒト動物細胞に導入することを含む、血管内皮細胞特異的に所望のRNAを産生させる方法を提供する。
That is, the present invention provides a vascular endothelial cell-specific promoter comprising any one of the following DNAs and having a promoter activity for specifically expressing a gene in vascular endothelial cells.
(a) A DNA comprising a region of 709 bases or more in the base sequence shown in SEQ ID NO: 1 and comprising a region of 4312nt to 5020nt.
(b) A DNA comprising a nucleotide sequence having 90% or more identity with (a) and having a promoter activity that allows a vascular endothelial cell to specifically express a gene.
The present invention also provides an expression vector for producing a desired RNA specifically for vascular endothelial cells, comprising the promoter of the present invention. Furthermore, the present invention provides a cell containing the expression vector of the present invention. Furthermore, the present invention provides a composition for producing a desired RNA specifically for vascular endothelial cells, comprising the expression vector of the present invention. Furthermore, the present invention provides a kit for producing desired RNA specifically for vascular endothelial cells, comprising the expression vector of the present invention. Furthermore, the present invention incorporates the above-described promoter of the present invention and a DNA encoding a desired RNA operably linked to the promoter into the genome to produce the desired RNA specifically for vascular endothelial cells. A transgenic non-human animal is provided. Furthermore, the present invention relates to a vascular endothelial cell-specific target comprising introducing the promoter of the present invention and a DNA encoding a desired RNA operably linked to the promoter into a non-human animal cell. Methods for producing RNA are provided.
本発明により、公知の血管内皮細胞特異的プロモーターよりも優れた血管内皮細胞特異的プロモーターが提供された。本発明のプロモーターは、公知のプロモーターよりも血管内皮細胞への特異性がはるかに高く、上皮系細胞及び間葉系細胞ではほとんど全く転写活性を示さない。また、特異性が高いのみならず、転写活性の強さにも極めて優れており、遺伝子の恒常的強発現に一般に用いられるCMVプロモーターと比べても、血管内皮細胞において約１０倍も強い転写活性を持つ。本発明のプロモーターを用いれば、血管内皮細胞で効率よく目的遺伝子を発現させることができるので、遺伝子治療等の臨床応用や基礎研究にも有用である。例えば、本発明のプロモーターを用いて血管内皮特異的に蛍光タンパク質を発現させ、血管内皮細胞を可視化した動物モデルを作出すれば、該モデルにより薬剤の血管に対する影響を評価することができるようになる。また、アンチセンス法やRNAiによる遺伝子ノックダウンや、Cre/LoxPシステム等を用いた遺伝子ノックアウトの手法が確立されているが、組織特異的プロモーターとして本発明のプロモーターを採用すれば、血管内皮特異的に目的遺伝子をノックダウン、ノックアウトした動物を作出することができる。こうしたノックダウンないしはノックアウト動物により、各種遺伝子の血管内皮細胞における機能を解析できる。以上の通り、本発明は、臨床及び基礎研究の双方に大いに貢献するものである。 The present invention provides a vascular endothelial cell-specific promoter that is superior to known vascular endothelial cell-specific promoters. The promoter of the present invention is much more specific for vascular endothelial cells than known promoters, and exhibits almost no transcriptional activity in epithelial and mesenchymal cells. In addition to its high specificity, it also excels in transcriptional activity and is about 10 times stronger in vascular endothelial cells than the CMV promoter generally used for the constant strong expression of genes. have. Since the target gene can be efficiently expressed in vascular endothelial cells by using the promoter of the present invention, it is useful for clinical applications such as gene therapy and basic research. For example, if an animal model in which a fluorescent protein is expressed specifically in the vascular endothelium using the promoter of the present invention and the vascular endothelial cells are visualized, the effect of the drug on the blood vessel can be evaluated by the model. . Furthermore, gene knockdown methods using antisense methods, RNAi, and Cre / LoxP systems have been established. If the promoter of the present invention is used as a tissue-specific promoter, vascular endothelium-specific methods are used. It is possible to create an animal in which the target gene is knocked down and knocked out. Such knockdown or knockout animals can analyze the function of various genes in vascular endothelial cells. As described above, the present invention greatly contributes to both clinical and basic research.
