JP2015506696A - ShRNA that suppresses TGF-β2 expression - Google Patents
ShRNA that suppresses TGF-β2 expression Download PDFInfo
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- JP2015506696A JP2015506696A JP2014555484A JP2014555484A JP2015506696A JP 2015506696 A JP2015506696 A JP 2015506696A JP 2014555484 A JP2014555484 A JP 2014555484A JP 2014555484 A JP2014555484 A JP 2014555484A JP 2015506696 A JP2015506696 A JP 2015506696A
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Abstract
本発明は、ＴＧＦ−β２発現を抑制するｓｈＲＮＡに関する。本発明によれば、ＴＧＦ−β２発現を抑制するｓｈＲＮＡを用いた抗腫瘍組成物を提供することができる。The present invention relates to a shRNA that suppresses TGF-β2 expression. According to the present invention, an antitumor composition using shRNA that suppresses TGF-β2 expression can be provided.
Description
本発明は、ＴＧＦ−β２発現を抑制するｓｈＲＮＡ及びこれを含む抗腫瘍組成物に関する。 The present invention relates to an shRNA that suppresses TGF-β2 expression and an antitumor composition comprising the same.
ＴＧＦ−β２は、ＴＧＦ−β１と同様に、細胞毒性Ｔ細胞、ナチュラルキラー細胞、そしてマクロファージなどの増殖と分化を抑制してますます大きくなる腫瘍に対する免疫応答を阻害するだけでなく、多機能の分泌タンパク質で細胞の形態（ｔｙｐｅ）と時期によって増殖抑制、複製、浸潤、転移、細胞死滅、免疫応答、そして血管生成など多様な役割を行うだけでなく、ＴＧＦ−β１のように、ＴＧＦ−β２も信号経路を非活性化させるか、あるいは細胞周期の非正常的な調節などに起因して、ＴＧＦ−β２による増殖抑制作用に抵抗性を生じる腫瘍が進行（ｐｒｏｇｒｅｓｓｉｏｎ）する後期に至ると、ＴＧＦ−β２は腫瘍をさらに発展させる役割をすることになる。よって、人体の免疫体系を克服して増殖した腫瘍細胞はＴＧＦ−β２を分泌することで、免疫監視から自由であり同時に増殖と浸潤転移及び血管生成にプラス要因として作用することになる。ＴＧＦ−β２がＴＧＦ−β１とは明白に異なった作用をする点は以下である。ＴＧＦ−β２がＦｏｘｐ３を誘導して免疫抑制誘導を著しく進行させるとのことと、腫瘍の転移、新生血管形成、そして増殖などにも影響を及ぼして悪性としての腫瘍進行を誘導することである。 Similar to TGF-β1, TGF-β2 not only inhibits the immune response to increasingly large tumors by suppressing the proliferation and differentiation of cytotoxic T cells, natural killer cells, macrophages, etc. It is a secreted protein that not only plays various roles such as growth inhibition, replication, invasion, metastasis, cell death, immune response, and angiogenesis depending on the cell type and timing, but also TGF-β2 like TGF-β1. When TGF-β2 progresses to the later stage of progression, the TGF-β2 is resistant to growth inhibition due to inactivation of the signal pathway or abnormal regulation of the cell cycle. -Β2 will play a role in further developing the tumor. Thus, tumor cells that have proliferated over the human immune system secrete TGF-β2, thereby being free from immune surveillance and at the same time acting as a positive factor on proliferation, invasion metastasis, and blood vessel formation. The point that TGF-β2 acts clearly different from TGF-β1 is as follows. TGF-β2 induces Foxp3 to significantly promote immunosuppression, and also affects tumor metastasis, neovascularization, proliferation, etc., and induces tumor progression as malignant.
ＴＧＦ−β２に係る先行研究である、非特許文献１には、人間ＴＧＦ−β２のコーディング配列に対する合成１８−ｍｅｒホスホチオエートアンチセンスオリゴヌクレオチド（ｐｈｏｓｐｈｏｔｈｉｏａｔｅ ａｎｔｉｓｅｎｓｅ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ）を用い、腫瘍の免疫抑制除去、腫瘍の大きさ減少、リンパ節への転移及び血管形成減少などが観察されたが、その効果は不十分であった。 Non-patent document 1, which is a prior study on TGF-β2, uses a synthetic 18-mer phosphothioate antisense oligonucleotide against the coding sequence of human TGF-β2 to remove tumor immunosuppression, Although tumor size reduction, lymph node metastasis, and angiogenesis reduction were observed, the effects were insufficient.
非特許文献２には、ｍｕｒｉｎｅのＴＧＦ−β２に対するｓｈＲＮＡを、ＴＧＣＴＧＴＴＧＡＣＡＧＴＧＡＧＣＧＣＧＧＴＧＴＡＴＡＡＡＴＣＧＡＧＡＣＣＡＡＡＴＴＡＧＴＧＴＧＡＡＧＣＣＡＣＡＧＡＴＧＴＡＴＴＴＧＧＴＣＴＣＧＡＴＴＴＡＴＡＣＡＣＣＴＴＧＣＣＣＣＴＡＣＴＧＣＣＴＣＧＧＡ（ｔａｒｇｅｔ）で製作し、さらにＴＧＦ−β２ ｓｈＲＮＡを生成するレンチウイルス（ｌｅｎｔｉｖｉｒｕｓ）を製作したが、レンチウイルスが染色体にインテグレーション（ｉｎｔｅｇｒａｔｉｏｎ）して癌がなくなった後にも正常細胞には引き続き伝達される副作用が大きな問題となった。 Non-patent document 2 describes murine TGF-β2 shRNA, TGCTGTTGAGAGTGAGCGCGGTGTATAAATCGAGACCAAATTTAGGTGTGAAGCCACGAGTGTATTTGGTCCTCGATTTATACACCTTGCCCCTACTGTCTCTGGA ) Side effects that continue to be transmitted to normal cells after cancer disappeared.
そこで、本発明者は、上記のような問題点を解決するために研究努力した結果、人間ＴＧＦ−β２またはマウスＴＧＦ−β２の沈黙（ｓｉｌｅｎｃｉｎｇ）を効果的に誘導するターゲットを選定してｓｈＲＮＡを製作し、これをアデノウイルスに搭載させて既存の非ウイルス性製剤によるｓｈＲＮＡの伝達能力を画期的に改善させることで、本発明が完成された。 Therefore, as a result of research efforts to solve the above problems, the present inventor selected a target that effectively induces silencing of human TGF-β2 or mouse TGF-β2 and selected shRNA. The present invention was completed by producing and mounting it on an adenovirus to dramatically improve the shRNA transmission ability of existing non-viral preparations.
したがって、本発明は、配列番号１または２と表される塩基配列を標的配列とし、ＴＧＦ−β２発現を抑制するｓｈＲＮＡを提供することにその目的がある。 Therefore, an object of the present invention is to provide an shRNA that suppresses TGF-β2 expression using the base sequence represented by SEQ ID NO: 1 or 2 as a target sequence.
本発明は、また、前記ｓｈＲＮＡを有効成分として含む抗腫瘍組成物を提供することに他の目的がある。 Another object of the present invention is to provide an antitumor composition comprising the shRNA as an active ingredient.
本発明は、また、前記ｓｈＲＮＡ発現する組換え発現ベクターを提供することにさらに他の目的がある。 Another object of the present invention is to provide a recombinant expression vector for expressing the shRNA.
本発明は、また、前記組換え発現ベクターを有効成分とする抗腫瘍組成物を提供することにさらに他の目的がある。 Another object of the present invention is to provide an antitumor composition comprising the recombinant expression vector as an active ingredient.
本発明は、また、前記組換え発現ベクターが導入されたアデノウイルスを提供することにさらに他の目的がある。 Another object of the present invention is to provide an adenovirus into which the recombinant expression vector has been introduced.
本発明は、前記課題を解決するための手段として、配列番号１または２に表される塩基配列を標的配列とし、ＴＧＦ−β２発現を抑制するｓｈＲＮＡを提供する。 The present invention provides a shRNA that suppresses the expression of TGF-β2 using the base sequence represented by SEQ ID NO: 1 or 2 as a target sequence as means for solving the above-mentioned problems.
本発明は、前記課題を解決するための他の手段として、前記ｓｈＲＮＡを有効成分で含む抗腫瘍組成物を提供する。 The present invention provides an antitumor composition comprising the shRNA as an active ingredient as another means for solving the above-mentioned problems.
本発明は、前記課題を解決するためのさらに他の手段として、前記ｓｈＲＮＡ発現用の組換え発現ベクターを提供する。 The present invention provides the recombinant expression vector for shRNA expression as still another means for solving the above-mentioned problems.
本発明は、前記課題を解決するためのさらに他の手段として、前記組換え発現ベクターを有効成分として含む抗腫瘍組成物を提供する。 The present invention provides an antitumor composition comprising the recombinant expression vector as an active ingredient as yet another means for solving the above-mentioned problems.
本発明は、前記課題を解決するためのさらに他の手段として、前記組換え発現ベクターが導入されたアデノウイルスを提供する。 The present invention provides an adenovirus into which the recombinant expression vector has been introduced, as yet another means for solving the above problems.
本発明は、ＴＧＦ−β２発現を抑制する新たなｓｈＲＮＡを製作し、遺伝子伝達体としてアデノウイルスを使用して感染率を増加させることで、従来技術と比較して特異性、伝達能及び発現抑制能を大きく向上させた。 The present invention produces a new shRNA that suppresses TGF-β2 expression and uses adenovirus as a gene carrier to increase the infection rate, thereby reducing specificity, transmission ability and expression suppression compared to the prior art. Greatly improved performance.
すなわち、本発明により、ＴＧＦ−β２発現を抑制するｓｈＲＮＡを含む抗腫瘍組成物が提供される。特に、大部分の癌細胞において伝達効率性が優れたアデノウイルスに標的性を付与することで、すべての癌に適用可能である。 That is, according to the present invention, an antitumor composition comprising shRNA that suppresses TGF-β2 expression is provided. In particular, it can be applied to all cancers by imparting targeting to adenoviruses with excellent transmission efficiency in most cancer cells.