本発明のプロモーターは、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有する血管内皮特異的プロモーターであり、下記いずれかのDNAからなる。
(a) 配列番号１に示す塩基配列中の連続する709塩基以上の領域からなり、4312nt〜5020ntの領域を含むDNA。
(b) (a)と９０％以上の同一性を有する塩基配列からなり、かつ、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有するDNA。
The promoter of the present invention is a vascular endothelium-specific promoter having promoter activity that causes a vascular endothelial cell to specifically express a gene, and consists of any of the following DNAs.
(a) A DNA comprising a region of 709 bases or more in the base sequence shown in SEQ ID NO: 1 and comprising a region of 4312nt to 5020nt.
(b) A DNA comprising a nucleotide sequence having 90% or more identity with (a) and having a promoter activity that allows a vascular endothelial cell to specifically express a gene.
(a)のDNAは、配列番号１に示す塩基配列中の連続する領域（部分領域又は全長領域）からなるDNAである。配列番号１に示す塩基配列は、BAZFの転写開始点より上流5530塩基の領域（-5529〜+1）であり、このうちの4312nt〜5020ntの709塩基の領域（-1218〜-510）がプロモーター活性に重要な領域である（図１）。(a)のDNAは、この4312nt〜5020ntの領域を少なくとも含む。 The DNA of (a) is a DNA consisting of a continuous region (partial region or full-length region) in the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 1 is a 5530 base region upstream from the transcription start point of BAZF (-5529 to +1), and a 709 base region from 4312 nt to 5020 nt (-1218 to -510) is the promoter. This is an important region for activity (FIG. 1). The DNA of (a) contains at least this 4312 nt to 5020 nt region.
(a)のDNAには、配列番号１に示す塩基配列中の連続する1094塩基以上の領域から成り、3927nt〜5020nt（-1603〜-510）の領域を含むDNAや、4312nt〜5530nt（-1218〜+1）の1219塩基の領域を含むDNAが包含される。具体例を挙げると、4312nt〜5530nt（-1218〜+1）からなるDNA、3927nt〜5530nt（-1603〜+1）からなるDNA等が挙げられるが、これらに限定されない。 The DNA of (a) is a DNA comprising a region of 1094 bases or more in the base sequence shown in SEQ ID NO: 1 and comprising a region of 3927 nt to 5020 nt (-1603 to -510), or 4312 nt to 5530 nt (-1218 DNA including the region of 1219 bases of ˜ + 1) is included. Specific examples include, but are not limited to, DNA consisting of 4312nt to 5530nt (-1218 to +1), DNA consisting of 3927nt to 5530nt (-1603 to +1), and the like.
(b)のDNAは、(a)のDNAにおいて、少数の塩基が置換、欠失及び／又は挿入された塩基配列からなり、かつ、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有するDNAである。(a)のDNAとの配列の同一性が高いDNAは、(a)のDNAと同様に血管内皮細胞特異的なプロモーター活性を発揮し得る。(b)のDNAの(a)との同一性は９０％以上、好ましくは９５％以上、より好ましくは９８％以上である。(b)のDNAの好ましい例としては、さらに、(a)のDNAにおいて１個又は数個の塩基が置換、欠失及び／又は挿入された塩基配列からなるDNAを挙げることができる。 The DNA of (b) consists of a base sequence in which a small number of bases are substituted, deleted, and / or inserted in the DNA of (a), and has a promoter activity that causes vascular endothelial cells to specifically express the gene. DNA. A DNA having a high sequence identity with the DNA of (a) can exhibit a vascular endothelial cell-specific promoter activity, like the DNA of (a). The identity of (b) DNA with (a) is 90% or more, preferably 95% or more, more preferably 98% or more. Preferable examples of the DNA of (b) further include DNA having a base sequence in which one or several bases are substituted, deleted and / or inserted in the DNA of (a).