ＲＮＡ干渉（ＲＮＡ ｉｎｔｅｒｆｅｒｅｎｃｅ、ＲＮＡｉ）は、標的遺伝子の発現を選択的に抑制する天然のメカニズムである。配列特異的なｍＲＮＡ分解の媒介者は、より長いｄｓＲＮＡからリボヌクレアーゼIIIの切断により生産された１９〜２３ヌクレオチドの小さな干渉ＲＮＡである。細胞質のＲＩＳＣ（ＲＮＡ−ｉｎｄｕｃｅｄ ｓｉｌｅｎｃｉｎｇ ｃｏｍｐｌｅｘ）はｓｉＲＮＡに結合し、そのｓｉＲＮＡのうち、１本鎖に相補的な配列を含むｍＲＮＡの分解を指示する。哺乳動物においてＲＮＡ干渉の適用は、治療遺伝子沈黙（ｓｉｌｅｎｃｉｎｇ）の効能を有する。ｓｉＲＮＡの長所を有しながらも、ｓｉＲＮＡは試験管内で製造しなければならなく、ノックダウン遺伝子を、通常的に６〜１０日間一時的形質感染によって伝達されるべきであるとのことから臨床適用に制限を有する。本発明に係るｓｈＲＮＡ（ｓｍａｌｌ−ｈａｉｒｐｉｎ ＲＮＡ）発現システムが上記のような短所を解決することができる。 RNA interference (RNAi) is a natural mechanism that selectively suppresses the expression of target genes. A mediator of sequence-specific mRNA degradation is a small interfering RNA of 19-23 nucleotides produced by cleavage of ribonuclease III from a longer dsRNA. Cytoplasmic RISC (RNA-induced silencing complex) binds to siRNA and directs degradation of mRNA containing a sequence complementary to a single strand of the siRNA. Application of RNA interference in mammals has the effect of therapeutic gene silencing. While having the advantages of siRNA, siRNA must be produced in vitro and the knockdown gene should normally be transmitted by transient trait infection for 6-10 days for clinical application Have a limit. The shRNA (small-hairpin RNA) expression system according to the present invention can solve the above disadvantages.
ｓｈＲＮＡは、１本鎖のＲＮＡにおいて部分的に回文状の塩基配列を含むことで、分子内で２本鎖の構造を有し、ヘアピンのような構造となる約２０塩基以上の分子である。 shRNA is a molecule of about 20 bases or more that has a double-stranded structure in the molecule and has a hairpin-like structure by including a partially palindromic base sequence in single-stranded RNA. .
本発明は、ＴＧＦ−β２発現を抑制するｓｈＲＮＡに関し、下記配列を標的配列とすることを特徴とする。 The present invention relates to a shRNA that suppresses TGF-β2 expression, and is characterized in that the following sequence is a target sequence.
マウス標的配列：５’−ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ−３’［配列番号１］ Mouse target sequence: 5'-GGATTGAACTGTATCAGATCCTTAA-3 '[SEQ ID NO: 1]
人間標的配列：５’−ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ−３’［配列番号２］ Human target sequence: 5'-GGATTGAGCTATATCAGATTCCAA-3 '[SEQ ID NO: 2]
本発明において、ＴＧＦ−β２発現を抑制するｓｈＲＮＡは、ＴＧＦ−β２遺伝子の一部に相補的な配列を有し、ＴＧＦ−β２遺伝子のｍＲＮＡを分解したり、翻訳を抑制したりすることができる。相補性が８０〜９０％の場合には、ｍＲＮＡの翻訳を抑制することができ、１００％の場合には、ｍＲＮＡを分解させることができる。 In the present invention, the shRNA that suppresses TGF-β2 expression has a sequence complementary to part of the TGF-β2 gene, and can degrade the mRNA of the TGF-β2 gene or suppress translation. . When complementarity is 80 to 90%, translation of mRNA can be suppressed, and when it is 100%, mRNA can be degraded.
したがって、本発明においてＴＧＦ−β２発現を抑制するｓｈＲＮＡは、マウスｍＲＮＡの４９４〜５１８番目のヌクレオチドに、人間ｍＲＮＡの５７８〜６０２番目のヌクレオチドに対する相補的な配列に対して８０％以上、好ましくは９０％以上、より好ましくは１００％相同性を有する塩基配列を含むことができる。 Therefore, in the present invention, the shRNA that suppresses TGF-β2 expression is 80% or more, preferably 90% of the sequence complementary to the nucleotides 494 to 518 of mouse mRNA and the nucleotides 578 to 602 of human mRNA. % Or more, more preferably 100% homologous base sequence can be included.
一態様として、マウスのｓｈＲＮＡは、配列番号１（標的配列）に示された塩基配列とその相補的な塩基配列からなっていて、人間のｓｈＲＮＡは配列番号２（標的配列）に示された塩基配列とその相補的な塩基配列からなることができる。前記のそれぞれの塩基配列とその相補的な塩基配列は、４〜１０ｂｐのループ領域により回文的に（ｐａｌｉｎｄｒｏｍ）連結されてヘアピン構造を形成することができる。 In one embodiment, the mouse shRNA is composed of the base sequence shown in SEQ ID NO: 1 (target sequence) and its complementary base sequence, and the human shRNA is the base shown in SEQ ID NO: 2 (target sequence). It can consist of a sequence and its complementary base sequence. Each of the aforementioned base sequences and its complementary base sequence can be palindromically linked by a 4 to 10 bp loop region to form a hairpin structure.
本発明のｓｈＲＮＡの具体的な例としては、下記配列を含むことができる： Specific examples of shRNAs of the invention can include the following sequences:
配列番号１のマウス標的配列とするｓｈＲＮＡ：５’−ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｔｃｔｃ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ−３’［配列番号３］ ShRNA to be used as the mouse target sequence of SEQ ID NO: 1: 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCATCC-3 '[SEQ ID NO: 3]
配列番号２の人間標的配列のためのｓｈＲＮＡ：５’−ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｔｃｔｃ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ−３’[配列番号４］。 ShRNA for the human target sequence of SEQ ID NO: 2: 5'-GGATTGAGCTCATATCAGATTTCCAA tctc TTGAGAATCTGATATAGCTCAATCC-3 '[SEQ ID NO: 4].
ＲＮＡｉによりＴＧＦ−β２の発現を抑制する物質としては、３’末端に突出部を有する短いヘアピン構造で構成されたｓｈＲＮＡ（ｓｈｏｒｔ ｈａｉｒｐｉｎ ＲＮＡ）を使用することができる。 As a substance that suppresses the expression of TGF-β2 by RNAi, shRNA (short hairpin RNA) composed of a short hairpin structure having a protruding portion at the 3 ′ end can be used.
ＲＮＡｉによりＴＧＦ−β２の発現を抑制する物質は、人工的に化学合成してもよく、センス鎖及びアンチセンス鎖のＤＮＡ配列を逆方向に連結したヘアピン構造のＤＮＡをＴ７ＲＮＡポリメラーゼにより実験室条件（ｉｎ ｖｉｔｒｏ）でＲＮＡを合成して製作してもよい。実験室条件で合成する場合、Ｔ７ＲＮＡポリメラーゼ及びＴ７プロモーターを用いて、鋳型ＤＮＡからアンチセンス及びセンスＲＮＡを合成することができる。これらを実験室条件でアニーリングした後に、細胞に導入するとＲＮＡｉが誘発され、ＴＧＦ−β２ ｍＲＮＡの分解を誘導する。細胞への導入は、例えば、リン酸カルシウム法、または各種トランスフェクション試薬（例えば、ｏｌｉｇｏｆｅｃｔａｍｉｎｅ、 ｌｉｐｏｆｅｃｔａｍｉｎｅ及びｌｉｐｏｆｅｃｔｉｏｎなど）を用いた方法によって行われる。 A substance that suppresses the expression of TGF-β2 by RNAi may be artificially chemically synthesized. A DNA having a hairpin structure in which the DNA sequences of the sense strand and the antisense strand are linked in the reverse direction is subjected to laboratory conditions ( In vitro) RNA may be synthesized and produced. When synthesized under laboratory conditions, antisense and sense RNA can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed under laboratory conditions and then introduced into cells, RNAi is induced and induces degradation of TGF-β2 mRNA. Introduction into cells is performed, for example, by a calcium phosphate method or a method using various transfection reagents (for example, oligofectamine, lipofectamine, lipofection, etc.).
ＲＮＡｉによりＴＧＦ−β２の発現を抑制する物質としては、ｓｈＲＮＡまたは前記ＤＮＡを含む発現ベクターを用いてもよく、前記発現ベクターを含む細胞を用いてもよい。前記発現ベクターや細胞の種類は特に限定しないが、既に医薬として使用されている発現ベクターや細胞が好ましい。 As a substance that suppresses the expression of TGF-β2 by RNAi, an expression vector containing shRNA or the DNA may be used, or a cell containing the expression vector may be used. The type of the expression vector or cell is not particularly limited, but an expression vector or cell that has already been used as a pharmaceutical is preferable.
本発明では、配列番号１または２に表される塩基配列を標的配列とするｓｈＲＮＡが用いられる。 In the present invention, shRNA having the base sequence represented by SEQ ID NO: 1 or 2 as a target sequence is used.
したがって、本発明は、前記ｓｈＲＮＡ発現用の組換え発現ベクターを含む。 Accordingly, the present invention includes the recombinant expression vector for expressing shRNA.
本発明の組換えベクターは、当該分野に公知された組換えＤＮＡ方法によって構成される。 The recombinant vector of the present invention is constructed by a recombinant DNA method known in the art.
本発明においてｓｈＲＮＡを伝達するのに有用なウイルス（またはウイルスベクター）としては、アデノウイルス、レトロウイルス、レンチウイルス、アデノ随伴ウイルスなどがあり、腫瘍のように制限的な発現誘導が必要な理由として、アデノウイルスが好ましい。 Examples of viruses (or viral vectors) useful for transmitting shRNA in the present invention include adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, and the like. Adenovirus is preferred.
アデノウイルスに前記ｓｈＲＮＡを導入するために、ｓｈＲＮＡ配列に基づいて下記のＤＮＡを製作することができる。 In order to introduce the shRNA into adenovirus, the following DNA can be prepared based on the shRNA sequence.
＜マウス標的配列に対するＤＮＡ＞
トップストランド：５’−ｇａｔｃｃ ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｔｃｔｃ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ ｔｔｔｔ ａ−３’［配列番号５］
<DNA for mouse target sequence>
Top strand: 5'-gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCATCC ttt a-3 '[SEQ ID NO: 5]
ボトムストランド：５’−ａｇｃｔｔ ａａａａ ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｇａｇａ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ ｇ−３’［配列番号６］ Bottom strand: 5'-agcttt aaaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCATCC g-3 '[SEQ ID NO: 6]
＜人間標的配列に対するＤＮＡ＞
トップストランド：５’−ｇａｔｃｃ ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｔｃｔｃ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ ｔｔｔｔ ａ−３’［配列番号７］
<DNA for human target sequence>
Top strand: 5′-gatcc GGATTGAGCTCATATCAGATTTCCAA tctc TTGAGAATCTGATATAGCTCAATCC ttt a-3 ′ [SEQ ID NO: 7]
ボトムストランド：５’−ａｇｃｔｔ ａａａａ ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｇａｇａ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ ｇ−３’［配列番号８］ Bottom strand: 5'-agcttt aaaaa GGATTGAGCTCATATCAGATTTCCAA gaga TTGAGAATCTGATATAGCTCAATCC g-3 '[SEQ ID NO: 8]
また、本発明においてｓｈＲＮＡを伝達するのに有用な非ウイルスベクターとしては、上述のウイルスベクターを除いた通常に遺伝子療法に用いるすべてのベクターを意味し、そのような例としては、真核細胞で発現可能な多様なプラスミド及びリボソームなどがある。 In addition, the non-viral vector useful for transmitting shRNA in the present invention means all vectors normally used for gene therapy except the above-mentioned viral vectors. Examples of such vectors include eukaryotic cells. There are various plasmids and ribosomes that can be expressed.
一方、本発明においてＴＧＦ−β２発現を抑制するｓｈＲＮＡは、伝達された細胞に適切に転写されるために、少なくともプロモーターに作動可能に連結されることが好ましい。前記プロモーターは真核細胞で機能するプロモーターであればどれでもよいが、Ｕ６プロモーターがＲＮＡ重合酵素IIIとしてｓｍａｌｌ ｓｉｚｅ ＲＮＡを生成するのに有利な理由として特に好ましい。ＴＧＦ−β２発現を抑制するｓｈＲＮＡの効率的な転写のために必要に応じてリーダー配列、ポリアデニル化配列、プロモーター、エンハンサー（ｅｎｈａｎｃｅｒ）、アップストリーム（ｕｐｓｔｒｅａｍ）活性化配列、シグナルペプチド配列及び転写終結因子を含む調節配列をさらに含むことができる。 On the other hand, in the present invention, the shRNA that suppresses TGF-β2 expression is preferably operably linked to at least a promoter in order to be appropriately transcribed into the transferred cell. The promoter may be any promoter that functions in eukaryotic cells, but the U6 promoter is particularly preferable as an advantageous reason for producing small size RNA as RNA polymerase III. Leader sequence, polyadenylation sequence, promoter, enhancer, upstream activation sequence, signal peptide sequence and transcription termination factor as necessary for efficient transcription of shRNA that suppresses TGF-β2 expression A regulatory sequence can be further included.
ここで、用いられる用語「作動可能に連結された」とは、核酸配列との間の結合が機能的に連関されていることを意味する。任意の核酸配列が作動可能に連結された場合は、任意の核酸配列が他の核酸配列と機能的に関連性を有するように位置している場合である。本発明において、任意の転写調節配列がｓｈＲＮＡの転写に影響を及ぼす場合、前記転写調節配列が前記ｓｈＲＮＡと作動可能に連結されているという。 As used herein, the term “operably linked” means that the binding between the nucleic acid sequences is functionally linked. When any nucleic acid sequence is operably linked, any nucleic acid sequence is positioned so as to be functionally related to another nucleic acid sequence. In the present invention, when any transcriptional regulatory sequence affects shRNA transcription, the transcriptional regulatory sequence is said to be operably linked to the shRNA.
また、本発明は、前記配列番号３または４のＴＧＦ−β２発現を抑制するｓｈＲＮＡ、配列番号５に表されるトップストランド（ｔｏｐ ｓｔｒａｎｄ）と、配列番号６に表されるボトムストランド（ｂｏｔｔｏｍ ｓｔｒａｎｄ）を含むＤＮＡまたは配列番号７に表されるトップストランド（ｔｏｐ ｓｔｒａｎｄ）と、配列番号８に表されるボトムストランド（ｂｏｔｔｏｍ ｓｔｒａｎｄ）を含むＤＮＡまたはこれを発現する組換え発現ベクターを有効成分として含む抗腫瘍組成物に関する。 The present invention also provides a shRNA that suppresses TGF-β2 expression of SEQ ID NO: 3 or 4, a top strand represented by SEQ ID NO: 5, and a bottom strand represented by SEQ ID NO: 6. An anti-DNA comprising a DNA comprising DNA or a top strand represented by SEQ ID NO: 7 and a DNA comprising a bottom strand represented by SEQ ID NO: 8 or a recombinant expression vector expressing the same as an active ingredient It relates to a tumor composition.
本発明の抗腫瘍組成物の投与経路は、特に限定されず、経口投与または非経口投与（例えば、静脈内投与、筋肉内投与、皮下投与、皮内投与、粘膜投与、直腸内投与、膣内投与、患者への局所投与、皮膚投与など）のいずれか１つの投与経路に投与してもよい。経口投与の適当な製剤形態としては、固形または液体の形態が可能であって、非経口投与の適当な製剤形態としては、注射剤、点滴剤、坐剤、外用剤、点眼剤、点鼻剤などの形態が可能である。本発明の抗腫瘍組成物は、その製剤形態によって、必要に応じて薬学的に許容可能な添加剤を含んでもよい。薬学的に許容可能な添加剤の具体的な例としては、例えば、賦形剤、結合剤、崩壊剤、滑択剤、抗酸化剤、保存剤、安定化剤、等張化剤、着色剤、矯味剤、希釈剤、乳化剤、懸濁化剤、溶媒、充填剤、増量剤、緩衝剤、送達担体、キャリア、賦形剤及び／または薬学的アジュバントなどがあげられる。 The administration route of the antitumor composition of the present invention is not particularly limited, and is oral or parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal Administration, topical administration to a patient, dermal administration, etc.). Suitable dosage forms for oral administration can be in solid or liquid form. Suitable dosage forms for parenteral administration include injections, drops, suppositories, external preparations, eye drops, and nasal drops. Etc. are possible. The antitumor composition of the present invention may contain a pharmaceutically acceptable additive as necessary depending on the preparation form. Specific examples of pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, preservatives, stabilizers, isotonic agents, and coloring agents. , Flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, fillers, buffers, delivery carriers, carriers, excipients and / or pharmaceutical adjuvants.
経口用の固形製剤形態の本発明の抗腫瘍組成物としては、例えば、有効成分に賦形剤を加え、それと共に、必要に応じて、結合剤、崩壊剤、滑択剤、着色剤または矯味剤などの製剤用添加物を加えた後、通常方法で、錠剤、顆粒剤、散剤、カプセル剤として調製することができる。経口用液体製剤形態の本発明の抗腫瘍組成物としては、有効成分として、矯味剤、安定化剤、または保存剤などの製剤用添加物１種または２種以上を加え、通常方法により、内服液剤、シロップ剤、エリキシル剤などとして調製することができる。 As the antitumor composition of the present invention in the form of an oral solid preparation, for example, an excipient is added to an active ingredient, and a binder, a disintegrating agent, a lubricant, a coloring agent, or a taste masking agent, if necessary. After adding a pharmaceutical additive such as an agent, it can be prepared as a tablet, granule, powder, or capsule by a conventional method. As an antitumor composition of the present invention in the form of an oral liquid preparation, one or more additives for preparation such as a corrigent, stabilizer or preservative are added as active ingredients, It can be prepared as a liquid, syrup, elixir or the like.
本発明の抗腫瘍組成物を液体製剤として処方するために用いる溶媒としては、水性または非水性のいずれでもよい。液体製剤は当該分野に周知の方法により調製することができる。例えば、注射剤は、生理食塩水、ＰＢＳのような緩衝液、滅菌水などの溶剤に溶解した後、濾過紙などに濾過滅菌し、続いて滅菌容器（例えば、アンプルなど）に充填して調製することができる。この注射剤には、必要に応じて、慣用の薬学的キャリアを含んでもよい。 The solvent used for formulating the antitumor composition of the present invention as a liquid preparation may be either aqueous or non-aqueous. Liquid formulations can be prepared by methods well known in the art. For example, an injection is prepared by dissolving in a physiological saline solution, a buffer solution such as PBS, or a solvent such as sterilized water, sterilizing by filtration on filter paper, and then filling in a sterilized container (for example, an ampoule). can do. This injection may contain a conventional pharmaceutical carrier, if necessary.
また、非侵襲的なカテーテルを用いる投与方法を使用してもよい。本発明で使用することができるキャリアとしては、中性、緩衝化生理食塩水、または血清アルブミンを含む生理食塩水などがあげられる。 Alternatively, administration methods using non-invasive catheters may be used. Carriers that can be used in the present invention include neutral, buffered saline, or saline containing serum albumin.
ＴＧＦ−β２発現を抑制するｓｈＲＮＡ発現ベクターなど遺伝子送逹については、適用される細胞内においてＴＧＦ−β２発現を抑制するｓｈＲＮＡまたはｓｈＲＮＡ発現ベクターを発現させている限り、特に方法は限定しないが、例えば、ウイルスベクター、リポソームを用いた遺伝子導入を利用することが可能である。ウイルスベクターとしては、例えば、レトロウイルス、ワクシニアウイルス、アデノウイルス、 レンチウイルス などの動物ウイルスがあげられる。 As for gene delivery such as shRNA expression vector that suppresses TGF-β2 expression, the method is not particularly limited as long as shRNA or shRNA expression vector that suppresses TGF-β2 expression is expressed in the applied cells. It is possible to utilize gene transfer using viral vectors and liposomes. Examples of viral vectors include animal viruses such as retroviruses, vaccinia viruses, adenoviruses, and lentiviruses.