ここで、塩基配列の「同一性」とは、比較すべき２つの塩基配列の塩基ができるだけ多く一致するように両配列を整列させ、一致した塩基数を、全塩基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する２つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化は、例えばBLAST、FASTA、CLUSTAL W等の周知のプログラムを用いて行なうことができる。ギャップが挿入される場合、上記全塩基数は、１つのギャップを１つの塩基として数えた塩基数となる。このようにして数えた全塩基数が、比較する２つの配列間で異なる場合には、同一性（％）は、長い方の配列の全塩基数で、一致した塩基数を除して算出される。 Here, the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of bases is the number of bases obtained by counting one gap as one base. When the total number of bases counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases. The
ただし、比較すべき配列が他の任意の配列と連結された状態にある場合には（例えば、発現ベクターに組み込まれた状態、発現させたい所望の遺伝子と連結させた状態など）、プロモーターに相当する領域のみを取り出して配列を対比し、同一性を算出する。 However, if the sequence to be compared is in a state linked to any other sequence (for example, a state incorporated in an expression vector, a state linked to a desired gene to be expressed, etc.), it corresponds to a promoter. Only the region to be extracted is taken out and the sequences are compared to calculate the identity.
プロモーターは、発現させたいRNAをコードするDNAと連結して用いられるものであり、また多くの場合発現ベクターに組み込まれた状態で市販されるため、通常、他の配列が付加された形態にある。本発明のプロモーターの一端又は両端に任意の配列が付加されたDNAは、本発明のプロモーターを利用したものといえるため、本発明の範囲に包含される。任意の他の配列としては、特に限定されないが、例えば、ベクター由来の配列、制限酵素認識配列、スペーサー配列、マーカー遺伝子配列、血管内皮細胞で特異的に発現させたい所望のRNAをコードする塩基配列等が挙げられる。 Promoters are used by linking to the DNA encoding the RNA to be expressed, and are often marketed in the state of being incorporated into an expression vector, and are usually in a form with other sequences added. . A DNA having an arbitrary sequence added to one or both ends of the promoter of the present invention is included in the scope of the present invention because it can be said to use the promoter of the present invention. Any other sequence is not particularly limited. For example, a sequence derived from a vector, a restriction enzyme recognition sequence, a spacer sequence, a marker gene sequence, a base sequence encoding a desired RNA to be specifically expressed in vascular endothelial cells Etc.
血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性とは、所望の遺伝子をプロモーターと機能的に連結させた形態で血管内皮細胞（例えばHUVEC等の血管内皮由来培養細胞）に導入した場合に、該血管内皮細胞内で所望の遺伝子のmRNAを転写させる活性をいう。「機能的に連結」とは、該プロモーターの支配を受けるようにその下流に遺伝子配列を連結することをいう。任意の塩基配列からなるDNAがそのようなプロモーター活性を有しているかどうかは、ルシフェラーゼ遺伝子等を利用した周知のレポーターアッセイにより容易に確認することができる。レポーターアッセイのためのキットや試薬類は市販されており、当業者であれば容易に実施できる。 Promoter activity that specifically expresses a gene in vascular endothelial cells means that when a desired gene is introduced into a vascular endothelial cell (for example, a cultured cell derived from vascular endothelium such as HUVEC) in a form functionally linked to the promoter, The activity to transcribe mRNA of a desired gene in the vascular endothelial cell. “Functionally linked” refers to linking a gene sequence downstream thereof so as to be controlled by the promoter. Whether or not DNA having an arbitrary base sequence has such promoter activity can be easily confirmed by a well-known reporter assay using a luciferase gene or the like. Kits and reagents for reporter assays are commercially available and can be easily implemented by those skilled in the art.
(a)のDNAは、公知の遺伝子工学的手法や市販の核酸合成機を用いた常法により容易に調製することができる。例えば、血管内皮細胞（例えばHUVEC等の血管内皮由来培養細胞）から抽出したゲノムDNAを鋳型としたPCRにより増幅して得ることができる。PCRに用いるプライマーは、配列番号１に示した塩基配列に基づいて適宜設計できる。 The DNA of (a) can be easily prepared by a known genetic engineering technique or a conventional method using a commercially available nucleic acid synthesizer. For example, it can be obtained by amplification by PCR using genomic DNA extracted from vascular endothelial cells (for example, cultured cells derived from vascular endothelium such as HUVEC) as a template. Primers used for PCR can be appropriately designed based on the base sequence shown in SEQ ID NO: 1.
(b)のDNAは、市販の核酸合成機を用いるほか、BAZFプロモーター領域に天然の変異を有する血管内皮細胞から抽出したゲノムDNAをPCRの鋳型として用いることで得ることができる。あるいは、(a)のDNAに常法により適宜変異を導入することで合成することもできる。 In addition to using a commercially available nucleic acid synthesizer, the DNA of (b) can be obtained by using genomic DNA extracted from vascular endothelial cells having a natural mutation in the BAZF promoter region as a PCR template. Alternatively, it can also be synthesized by appropriately introducing mutations into the DNA of (a) by a conventional method.
本発明のプロモーターは、マルチクローニングサイト等を適宜具備する発現ベクターに組み込まれた形態で提供できる。ベクターはプラスミドでもウイルスでもよい。各種のプラスミド発現ベクター及びウイルス発現ベクターが公知であり、使用対象となる宿主細胞の種類ないしは動物種に応じて適宜選択することができる。 The promoter of the present invention can be provided in the form of being incorporated into an expression vector having a multicloning site or the like as appropriate. The vector may be a plasmid or a virus. Various plasmid expression vectors and virus expression vectors are known, and can be appropriately selected according to the type of host cell or animal species to be used.
本発明のプロモーターを含む発現ベクターは、さらに、該プロモーターに機能的に連結された、所望のRNAをコードするDNAを含み得る。所望のRNAをコードするDNAを、本発明のプロモーターに機能的に連結するようにして発現ベクターに組み込み、これを非ヒト動物細胞に導入してトランスジェニック動物を作出すれば、血管内皮特異的に所望のRNAを産生するトランスジェニック動物が得られる。こうしたトランスジェニック動物は、基礎研究のツールとして有用である。また、本発明のプロモーターを含む発現ベクターは、後述する通り、医薬としても有用である。 The expression vector containing the promoter of the present invention may further contain DNA encoding the desired RNA operably linked to the promoter. When a DNA encoding a desired RNA is incorporated into an expression vector so as to be operably linked to the promoter of the present invention and introduced into a non-human animal cell to produce a transgenic animal, the vascular endothelium is specifically identified. Transgenic animals that produce the desired RNA are obtained. Such transgenic animals are useful as basic research tools. Moreover, the expression vector containing the promoter of the present invention is also useful as a medicament as described later.
本発明のプロモーターに機能的に連結させる所望のRNAをコードするDNAは、例えば、タンパク質をコードする遺伝子のcDNAのほか、アンチセンス核酸、shRNA、miRNA等の遺伝子発現を調節するRNAをコードするDNAであり得る。タンパク質をコードする遺伝子は特に限定されず、内在性遺伝子でも外来遺伝子でもよい。 The DNA encoding the desired RNA that is operably linked to the promoter of the present invention includes, for example, a cDNA encoding a gene encoding a protein and an RNA that regulates gene expression such as an antisense nucleic acid, shRNA, miRNA, etc. It can be. The gene encoding the protein is not particularly limited, and may be an endogenous gene or a foreign gene.
本発明の基礎研究への応用の具体例としては、血管内皮細胞特異的に特定の遺伝子を発現させ、又は特定の遺伝子の発現をノックアウト若しくはノックダウンさせたトランスジェニック動物が挙げられる。 Specific examples of the application of the present invention to basic research include transgenic animals in which a specific gene is expressed specifically in vascular endothelial cells, or expression of a specific gene is knocked out or knocked down.