ＲＮＡｉによりＴＧＦ−β２発現を抑制する物質は、細胞に直接注入してもよい。 A substance that suppresses TGF-β2 expression by RNAi may be directly injected into cells.
本発明の抗腫瘍組成物の有効成分は治療学的有効量として用い、前記組成物の投与量は、使用目的、疾患の中毒度、患者の年齢、体重、性別、既往歴、または有効成分として用いる物質の種類などを考慮して当業者が決定することができる。例えば、有効成分として、大人１ｋｇ当たり約１×１０１０〜１×１０１２ｐａｒｔｉｃｌｅｓである。本発明の抗腫瘍組成物の投与頻度は、例えば、１日１回〜数ヶ月に１回であればよい。 The active ingredient of the antitumor composition of the present invention is used as a therapeutically effective amount. Those skilled in the art can determine the type of substance to be used in consideration. For example, the active ingredient is about 1 × 10 10 to 1 × 10 12 particles per kg adult. The administration frequency of the antitumor composition of the present invention may be, for example, once a day to once every several months.
本発明のｓｈＲＮＡは、ＴＧＦ−β２発現を抑制するので、本発明の薬剤学的組成物は、腫瘍と係る多様な疾病または疾患、例えば癌、具体的には、脳癌、胃癌、肺癌、乳房癌、卵巣癌、肝臓癌、気管支癌、鼻咽頭癌、喉頭癌、食道癌、膵臓癌、膀胱癌、前立腺癌、大腸癌、結腸癌及び子宮頸部癌などの予防及び治療に利用される。本明細書において用語「治療」は（ｉ）腫瘍細胞形成の予防；（ｉｉ）腫瘍細胞の除去による腫瘍と係る疾病または疾患の抑制；及び（ｉｉｉ）腫瘍細胞の除去による腫瘍と係る疾病または疾患の軽減を意味する。よって、本明細書において用語「治療学的有効量」は上記の薬理学的効果を達成するのに十分な量を意味する。 Since the shRNA of the present invention suppresses TGF-β2 expression, the pharmaceutical composition of the present invention can be used for various diseases or diseases related to tumors such as cancer, specifically brain cancer, stomach cancer, lung cancer, breast cancer. It is used for prevention and treatment of cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, colon cancer, cervical cancer and the like. As used herein, the term “treatment” refers to (i) prevention of tumor cell formation; (ii) suppression of a disease or disorder associated with the tumor by removal of tumor cells; and (iii) a disease or disorder associated with the tumor by removal of tumor cells. Means lessening. Thus, as used herein, the term “therapeutically effective amount” means an amount sufficient to achieve the pharmacological effect described above.
以下、本発明に係る実施例で本発明をより詳しく説明されるが、本発明の範囲が下記実施例によって制限されない。
実施例１：ｓｈＲＮＡ製造−ＴＧＦ−β２の沈黙（ｓｉｌｅｎｃｉｎｇ）を効果的に誘導するターゲット選定
Hereinafter, the present invention will be described in more detail by way of examples according to the present invention, but the scope of the present invention is not limited by the following examples.
Example 1: shRNA production-Target selection that effectively induces TGF-β2 silencing
本発明は、ＴＧＦ−β２の沈黙（ｓｉｌｅｎｃｉｎｇ）を誘導するために、センス２５ｍｅｒ／アンチセンス２５ｍｅｒ（４個塩基を有するループを中に含む）に基づいたｓｈＲＮＡを製作し、アデノウイルスで発現させるためにシャトルベクターに導入し、相同組換え（ｈｏｍｏｌｏｇｏｕｓ ｒｅｃｏｍｂｉｎａｔｉｏｎ）ウイルスを製作した。 In order to induce silencing of TGF-β2, the present invention creates a shRNA based on sense 25mer / antisense 25mer (including a loop with 4 bases) and expresses it in adenovirus. Into a shuttle vector to produce a homologous recombination virus.
ｓｈＲＮＡ ＴＧＦ−β２の特異性検証のために、スクランブルド（ｓｃｒａｍｂｌｅｄ）ｓｈＲＮＡを有するシャトルベクターも同時に製作した。従来方法と比較して特異性と発現抑制能が大きく向上した。 For verification of the specificity of shRNA TGF-β2, a shuttle vector having scrambled shRNA was also produced at the same time. Compared with the conventional method, specificity and expression suppression ability were greatly improved.
このために、ＴＧＦ−β２のｓｈＲＮＡ最小１０ｎＭでマウスのＴＧＦ−β２ ｍＲＮＡを７５％以上抑制する効果があるｓｈＲＮＡを、リアルタイムＰＣＲ方法を介して確保した。 For this purpose, shRNA having an effect of suppressing 75% or more of mouse TGF-β2 mRNA with a minimum of 10 nM shRNA of TGF-β2 was secured through a real-time PCR method.
このために、マウス用ｓｈＲＮＡは、皮膚癌細胞であるＢ１６Ｆ１０にトランスフェクションさせ、２４時間が過ぎた後に減少される程度を調査した。 For this purpose, mouse shRNA was transfected into skin cancer cells, B16F10, and the extent to which it was reduced after 24 hours was investigated.
次は、実験方法である。
リアルタイムＲＴ−ＰＣＲで多様な種類の候補ｓｈＲＮＡ １０ｎＭを３０％ Ｂ１６Ｆ１０にトランスフェクションし、２４時間培養後、確認（ｖａｌｉｄａｔｉｏｎ）を介してマウスＴＧＦ−β２に対して５個のｓｈＲＮＡスクリーニングの結果、ターゲットに該当されるｓｈＲＮＡにおいて７３．７５％の沈黙（ｓｉｌｅｎｃｉｎｇ）効果を確認した。
Next is the experimental method.
Various types of candidate shRNA 10 nM were transfected into 30% B16F10 by real-time RT-PCR, cultured for 24 hours, and then subjected to 5 shRNA screening against mouse TGF-β2 via validation. A 73.75% silencing effect was confirmed in the corresponding shRNA.
リアルタイムＲＴ−ＰＣＲのために、正方向プライマーとしては、５’−ＧＴＧＡＡＴＧＧＣＴＣＴＣＣＴＴＣＧＡＣ−３’［配列番号９］と逆方向プライマーとしては、５’−ＣＣＴＣＧＡＧＣＴＣＴＴＣＧＣＴＴＴＴＡ−３’［配列番号１０］であって、反応条件は、次のように行った。 For real-time RT-PCR, the forward primer is 5′-GTGAATGGCTCTCCTCTGAC-3 ′ [SEQ ID NO: 9] and the reverse primer is 5′-CCTCGAGCTCTTCGCTTTTA-3 ′ [SEQ ID NO: 10] The conditions were as follows.
１段階：逆転写（４２℃ ５ｍｉｎ、９５℃ １０ｓｅｃ）、
２段階：ＰＣＲ反応（９５℃ ５ｓｅｃ、６０℃ ２０ｓｅｃ）５０ｃｙｃｌｅｓ、
３段階：分離（６０℃−＞９５℃）で行った。
1st step: reverse transcription (42 ° C. 5 min, 95 ° C. 10 sec),
Two steps: PCR reaction (95 ° C. 5 sec, 60 ° C. 20 sec) 50 cycles,
Three steps: performed in separation (60 ° C .-> 95 ° C.).
確認（ｖａｌｉｄａｔｉｏｎ）実験結果、１０個の標的配列候補のうち、下記表１に基づいて下記ターゲットを選定した。 As a result of the validation experiment, the following targets were selected based on Table 1 below among the 10 target sequence candidates.
マウス標的配列：５’−ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ−３’［配列番号１］ Mouse target sequence: 5'-GGATTGAACTGTATCAGATCCTTAA-3 '[SEQ ID NO: 1]
注）ｃｔ：ｃｙｃｌｅ ｔｈｒｅｓｈｏｌｄ， ｓａｔｕｒａｔｉｏｎに到達するのに所要されたサイクル数、小さいほど元のｍＲＮＡ量は多くなる。 Note) ct: The number of cycles required to reach cycle threshold, saturation, the smaller the original mRNA amount, the smaller.
△ｃｔ：ＴＧＦ−β２用ｃｔからａｃｔｉｏｎ用ｃｔを引いた値。
△△ｃｔ：ＴＧＦ−β２ ｓｈＲＮＡ処理サンプルの△ｃｔから対照群のＴＧＦ−β２の△ｃｔを引いた値。
２−△△ｃｔ：２のマイナス指数の△△ｃｔ値
発現抑制率：２−△△ｃｔを百分率で表したものである。
Δct: value obtained by subtracting ct for action from ct for TGF-β2.
ΔΔct: a value obtained by subtracting Δct of TGF-β2 of the control group from Δct of the TGF-β2 shRNA-treated sample.
2-ΔΔct: ΔΔct value of minus index of 2 Expression suppression rate: 2-ΔΔct expressed as a percentage.
前記標的配列に対して２５／２５＋４ループを有するｓｈＲＮＡを合成し、これらの標的配列に対する抑制効果をリアルタイムＰＣＲで確認した。 ShRNA having a 25/25 + 4 loop with respect to the target sequence was synthesized, and the inhibitory effect on these target sequences was confirmed by real-time PCR.
マウス標的配列（配列番号１）とするｓｈＲＮＡ：５’−ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｔｃｔｃ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ−３’［配列番号３］ ShRNA as mouse target sequence (SEQ ID NO: 1): 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCATCC-3 '[SEQ ID NO: 3]
上述のリアルタイムＰＣＲによって選定された配列番号３の塩基配列をアデノウイルスで発現させるために、両端にＢａｍＨＩとＨｉｎｄIII塩基サイトを挿入し、中間にｔｃｔｃの４個の塩基を有するループ（ｌｏｏｐ）を有するように製作した。すなわち、マウスｓｈＲＮＡの基本的な構造は５’−２５ｍｅｒ−ループ（４ｍｅｒ）−２５ｍｅｒ−３’で構成されている。 In order to allow the adenovirus to express the nucleotide sequence of SEQ ID NO: 3 selected by the above-mentioned real-time PCR, BamHI and HindIII base sites are inserted at both ends, and a loop having four tctc bases in between is inserted. Was produced as follows. That is, the basic structure of mouse shRNA is composed of 5'-25mer-loop (4mer) -25mer-3 '.