例えば、GFP等の蛍光タンパク質を本発明のプロモーターの制御下で動物（ヒトを除く）に導入すれば、血管内皮細胞を可視化した動物モデルを作出できる。この動物モデルに薬剤を投与し、血管に対する該薬剤の影響を調べることができる。 For example, if a fluorescent protein such as GFP is introduced into an animal (excluding humans) under the control of the promoter of the present invention, an animal model in which vascular endothelial cells are visualized can be created. A drug can be administered to this animal model, and the influence of the drug on blood vessels can be examined.
動物において特定の遺伝子をノックダウン又はノックアウトする手法も確立している。遺伝子ノックダウンは、アンチセンス法のほか、shRNAやmiRNAを用いたRNAiにより行なうことができる。目的の遺伝子に対するアンチセンスRNA、shRNA又はmiRNAを産生できるDNAを本発明のプロモーターに機能的に連結し、これを動物に導入すれば、血管内皮において特異的に目的遺伝子の発現が低減したノックダウン動物を得ることができる。遺伝子ノックアウトは、近年ではCre/LoxPシステムが主に使用されている。欠損させたい遺伝子がゲノム中でLoxPに挟み込まれた状態にあるトランスジェニック動物を作出し、この動物内でCreリコンビナーゼを発現させると、LoxPに挟まれたゲノム領域が切り出される。別途、本発明のプロモーター制御下でCreを発現するトランスジェニック動物を作出しておき、前述のLoxPトランスジェニック動物と交配させれば、目的遺伝子の発現が血管内皮において特異的にノックアウトされたトランスジェニック動物を得ることができる。こうしたノックアウト、ノックダウン動物は、血管内皮における遺伝子の機能解析に有用である。 Methods have also been established for knocking down or knocking out specific genes in animals. In addition to the antisense method, gene knockdown can be performed by RNAi using shRNA or miRNA. When DNA that can produce antisense RNA, shRNA, or miRNA for the target gene is operably linked to the promoter of the present invention and introduced into an animal, knockdown in which expression of the target gene is specifically reduced in the vascular endothelium Animals can be obtained. In recent years, Cre / LoxP system is mainly used for gene knockout. When a transgenic animal is created in which the gene to be deleted is sandwiched between LoxPs in the genome and Cre recombinase is expressed in the animal, the genomic region sandwiched between LoxPs is excised. Separately, by creating a transgenic animal that expresses Cre under the control of the promoter of the present invention and mating with the aforementioned LoxP transgenic animal, the transgenic gene whose expression of the target gene is specifically knocked out in the vascular endothelium Animals can be obtained. Such knockout and knockdown animals are useful for functional analysis of genes in vascular endothelium.
また、本発明のプロモーターを含む発現ベクターは、医薬としても有用であり得る。例えば、血管内皮において特定の遺伝子産物が減少ないしは欠失することが原因で生じる疾患の場合、その遺伝子を本発明のプロモーター下流に連結した発現ベクターが治療薬として有用であり得る。このような疾患の具体例としては、例えば、血友病、第II、第V、第VII、第X因子欠乏症等を挙げることができる。また、これとは逆に、血管内皮において特定の遺伝子が過剰発現していることが原因で生じる疾患の場合、該遺伝子に対するアンチセンスRNA、shRNA、miRNA等の、対象遺伝子の発現量を低減できるRNAをコードするDNAを本発明のプロモーター下流に連結した発現ベクターが治療薬として有用であり得る。こうした疾患の具体例としては、例えば、癌、加齢黄斑変性、糖尿病性網膜症等をあげることができる。核酸医薬自体はこの分野で周知であり、そのためのプラスミドベクターやウイルスベクターが各種研究開発されている。 Moreover, the expression vector containing the promoter of the present invention may be useful as a pharmaceutical. For example, in the case of a disease caused by a decrease or deletion of a specific gene product in the vascular endothelium, an expression vector in which that gene is linked downstream of the promoter of the present invention may be useful as a therapeutic agent. Specific examples of such diseases include hemophilia, II, V, VII, and X deficiency. In contrast, in the case of a disease caused by the overexpression of a specific gene in the vascular endothelium, the expression level of the target gene such as antisense RNA, shRNA, miRNA, etc. can be reduced. An expression vector in which a DNA encoding RNA is linked downstream of the promoter of the present invention may be useful as a therapeutic agent. Specific examples of such diseases include cancer, age-related macular degeneration, diabetic retinopathy and the like. Nucleic acid drugs themselves are well known in the art, and various plasmid vectors and virus vectors have been researched and developed for this purpose.
本発明のプロモーターを含む発現ベクターは、適宜他の成分と組み合わせて、血管内皮細胞特異的に所望のRNAを産生させるための組成物（医薬組成物、研究用試薬組成物など）として提供することができる。あるいは、該発現ベクターは、適宜他の試薬類、ポジティブコントロールベクター等と組み合わせて、血管内皮細胞特異的に所望のRNAを産生させるためのキットとして提供することができる。 The expression vector containing the promoter of the present invention is provided as a composition (pharmaceutical composition, research reagent composition, etc.) for producing desired RNA specifically for vascular endothelial cells in combination with other components as appropriate. Can do. Alternatively, the expression vector can be provided as a kit for producing desired RNA specifically for vascular endothelial cells by appropriately combining with other reagents, positive control vectors and the like.
本発明のプロモーターは、血管内皮細胞への特異性が極めて高く、上皮系細胞（例えばHEK293T, PC-9, Hela, HT-29）及び間葉系細胞（例えばFibroblast, HT1080, Huh-7, HepG2）内では実質的にプロモーター活性を発揮しない。下記実施例では、(a)の一例である、配列番号１の4312nt〜5530nt（-1218〜+1）からなるプロモーターを用いて、上皮系細胞及び間葉系細胞ではほとんど全く転写活性を示さず、血管内皮細胞に極めて特異的に転写活性を示すことが確認されている。また、本発明のプロモーターは、特異性のみならず、転写活性にも優れている。例えば、公知の強発現用プロモーターであるCMVプロモーターと比較しても、転写活性が数倍以上高い。このように、本発明のプロモーターによれば、所望のRNAを血管内皮細胞で特異的に高生産させることができるので、臨床、基礎研究の双方に非常に有用である。 The promoter of the present invention has very high specificity for vascular endothelial cells, and epithelial cells (for example, HEK293T, PC-9, Hela, HT-29) and mesenchymal cells (for example, Fibroblast, HT1080, Huh-7, HepG2). ) Does not substantially exhibit promoter activity. In the following examples, using a promoter consisting of 4312nt to 5530nt (-1218 to +1) of SEQ ID NO: 1, which is an example of (a), almost no transcriptional activity is shown in epithelial cells and mesenchymal cells. It has been confirmed that vascular endothelial cells exhibit transcription activity very specifically. The promoter of the present invention is excellent not only in specificity but also in transcription activity. For example, the transcriptional activity is several times higher than the CMV promoter, which is a known strong expression promoter. As described above, according to the promoter of the present invention, a desired RNA can be specifically and highly produced in vascular endothelial cells, so that it is very useful for both clinical and basic research.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
血管内皮特異的プロモーター候補のスクリーニング
通常の方法で培養したヒト臍帯静脈内皮細胞(HUVEC)と、内皮細胞増殖因子(VEGF-A)で刺激したHUVECから、常法によりRNAを抽出してcDNAを合成した。これらcDNAをオリジナルのジーンチップを用いて解析し、VEGF-A刺激にて発現誘導される遺伝子を選択した。ここで得られた候補遺伝子について定量PCR法で確認した結果、最も強く誘導される遺伝子がBAZF(B-cell lymphoma 6 associated zinc finger protein)であった。またデータベース（東京大学SBMシステム生物医学データベースRefExA http://157.82.78.238/refexa/main_search.jsp）にてその発現特性を確認したところ、血管内皮細胞特異的な発現をする分子であることがわかった。
Screening for candidate vascular endothelium-specific promoters Extracting RNA from conventional human umbilical vein endothelial cells (HUVEC) and HUVECs stimulated with endothelial growth factor (VEGF-A) to synthesize cDNA by conventional methods did. These cDNAs were analyzed using the original gene chip, and the genes whose expression was induced by VEGF-A stimulation were selected. As a result of confirming the candidate gene obtained here by quantitative PCR, the most strongly induced gene was BAZF (B-cell lymphoma 6 associated zinc finger protein). In addition, when its expression characteristics were confirmed in a database (University of Tokyo SBM system biomedical database RefExA http://157.82.78.238/refexa/main_search.jsp), it was found to be a molecule that specifically expresses vascular endothelial cells. It was.