これに基づいて、アデノウイルスに導入するための下記の２本鎖のＤＮＡストランド（ｓｔｒａｎｄ）を製作してｓｈＲＮＡ生成を誘導した。 Based on this, the following double-stranded DNA strand for introduction into adenovirus was prepared to induce shRNA production.
トップストランド：５’−ｇａｔｃｃ ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｔｃｔｃ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ ｔｔｔｔ ａ−３’［配列番号５］ Top strand: 5'-gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCATCC ttt a-3 '[SEQ ID NO: 5]
ボトムストランド：５’−ａｇｃｔｔ ａａａａ ＧＧＡＴＴＧＡＡＣＴＧＴＡＴＣＡＧＡＴＣＣＴＴＡＡ ｇａｇａ ＴＴＡＡＧＧＡＴＣＴＧＡＴＡＣＡＧＴＴＣＡＡＴＣＣ ｇ−３’［配列番号６］ Bottom strand: 5'-agcttt aaaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCATCC g-3 '[SEQ ID NO: 6]
人間ＴＧＦ−β２抑制のためのリアルタイムＰＣＲ用プライマーは次のようである。 The primers for real-time PCR for human TGF-β2 suppression are as follows.
正方向プライマー：５’−ＧＣＴＧＣＣＴＡＣＧＴＣＣＡＣＴＴＴＡＣＡＴ−３’［配列番号１１］ Forward primer: 5'-GCTGCCCTCGTCACTTTATACAT-3 '[SEQ ID NO: 11]
逆方向プライマー：５’−ＡＴＡＴＡＡＧＣＴＣＡＧＧＡＣＣＣＴＧＣＴＧ−３’［配列番号１２］ Reverse primer: 5'-ATATAAGCTCAGGACCCTGCTG-3 '[SEQ ID NO: 12]
反応条件は、１段階：逆転写（４２℃ ５ｍｉｎ、９５℃ １０ｓｅｃ）、２段階：ＰＣＲ反応（９５℃ ５ｓｅｃ、６０℃ ２０ｓｅｃ）５０ｃｙｃｌｅｓ、３段階：分離（６０℃−＞９５℃）で行った。 The reaction conditions were 1 step: reverse transcription (42 ° C 5 min, 95 ° C 10 sec), 2 steps: PCR reaction (95 ° C 5 sec, 60 ° C 20 sec), 50 cycles, 3 steps: separation (60 ° C-> 95 ° C). .
確認（ｖａｌｉｄａｔｉｏｎ）実験結果、３個の標的配列候補のうち、下記表２に基づいて下記ターゲットを選定した。 As a result of the validation experiment, the following targets were selected from the three target sequence candidates based on Table 2 below.
人間標的配列：５’−ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ−３’［配列番号２］ Human target sequence: 5'-GGATTGAGCTATATCAGATTCCAA-3 '[SEQ ID NO: 2]
前記標的配列に対して２５／２５＋４ループを有するｓｈＲＮＡを合成し、これらの標的配列に対する抑制効果をリアルタイムＰＣＲで確認した。 ShRNA having a 25/25 + 4 loop with respect to the target sequence was synthesized, and the inhibitory effect on these target sequences was confirmed by real-time PCR.
人間標的配列（配列番号２）のためのｓｈＲＮＡ：５’−ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｔｃｔｃ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ−３’［配列番号４］ ShRNA for human target sequence (SEQ ID NO: 2): 5'-GGATTGAGCTCATATCAGATTTCCAA tctc TTGAGAATCTGATATAGCTCAATCC-3 '[SEQ ID NO: 4]
上述のリアルタイムＰＣＲにより選定された配列番号４の塩基配列をアデノウイルスで発現させるために、両端にＢａｍＨＩとＨｉｎｄIII塩基サイトを挿入し、中間にｔｃｔｃの４個の塩基を有するループを有するように製作した。すなわち、人間ｓｈＲＮＡの基本構造は、５’−２５ｍｅｒ−ループ（４ｍｅｒ）−２５ｍｅｒ−３’で構成されている。 In order to express the base sequence of SEQ ID NO: 4 selected by the above-mentioned real-time PCR in adenovirus, BamHI and HindIII base sites are inserted at both ends, and a loop having 4 bases of tctc in the middle is produced. did. That is, the basic structure of human shRNA is composed of 5'-25mer-loop (4mer) -25mer-3 '.
これに基づいて、アデノウイルスに導入させるための下記２本鎖のＤＮＡを製作した。 Based on this, the following double-stranded DNA for introduction into adenovirus was prepared.
トップストランド：５’−ｇａｔｃｃ ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｔｃｔｃ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ ｔｔｔｔ ａ−３’［配列番号７］ Top strand: 5'-gatcc GGATTGAGCTCATATCAGATTCCAA tctc TTGAGAATCTGATATAGCTCAATCC ttt a-3 '[SEQ ID NO: 7]
ボトムストランド：５’−ａｇｃｔｔ ａａａａ ＧＧＡＴＴＧＡＧＣＴＡＴＡＴＣＡＧＡＴＴＣＴＣＡＡ ｇａｇａ ＴＴＧＡＧＡＡＴＣＴＧＡＴＡＴＡＧＣＴＣＡＡＴＣＣ ｇ−３’［配列番号８］
実施例２：ターゲット配列に対するｓｈＲＮＡ発現する複製不能アデノウイルスベクター製作
Bottom strand: 5′-agcttt aaaaa GGATTGAGCTATATCAGATTTCCAA gaga TTGAGAATCTGATATAGCTCAATCC g-3 ′ [SEQ ID NO: 8]
Example 2: Construction of a replication-incompetent adenovirus vector expressing shRNA against a target sequence
リアルタイムＲＴ−ＰＣＲを介して確認された最も効果的に発現を抑制するｓｈＲＮＡ塩基配列をセンスとアンチセンス配列がｔｃｔｃあるいはｔｃｔｃｔｃを間に置いて位置するようにし、両端にＢａｍＨＩとＨｉｎｄIII制限酵素塩基配列を有した塩基で構成された、オリゴヌクレオチドと相補的なオリゴヌクレオチドをそれぞれ合成して合体（ａｎｎｅａｌｉｎｇ）させた後、Ｅ３シャトルベクターであるｐＳＰ７２ΔＥ３／ｓｉ−ｎｅｇａｔｉｖｅベクター［図１、ｐＳＰ７２ｃｌｏｎｉｎｇベクター（Ｐｒｏｍｅｇａ）にアデノウイルスＥ３Ｌ（２６５９１−２８５８８）とＥ３Ｒ（３０５０４−３１０５７）を挿入し、Ａｍｂｉｏｎ社のｐｓｉｌｅｎｃｅｒ ２．１−Ｕ６ ｈｙｇｒｏで−ＥｃｏＲＩ−Ｕ６ ｐｒｏｍｏｔｅｒ＋−ＢａｍＨＩ−ｎｏｎｓｅｎｓｅ ｓｈＲＮＡ用塩基配列であるａｃｔａｃｃｇｔｔｇｔｔａｔａｇｇｔｇｔｔｃａａｇａｇａｃａｃｃｔａｔａａｃａａｃｇｇｔａｇｔｔｔｔｔｔｇｇａａ−ＨｉｎｄIIIが入った形態のｐＳＰ７２ΔＥ３／ｓｉ−ｎｅｇａｔｉｖｅ（ｓｃｒａｍｂｌｅｄ）］を製作した。 The shRNA base sequence confirmed through real-time RT-PCR most effectively suppresses the expression so that the sense and antisense sequences are located with tctc or tctctc in between, and BamHI and HindIII restriction enzyme base sequences at both ends. After synthesizing and annealing oligonucleotides complementary to the oligonucleotides composed of bases having an E3, an E3 shuttle vector pSP72ΔE3 / si-negative vector [FIG. 1, pSP72 cloning vector (Promega) Adenovirus E3L (26591-28588) and E3R (30504-31057) were inserted into Ambion's psilencer 2.1-U6 hygro -EcoRI-U6 promoter +- BamHI-nonsense shRNA for a nucleotide sequence actaccgttgttataggtgttcaagagacacctataacaacggtagttttttggaa-HindIII is entered form of pSP72ΔE3 / si-negative (scrambled)] was manufactured.
人間またはマウスのｓｈＲＮＡ ＴＧＦ−β２導入のために、まず、上記のｐＳＰ７２ΔＥ３／ｓｉ−ｎｅｇａｔｉｖｅプラスミドをＢａｍＨＩとＨｉｎｄIIIで処理した後、人間またはマウスのｓｈＲＮＡ ＴＧＦ−β２を挿入させてｐＳＰ７２ΔＥ３−ｓｈ−ｈｕｍａｎ ＴＧＦ−β２またはｐＳＰ７２ΔＥ３−ｓｈ−ｍｏｕｓｅ ＴＧＦ−β２を製作した［図２］。陰性対照群アデノウイルスとしては、両端にＢａｍＨＩとＨｉｎｄIIIを有するようにし、スクランブルド（ｓｃｒａｍｂｌｅｄ）塩基配列（ａｃｔａｃｃｇｔｔｇｔｔａｔａｇｇｔｇ）とｌｏｏｐ（ｔｔｃａａｇａｇａ）を製作した。 In order to introduce human or mouse shRNA TGF-β2, first, the above-mentioned pSP72ΔE3 / si-negative plasmid was treated with BamHI and HindIII, and human or mouse shRNA TGF-β2 was then inserted into pSP72ΔE3-sh-human TGF. -Β2 or pSP72ΔE3-sh-mouse TGF-β2 was produced [Figure 2]. As a negative control group adenovirus, BamHI and HindIII were present at both ends, and a scrambled nucleotide sequence (actaccgtttgttataggt) and loop (ttcaagaga) were prepared.
アデノウイルスのＥ３部位ＰＣＲで養成クローン（＃１、２、５、６、７、８、９）だけを選択した後、［図３の（ａ）：ｄｌ３２４／ＩＸアデノウイルスバックボーン（ｂａｃｋｂｏｎｅ）ゲノム（ｇｅｎｏｍｉｃ）ＤＮＡとｐＳＰ７２−ｓｈ−ｈＴＧＦ−β２シャトルベクターとの相同組換え後、ｓｈ−ｈＴＧＦ−β２が含まれたクローンを選択するＰＣＲ結果、図３の（ｂ）：ｄｌ３２４／ＩＸアデノウイルスバックボーンゲノムＤＮＡとｐＳＰ７２−ｓｈ−ｈＴＧＦ−β２シャトルベクターとの相同組換え後、ＩＸ遺伝子有無を介して図３の（ａ）で確認されたｓｈ−ｈＴＧＦ−β２が含まれたクローンのうちからゲノムＤＮＡも含まれたクローンを再び選択するＰＣＲ結果］、図３の（ｃ）で示すように、ＨｉｎｄIII切断パターン（ｄｉｇｅｓｔｉｏｎｐａｔｔｅｒｎ）で最終の相同組換え体を選択した［図４］。 After selecting only the trained clones (# 1, 2, 5, 6, 7, 8, 9) by E3 site PCR of adenovirus, [Fig. 3 (a): dl324 / IX adenovirus backbone genome ( Genomic) DNA and pSP72-sh-hTGF-β2 shuttle vector followed by homologous recombination, followed by PCR to select clones containing sh-hTGF-β2, FIG. 3 (b): dl324 / IX adenovirus backbone genome After homologous recombination between the DNA and the pSP72-sh-hTGF-β2 shuttle vector, genomic DNA was selected from the clones containing sh-hTGF-β2 confirmed in (a) of FIG. PCR results for selecting the included clones again], as shown in FIG. The final homologous recombination were selected igestionpattern) [4].
次に、図３及び図４を詳細に説明する。 Next, FIGS. 3 and 4 will be described in detail.
図３の（ａ）において、ｄｌ３２４／ＩＸレーンはｄｌ３２４バックボーンであり；シャトルレーンはｐＳＰ７２−ｓｈ−ｈＴＧＦ−β２である。レーン１〜１０は、ｄｌ３２４バックボーンとｐＳＰ７２−ｓｈ−ｈＴＧＦ−β２との間の相同組換え後、バクテリアクローン（ｂａｃｔｅｒｉａｌ ｃｌｏｎｅ）から得たプラスミドをＥ３部位に増幅させた結果を示すもので、約２ｋｂに相当するバンドが示されればｐｏｓｉｔｉｖｅである。Ｅ３部分をＰＣＲしたとき、Ｅ３部分のないｄｌ３２４バックボーンでは２ｋｂに相当するバンドが示されないが、Ｅ３部分に、Ｕ６プロモーターとｓｈ−ｈＴＧＦ−β２とのｓｈコンストラクト（ｃｏｎｓｔｒｕｃｔ）が挿入されたシャトルベクターの場合、ＰＣＲすると２ｋｂのプロダクト（ｐｒｏｄｕｃｔ）の大きさ（ｓｉｚｅ）が示されることにより相同組換えされたか否かを確認することができる。 In FIG. 3 (a), the dl324 / IX lane is the dl324 backbone; the shuttle lane is pSP72-sh-hTGF-β2. Lanes 1 to 10 show the results of amplification of a plasmid obtained from a bacterial clone after homologous recombination between the dl324 backbone and pSP72-sh-hTGF-β2 to the E3 site, about 2 kb. If a band corresponding to is shown, it is positive. When PCR was performed on the E3 part, a band corresponding to 2 kb was not shown in the dl324 backbone without the E3 part, but the shuttle vector in which the sh construct of the U6 promoter and sh-hTGF-β2 was inserted into the E3 part. In this case, it can be confirmed whether or not homologous recombination has been achieved by showing the size of the 2 kb product when PCR is performed.
図３の（ｂ）において、ｄｌ３２４／ＩＸレーンはｄｌ３２４バックボーンであり；シャトルベクターはｐＳＰ７２−ｈＴＧＦ−β２である。図３の（ａ）で確認されたｓｈ−ｈＴＧＦ−β２が含まれたクローン（＃１、２、５、６、７、８、９）のうちからゲノムＤＮＡも含まれたクローンを再び選択するＰＣＲ結果で相同組換えされたことを確認する、図３の（ａ）に続いて連続的な選択実験により両方でｐｏｓｉｔｉｖｅしたクローンが相同組換えされたことを意味する。ＩＸ遺伝子部分をＰＣＲしたとき、ＩＸ遺伝子を有するｄｌ３２４バックボーンとＩＸ遺伝子を有しないシャトルベクターとの差を用いて相同組換えされたか否かを確認した。その結果、＃１、２、６、７だけが再び選択された。 In FIG. 3 (b), the dl324 / IX lane is the dl324 backbone; the shuttle vector is pSP72-hTGF-β2. Among the clones (# 1, 2, 5, 6, 7, 8, 9) containing sh-hTGF-β2 confirmed in (a) of FIG. 3, clones containing genomic DNA are again selected. It means that the positive clones in both cases were homologously recombined by a continuous selection experiment following (a) in FIG. 3 confirming that the PCR results were homologous recombination. When PCR was performed on the IX gene portion, it was confirmed whether homologous recombination was performed using the difference between the dl324 backbone having the IX gene and the shuttle vector not having the IX gene. As a result, only # 1, 2, 6, and 7 were selected again.
図３の（ｃ）は、バックボーンとサンプルとをＨｉｎｄIIIでカットティング（ｃｕｔｔｉｎｇ）したときに変化するパターンとの差により相同組換えされたか否かを最終的に確認したものである。レーン１〜３は前記＃１クローン由来ＤＮＡであり、レーン４〜６は＃２クローン、レーン７〜９は＃６クローンで得たＤＮＡをＤＨ５ａというコンピテントセル（ｃｏｍｐｅｔｅｎｔ ｃｅｌｌ）に再びトランスフォーメーション（ｔｒａｎｓｆｏｒｍａｔｉｏｎ）して得た子孫（ｐｒｏｇｅｎｙ）クローンで各母体（ｐａｒｅｎｔａｌ）クローンから来由されたそれぞれの３個のＤＮＡのうち＃１クローンだけが既存のｄｌ３２４−ＩＸ（もっぱら左側一番目ｌａｎｅ）とは異なったＨｉｎｄIIIパターンを示した。これは、バックボーンアデノウイルスＤＮＡがシャトルベクターと相同組換えをなしたことを意味し、よって、本発明は＃１クローンに基づいている。 (C) of FIG. 3 finally confirms whether or not homologous recombination has occurred due to a difference with a pattern that changes when the backbone and the sample are cut with HindIII. Lanes 1 to 3 are the DNAs derived from the # 1 clone, lanes 4 to 6 are the # 2 clones, and lanes 7 to 9 are the DNAs obtained from the # 6 clones again transformed into a competent cell called DH5a ( What is the existing dl324-IX (exclusively the first lane on the left side) of the three progeny clones obtained from the transformation and only the # 1 clone out of the three DNAs derived from each parental clone? A different HindIII pattern was shown. This means that the backbone adenovirus DNA has undergone homologous recombination with the shuttle vector, and thus the present invention is based on the # 1 clone.
図４は、ｐＰｏｌｙ２というプラスミドにＰａｃＩ部位に挿入されているＡｄ−ｄｌ３２４−ＩＸ−ｓｈ−ｈＴＧＦ−β２をＰａｃＩで切断してｐＰｏｌｙ２がまともに切断されたか否かを確認することで、ウイルス生産に要求される最終コンストラクト（ｃｏｎｓｔｒｕｃｔ）を決定する実験である。図３の（ｃ）から確認した＃１クローンに属する３個のＤＮＡは、ＰａｃＩで切断時すべて約２ｋｂに相当するｐＰｏｌｙ２バックボーンＤＮＡが全部出てしまった。これらが、それぞれのｓｈＲＮＡ ｈＴＧＦ−β２塩基配列を有しているか否かを配列分析して確認した結果、すべてのクローンで同一のｓｈＲＮＡ ｈＴＧＦ−β２塩基配列を有することを確認した（図５）。その後、これらをＰａｃＩ切断後に一緒に２９３Ａ細胞にトランスフェクション（ｔｒａｎｓｆｅｃｔｉｏｎ）してアデノウイルスを生産した。 FIG. 4 shows that the production of virus was confirmed by cutting Ad-dl324-IX-sh-hTGF-β2 inserted in the PacI site into a plasmid called pPoly2 with PacI and confirming that pPoly2 was cut correctly. This is an experiment to determine the required final construct. As for the three DNAs belonging to the # 1 clone confirmed from FIG. 3 (c), all of the pPoly2 backbone DNA corresponding to about 2 kb appeared when cut with PacI. As a result of confirming whether or not these had the respective shRNA hTGF-β2 base sequences by sequence analysis, it was confirmed that all clones had the same shRNA hTGF-β2 base sequences (FIG. 5). Thereafter, these were cotransfected into 293A cells after PacI digestion to produce adenovirus.
すなわち、前記方法で製作されたＥ３シャトルベクターをそれぞれのＸｍｎＩ制限酵素で処理して単一鎖に作った後、ＳｐｅＩ制限酵素を処理して単一鎖となった複製不能アデノウイルスであるｄｌ３２４とともに、大膓菌ＢＪ５１８３で同時に形質転換させて遺伝子相同組換えを誘導した。相同組換えされたプラスミドＤＮＡを収得してＨｉｎｄIII制限酵素で処理してＤＮＡパターンの変化を確認し、最終的に配列分析して相同組換え有無を確認した後、確認されたプラスミドをＰａｃＩで切断した後、２９３細胞株に形質転換してｓｈＲＮＡ ＴＧＦ−β２を発現する複製不能アデノウイルスを製作した（複製可能アデノウイルスにｓｈＲＮＡを製作する場合には、ｓｈＲＮＡによる抑制効果と細胞リシース（ｌｙｓｉｓ）効果とが混在されていて、抑制効果だけを明確に確認することができなかったので、複製不能アデノウイルスを製作した）。このアデノウイルスは、２９３細胞株で増殖させてＣｓＣｌ変化度（ｇｒａｄｉｅｎｔ）で濃縮して限界希釈培養法（ｌｉｍｉｔｉｎｇ ｄｉｌｕｔｉｏｎ）または溶菌斑検査（ｐｌａｑｕｅ ａｓｓａｙ）でウイルス力価を決定した。 That is, the E3 shuttle vector prepared by the above method was treated with each XmnI restriction enzyme to make a single strand, and then treated with SpeI restriction enzyme to make a single strand, along with dl324, a non-replicatable adenovirus. The gene homologous recombination was induced by simultaneous transformation with B. gonorrhoeae BJ5183. The homologous recombination plasmid DNA is obtained and treated with HindIII restriction enzyme to confirm the DNA pattern change, and finally the sequence analysis is performed to confirm the presence or absence of homologous recombination, and then the confirmed plasmid is cleaved with PacI. After that, the 293 cell line was transformed to produce a non-replicatable adenovirus expressing shRNA TGF-β2 (in the case of producing shRNA in a replicable adenovirus, the suppressive effect by shRNA and the cell lysis effect) ), And it was not possible to clearly confirm only the suppressive effect. The adenovirus was grown in the 293 cell line, concentrated with the CsCl gradient, and the virus titer was determined by limiting dilution or plaque assay.