BAZFのプロモーター領域の同定
ヒト臍帯静脈内皮細胞(HUVEC)よりゲノムDNAを抽出し、これを鋳型としたPCRにより、BAZFの転写開始点より上流5530塩基の領域（配列番号１、-5529〜+1）を増幅した。luciferase(蛍由来）遺伝子を含むプラスミド（pGL4.1、Promega）に増幅断片を挿入し、luciferase遺伝子の上流に上記増幅断片が挿入されたプラスミドを構築した。また上流領域の様々な長さに短縮した変異体の作成もおこなった（-2548/+1, -2033/+1, -1603/+1, -1218/+1, -510/+1)。
Identification of the BAZF promoter region Genomic DNA was extracted from human umbilical vein endothelial cells (HUVEC), and the PCR was used as a template for a region of 5530 bases upstream from the transcription start point of BAZF (SEQ ID NO: 1, -5529 to +1). ) Was amplified. The amplified fragment was inserted into a plasmid (pGL4.1, Promega) containing a luciferase (firefly-derived) gene, and a plasmid was constructed in which the amplified fragment was inserted upstream of the luciferase gene. In addition, mutants were shortened to various upstream lengths (-2548 / + 1, -2033 / + 1, -1603 / + 1, -1218 / + 1, -510 / + 1).
構築したプラスミドをHUVECに遺伝子導入し、その転写活性をLuciferase活性を測定することにより解析した。Luciferase活性の測定は、Dual-Luciferase Reporter Assay System（Promega）を用いて行なった。実験の際、遺伝子導入効率や細胞数のぶれを補正するために、チミジンキナーゼプロモーターの下流にluciferase(ウミシイタケ由来）を挿入したプラスミド（pRL-TK、Promega）を解析対象プラスミドと共に遺伝子導入し、そのluciferase活性を測定して解析対象プラスミドの測定値を補正した。 The constructed plasmid was introduced into HUVEC and its transcriptional activity was analyzed by measuring Luciferase activity. The measurement of Luciferase activity was performed using Dual-Luciferase Reporter Assay System (Promega). During the experiment, a plasmid (pRL-TK, Promega) in which luciferase (derived from Renilla) was inserted downstream of the thymidine kinase promoter was introduced together with the analysis target plasmid in order to correct for gene transfer efficiency and cell number fluctuation. The measured value of the analysis target plasmid was corrected by measuring the luciferase activity.
結果を図１に示す。-1218/+1では転写活性が存在するが-510/+1では転写活性がないことから、BAZFプロモーターは-1218/-510に含まれることが示された。 The results are shown in FIG. Since -1218 / + 1 has transcriptional activity but -510 / + 1 has no transcriptional activity, it was shown that the BAZF promoter is included in -1218 / -510.
BAZFプロモーターの細胞種特異性の解析
上記で構築した-1218/+1の領域を含むプラスミドを血管内皮細胞(HUVEC, CAEC, Dermal MVEC, Retinal MVEC)、上皮系細胞(HEK293T, PC-9, Hela, HT-29)、間葉系細胞(Fibroblast, HT1080, Huh-7, HepG2)に遺伝子導入し、その転写活性を測定した。
Analysis of cell type specificity of BAZF promoter Plasmids containing the -218 / + 1 region constructed above were expressed as vascular endothelial cells (HUVEC, CAEC, Dermal MVEC, Retinal MVEC), epithelial cells (HEK293T, PC-9, Hela). , HT-29) and mesenchymal cells (Fibroblast, HT1080, Huh-7, HepG2), and their transcriptional activity was measured.
結果を図２に示す。HUVEC, CAEC, Dermal MVEC, Retinal MVECにおいて転写活性が確認できたが、他の細胞では転写活性を示さなかった。以上より、BAZFプロモーターが血管内皮細胞に極めて特異的な転写活性をもつことが示された。 The results are shown in FIG. Transcriptional activity was confirmed in HUVEC, CAEC, Dermal MVEC, and Retinal MVEC, but other cells did not exhibit transcriptional activity. From the above, it was shown that the BAZF promoter has very specific transcriptional activity on vascular endothelial cells.
BAZFプロモーターの転写活性の解析
BAZFプロモーターの転写活性と、遺伝子の強発現に通常用いられるCMV由来プロモーターの転写活性を、HUVECにおいて比較した。CMVプロモーター領域は、公知のプラスミド（pcDNA3.1）からPCR増幅して調製し、上記で用いたluciferase(蛍由来）遺伝子を含むプラスミドに該増幅断片を挿入し、CMVプロモーターを含むプロモーターアッセイ用プラスミドを構築した。このCMVプロモータープラスミドと、BAZFの-1218/+1の領域を含むプラスミドを、それぞれHUVECに導入し、そのLuciferase活性を測定した。
Analysis of transcriptional activity of BAZF promoter
HUVEC compared the transcriptional activity of the BAZF promoter with the transcriptional activity of the CMV-derived promoter normally used for strong gene expression. The CMV promoter region is prepared by PCR amplification from a known plasmid (pcDNA3.1), the amplified fragment is inserted into the plasmid containing the luciferase (firefly-derived) gene used above, and a plasmid for promoter assay containing the CMV promoter Built. This CMV promoter plasmid and a plasmid containing the BAZF-1218 / + 1 region were each introduced into HUVEC, and their Luciferase activity was measured.
結果を図３に示す。BAZFプロモーターはCMVプロモーターと比較して強い転写活性を有することが示された。 The results are shown in FIG. The BAZF promoter was shown to have a strong transcriptional activity compared to the CMV promoter.
Claims (14)
(a) 配列番号１に示す塩基配列中の連続する709塩基以上の領域からなり、4312nt〜5020ntの領域を含むDNA。
(b) (a)と９０％以上の同一性を有する塩基配列からなり、かつ、血管内皮細胞に特異的に遺伝子を発現させるプロモーター活性を有するDNA。 A vascular endothelial cell-specific promoter consisting of any of the following DNAs and having a promoter activity that causes a vascular endothelial cell to specifically express a gene.
(a) A DNA comprising a region of 709 bases or more in the base sequence shown in SEQ ID NO: 1 and comprising a region of 4312nt to 5020nt.
(b) A DNA comprising a nucleotide sequence having 90% or more identity with (a) and having a promoter activity that allows a vascular endothelial cell to specifically express a gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011250591A JP2013102747A (en) | 2011-11-16 | 2011-11-16 | Vascular endothelial cell-specific promotor |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001504708A (en) * | 1996-12-03 | 2001-04-10 | コミサリア・ア・レネルジー・アトミーク−シー・イー・エー | VE cadherin promoter and its use |
| JP2001519145A (en) * | 1997-10-02 | 2001-10-23 | イムクローン システムズ インコーポレーテッド | Transgenic animal having knock-in VEC receptor gene and use thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001504708A (en) * | 1996-12-03 | 2001-04-10 | コミサリア・ア・レネルジー・アトミーク−シー・イー・エー | VE cadherin promoter and its use |
| JP2001519145A (en) * | 1997-10-02 | 2001-10-23 | イムクローン システムズ インコーポレーテッド | Transgenic animal having knock-in VEC receptor gene and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| JPN6015047482; ＢＭＣ Ｇｅｎｏｍｉｃｓ，２０１０ Ｍａｙ ２８，１１，３４２ * |
| JPN6015047483; Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ．，２００２ Ｍａｒ １，２９１（３），ｐ．５６７-７３ * |
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