最終ウイルス力価（ｖｉｒｕｓ ｔｉｔｅｒ）は、限界希釈適正法（ｌｉｍｉｔｉｎｇ ｄｉｌｕｔｉｏｎ ｔｉｔｒａｔｉｏｎ）により２．５×１０９ｐｆｕ／ｍｌであった。
実施例３：癌細胞での効果確認−ｓｈＲＮＡ発現するアデノウイルスによるＴＧＦ−β２発現抑制確認
The final virus titer was 2.5 × 10 9 pfu / ml by the limiting dilution titration.
Example 3: Confirmation of effect in cancer cells-Confirmation of suppression of TGF-β2 expression by adenovirus expressing shRNA
１）リアルタイムＲＴ−ＰＣＲで確認
ＴＧＦ−β２発現抑制確認は、人間の場合、人間前立腺癌細胞であるＤＵ−１４５に実施例２のアデノウイルス１〜１００ｍｏｉで感染させて２日後、トリゾール（Ｔｒｉｚｏｌ）で細胞をリーシス（ｌｙｓｉｓ）させ、クロロホルム、イソプロパノール、エタノールなどを連続的に処理してＲＮＡを収穫した後、ＴＧＦ−β２ ｍＲＮＡ発現抑制の程度をリアルタイムＰＣＲで確認した。
1) Confirmation by real-time RT-PCR In the case of humans, TGF-β2 expression inhibition confirmation was confirmed by infecting human prostate cancer cells DU-145 with the adenovirus 1 to 100 moi of Example 2 and two days later, Trizol. The cells were lysed and treated with chloroform, isopropanol, ethanol, and the like to harvest RNA, and the extent of TGF-β2 mRNA expression suppression was confirmed by real-time PCR.
マウスの場合、マウス黒色腫細胞であるＢ１６Ｆ１０に、実施例２のアデノウイルス１００、５００、１０００ｍｏｉで感染させ、その後の過程は人間の場合と同様に実施した。 In the case of mice, B16F10, which is a mouse melanoma cell, was infected with the adenovirus 100, 500, 1000 moi of Example 2, and the subsequent process was performed in the same manner as in humans.
人間ＴＧＦ−β２抑制のためのリアルタイムＰＣＲ用プライマーは、正方向プライマー：５’−ＧＣＴＧＣＣＴＡＣＧＴＣＣＡＣＴＴＴＡＣＡＴ−３’［配列番号１１］と逆方向プライマー：５’−ＡＴＡＴＡＡＧＣＴＣＡＧＧＡＣＣＣＴＧＣＴＧ−３’［配列番号１２］を用い、ＡＢ ｐｏｗｅｒＳＹＢＲ Ｇｒｅｅｎ ＲＮＡ−ｔｏ−Ｃｔ １ｓｔｅｐ ｋｉｔを用いてＲＴ ｅｎｚｙｍｅ ｍｉｘ（１２５Ｘ）０．２μｌ、ＲＴ−ＰＣＲ Ｍｉｘ（２ｘ）１２．５μｌ、Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ（１００ｐＭ）０．５μｌ、 ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ（１００ｐＭ）０．５μｌ、 ＲＮＡ（１０ｎｇ／μｌ）５μｌ、 Ｎｕｃｌｅａｓｅ−ｆｒｅｅ ｗａｔｅｒ ６．３μｌで総体積は２５μｌとなるようにし、反応条件は、次の表３のようである。 As primers for real-time PCR for human TGF-β2 suppression, forward primer: 5′-GCTGCCCTCGTCACTTTATACAT-3 ′ [SEQ ID NO: 11] and reverse primer: 5′-ATATAGCTCAGGACCCTGCTG-3 ′ [SEQ ID NO: 12] RT energy mix (125X) 0.2 μl, RT-PCR Mix (2 ×) 12.5 μl, Forward Primer (100 pM) 0.5 μl, reverse Primer (100 pM) using AB powerSYBR Green RNA-to-Ct 1step kit 5 μl, RNA (10 ng / μl) 5 μl, Nuclease-free water 6.3 μl so that the total volume is 25 μl. The reaction conditions are as shown in Table 3 below. It is.
マウスＴＧＦ−β２抑制のためのリアルタイムＰＣＲ用プライマーは、正方向プライマー：５’−ＧＴＧＡＡＴＧＧＣＴＣＴＣＣＴＴＣＧＡＣ−３’［配列番号９］と逆方向プライマー：５’−ＣＣＴＣＧＡＧＣＴＣＴＴＣＧＣＴＴＴＴＡ−３’［配列番号１０］を用い、ＡＢ ｐｏｗｅｒＳＹＢＲ Ｇｒｅｅｎ ＲＮＡ−ｔｏ−Ｃｔ １ｓｔｅｐ ｋｉｔを用いてＲＴ ｅｎｚｙｍｅ ｍｉｘ（１２５Ｘ）０．２μｌ、ＲＴ−ＰＣＲ Ｍｉｘ（２ｘ）１２．５μｌ、Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ（１００ｐＭ）０．５μｌ、ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ（１００ｐＭ）０．５μｌ、ＲＮＡ（１０ｎｇ／μｌ）５μｌ、Ｎｕｃｌｅａｓｅ−ｆｒｅｅ ｗａｔｅｒ ６．３μｌで総体積は２５μｌとなるようにし、反応条件は、次の表３のようである。 As primers for real-time PCR for suppressing mouse TGF-β2, forward primer: 5′-GTGAATGGCTCTCTTCGAC-3 ′ [SEQ ID NO: 9] and reverse primer: 5′-CCTCGAGCTCTTCGCTTTTA-3 ′ [SEQ ID NO: 10] RT energy mix (125X) 0.2 μl, RT-PCR Mix (2 ×) 12.5 μl, Forward Primer (100 pM) 0.5 μl, reverse Primer (100) using AB powerSYBR Green RNA-to-Ct 1step kit The total volume is 5 μl, RNA (10 ng / μl) 5 μl, Nuclease-free water 6.3 μl, and the total volume is 25 μl. The reaction conditions are as shown in Table 3 below.
人間ＴＧＦ−β２のｓｈＲＮＡ確認結果、１ｍｏｉのアデノウイルスで７３％の沈黙（ｓｉｌｅｎｃｉｎｇ）効果を示すなど、５０ｍｏｉのアデノウイルスで９０％以上のＴＧＦ−β２発現抑制が観察された［表４、図６］。 As a result of confirming the shRNA of human TGF-β2, 90% or more suppression of TGF-β2 expression was observed with 50 moi adenovirus, such as 73% silencing effect with 1 moi adenovirus [Table 4, FIG. ].
マウスＴＧＦ−β２のｓｈＲＮＡ確認結果、１０００ｍｏｉのアデノウイルスで５０％の沈黙（ｓｉｌｅｎｃｉｎｇ）効果を示した［図７］。人間と比較して相対的に低い抑制率は、マウス細胞に対するアデノウイルスの低い感染率に起因したものと見られる。これに対する確認は、マウスｓｈＴＧＦ−β２が発現するプラスミドをトランスフェクション（ｔｒａｎｓｆｅｃｔｉｏｎ）することで,発現されたｓｈＲＮＡが効果的にＴＧＦ−β２ ｍＲＮＡを抑制するものと確認した［図８］。 As a result of confirming the shRNA of mouse TGF-β2, it showed a silencing effect of 50% with 1000 moi adenovirus [FIG. 7]. The relatively low inhibition rate compared to humans is likely due to the low infection rate of adenovirus on mouse cells. Confirmation of this confirmed that the expressed shRNA effectively suppressed TGF-β2 mRNA by transfecting a plasmid expressing mouse shTGF-β2 [FIG. 8].
２）ＥＬＩＳＡで確認
上述のアデノウイルス１、５、１０、５０ｍｏｉ感染後、２日間人間の実施例２の前立腺癌細胞に培養しながら最後の２４時間は無血清バッジに分泌されたＴＧＦ−β２量を測定した。
2) Confirmation by ELISA The amount of TGF-β2 secreted in the serum-free badge for the last 24 hours while being cultured in human prostate cancer cells of Example 2 for 2 days after infection with adenovirus 1, 5, 10, 50moi described above Was measured.
ＴＧＦ−β２のｓｈＲＮＡを発現するアデノウイルスを１ｍｏｉだけ感染しても一日間に分泌されるＴＧＦ−β２はほとんど検出されなかった［表５、図９］。これは、ＴＧＦ−β２のｓｈＲＮＡが非常に効果的にＴＧＦ−β２のｍＲＮＡを分解していることを意味する。 TGF-β2 secreted during one day was hardly detected even when only 1 moi of the adenovirus expressing shRNA of TGF-β2 was infected [Table 5, FIG. 9]. This means that the TGF-β2 shRNA is very effectively degrading the TGF-β2 mRNA.
複製不能なアデノウイルスでＴＧＦ−β２に対するｓｈＲＮＡの効果を確認した後、このｓｈＲＮＡを発現しながら腫瘍選択的に細胞を殺傷するアデノウイルスを製作した。腫瘍選択的殺傷複製可能なアデノウイルスを作る前にアデノウイルスのＥ１Ａ部分に多様な遺伝子を入れることができるシャトルベクターを製作しようと、ｐＢＳＫＩＩプラスミド［Ｓｔｒａｔａｇｅｎｅ、 ＵＳＡ］にＥ１ＡとＥ１Ｂ５５ｋＤａ遺伝子を含み、多様な酵素部位（Ｅｎｚｙｍｅ ｓｉｔｅ）を含むｐＢＳＫII−３４８４合成遺伝子を製作した［図１０の（ａ）］。合成された遺伝子を相同組換え確認を容易にするためのｐＣＡ１４シャトルベクターに導入するための形態に変えるためにｐＢＳＫII−３４８４をＰＣＲしてＦｓｐIで制限酵素を処理した後に、ブランティング（ｂｌｕｎｔｉｎｇ）酵素でブラントエンド（ｂｌｕｎｔ ｅｎｄ）を作って再びＢａｍＨIで処理した。ｐＣＡ１４［Ｍｉｃｒｏｂｉｘ ＢｉｏｓｙｓｔｅｍｓＩｎｃ、 Ｃａｎａｄａ］は、ＳｓｐIで制限酵素を用いて切った後、ブランティング酵素を用いてブラントエンドを作った後に、ＢｇｌIIを処理して同一伝達制限酵素（Ｉｓｏｓｃｈｉｚｏｍｅｒ）であるＢａｍＨIとＢｇｌII、そして両端のブラントエンドを介して合成した遺伝子を挿入してシャトルベクターｐＣＡ１４−３４８４を製作した［図１０の（ｂ）］。その後に、ＣＭＶプロモーター遺伝子をＫｐｎIとＸｈｏIでｐＣＡ１４−３４８４に挿入してｐＣＡ１４−ＣＭＶ−３４８４を製作した［図１０の（ｃ）］。そして、ｐＣＡ１４−ＣＭＶ−３４８４でＥｃｏＲＩとＳａｌＩ制限酵素によりＥ１Ｂ５５ｋＤａ部分を切断し、ブランティング（ｂｌｕｎｔｉｎｇ）した後、再び連結（ｌｉｇａｔｉｏｎ）されたｐＣＡ１４−ＣＭＶ−３４８４−ΔＥ１Ｂ５５を得た［図１０の（ｄ）］。製作されたシャトルベクターｐＣＡ１４−ＣＭＶ−３４８４−ΔＥ１Ｂ５５をＸｍｎＩで切断してｌｉｎｅａｒｉｚａｔｉｏｎさせた後、ＩＸ遺伝子のないｄｌ３２４−ＢｓｔＢI−ｈｕｍａｎ ｓｈＴＧＦ−β２（または、ｍｏｕｓｅ ｓｈＴＧＦ−β２）をＢｓｔＢＩで切断した後、大膓菌ＢＪ５１８３で同時に形質転換させて相同組換えを誘導した。 After confirming the effect of shRNA on TGF-β2 with a non-replicatable adenovirus, an adenovirus was produced that selectively kills cells while expressing this shRNA. In order to construct a shuttle vector that can put various genes into the E1A part of adenovirus before making tumor-selective killing and replicating adenovirus, pBSKII plasmid [Stratagene, USA] contains E1A and E1B55kDa genes A synthetic gene for pBSKII-3484 containing an enzyme site (Enzyme site) was produced [Fig. 10 (a)]. In order to change the synthesized gene into a form for introduction into a pCA14 shuttle vector for facilitating confirmation of homologous recombination, pBSKII-3484 is subjected to PCR and treated with a restriction enzyme with FspI, followed by a blunting enzyme. A blunt end was made and treated with BamHI again. pCA14 [Microbix Biosystems Inc, Canada] was prepared by cutting a restriction enzyme with SspI and then creating a blunt end with a blunting enzyme, and then treating BglII with BamHI and BglII, which are the same transfer restriction enzymes (Isoschizomer). Then, a shuttle vector pCA14-3484 was produced by inserting a gene synthesized through the blunt ends at both ends [(b) of FIG. 10]. Thereafter, the CMV promoter gene was inserted into pCA14-3484 with KpnI and XhoI to produce pCA14-CMV-3484 [(c) of FIG. 10]. Then, the E1B55 kDa part was cleaved with EcoRI and SalI restriction enzymes with pCA14-CMV-3484, blunted, and then ligated again to obtain pCA14-CMV-3484-ΔE1B55 [FIG. d)]. After the prepared shuttle vector pCA14-CMV-3484-ΔE1B55 was cleaved with XmnI and linearized, dl324-BstBI-human shTGF-β2 (or mouse shTGF-β2) without IX gene was cleaved with BstBI. Homologous recombination was induced by simultaneous transformation with O. koji BJ5183.
相同組換えされたプラスミドＤＮＡを収得してＨｉｎｄIII制限酵素で処理してＤＮＡパターンの変化を確認し、最終的に配列分析して相同組換え有無を確認した後、確認されたプラスミドをＰａｃＩで切断した後、２９３細胞株に形質転換して腫瘍を選択的に殺傷しながら人間（またはマウス）ＴＧＦ−β２の発現を抑制するｄｌ３２４−ＣＭＶ−３４８４−ｓｈＴＧＦ−β２アデノウイルスを製作した［図１１］。 The homologous recombination plasmid DNA is obtained and treated with HindIII restriction enzyme to confirm the DNA pattern change, and finally the sequence analysis is performed to confirm the presence or absence of homologous recombination, and then the confirmed plasmid is cleaved with PacI. After that, the 293 cell line was transformed to produce dl324-CMV-3484-shTGF-β2 adenovirus that suppresses the expression of human (or mouse) TGF-β2 while selectively killing the tumor [FIG. 11]. .
図１２は、実際マウスｓｈＴＧＦ−β２が含まれた腫瘍選択的複製可能アデノウイルス製作のための相同組換え過程を示すものであって、Ｅ３スクリーニング結果、１、２、４、５、６クローンがｐｏｓｉｔｉｖｅ ｃｌｏｎｅで１次選択され（ａ）、ＨｉｎｄIII切断パターン（ｄｉｇｅｓｔｉｏｎ ｐａｔｔｅｒｎ）で相同組換えされたコロニーを選択し、１、２、４番コロニーのすべてを対照区とパターン比較した結果、組換えされたものと確認され（ｂ）、ＰａｃＩ切断後、１、２、４コロニーのうちの１番だけＰａｃＩで切断して２ｋｂ程度のバンドを確認することで、１番コロニーのＤＮＡが相同組換えされたｄｌ３２４−ＣＭＶ−３４８４−△Ｅ１Ｂ５５−△Ｅ３−ｓｈ−ｍＴＧＦ−β２のＤＮＡであることを確認することができた（ｃ）。 FIG. 12 shows the process of homologous recombination for producing a tumor-selective replicable adenovirus that actually contains mouse shTGF-β2, and the E3 screening results show that 1, 2, 4, 5, and 6 clones As a result of selecting a colony that was primarily selected by positive clone (a) and homologously recombined with HindIII digestion pattern, and all of the first, second, and fourth colonies were compared with the control group, (B) After cutting with PacI, only 1st of 1, 2, and 4 colonies were cut with PacI and a band of about 2 kb was confirmed, so that the DNA of the 1st colony was homologously recombined. Dl324-CMV-3484-ΔE1B55-ΔE3-sh-mTGF-β2 can be confirmed. (C).
図１３は、人間ｓｈＴＧＦ−β２が含まれた腫瘍選択的複製可能アデノウイルス製作のための相同組換え過程を示すものであって、ＨｉｎｄIII切断パターンによる相同組換えされたクローン（１、２、３）が選択され（ａ）、前記クローン１、２、３のＤＮＡをＰａｃＩで切断した場合、クローンＤＮＡ１、２、３すべてが切断されて２ｋｂ程度のバンドが確認されており、これは、１、２、３コロニーのＤＮＡが相同組換えされたｄｌ３２４−ＣＭＶ−３４８４−ΔＥ１Ｂ５５−ΔＥ３−ｓｈ−ｈＴＧＦ−β２のＤＮＡを確認することができた（ｂ）。
実施例５：細胞溶血確認
FIG. 13 shows a homologous recombination process for producing a tumor-selective replicable adenovirus containing human shTGF-β2, which is a homologous recombination clone (1, 2, 3) using a HindIII cleavage pattern. ) Is selected and when the DNAs of clones 1, 2, and 3 are cleaved with PacI, the clone DNAs 1, 2, and 3 are all cleaved to confirm a band of about 2 kb. It was possible to confirm the DNA of dl324-CMV-3484-ΔE1B55-ΔE3-sh-hTGF-β2 in which DNA of a few colonies was homologously recombined (b).
Example 5: Confirmation of cell hemolysis
複製可能なアデノウイルスの細胞殺傷能を確認するために、２４ウェルプレート（ｗｅｌｌ ｐｌａｔｅ）に各種類の細胞を４×１０４から１×１０５まで細胞の大きさに応じて細胞数を定めた後、分株して培養した後、翌日ｓｕｒｖｉｖｉｎ ｐｒｏｍｏｔｅｒとＣＭＶプロモーターを有した複製可能なアデノウイルスをＭＯＩごとに感染させて養成対照群である２９３Ａ細胞株から最も低いＭＯＩで細胞がウイルスによってすべて死んだときに実験を終了して、各プレートに死なないで生きていた細胞をクリスタルバイオレットで染色した。３．７％パラホルムアルデヒド（Ｐａｒａｆｏｒｍ ａｌｄｅｈｙｄｅ）で細胞を５分間常温で固定した後、０．０５％のクリスタルバイオレットにより常温で３０分間染色した後、水で洗浄して染色された細胞を観察した。２つの種類の腫瘍殺傷ウイルスでウイルスの腫瘍殺傷効果を比較した結果、プロモーターによる殺傷効果との差は大きくないものと示され、２つとも腫瘍選択性が優れるものとして確認された。 In order to confirm the cell killing ability of replicable adenovirus, the number of cells was determined according to the cell size from 4 × 10 4 to 1 × 10 5 in a 24-well plate (well plate). Then, after dividing and culturing, the cells were killed by the virus at the lowest MOI from the 293A cell line, which was a control group, by infecting each MOI with a replicable adenovirus having survivin promoter and CMV promoter the next day. At that time, the experiment was terminated, and cells that did not die on each plate were stained with crystal violet. The cells were fixed with 3.7% paraformaldehyde for 5 minutes at room temperature, then stained with 0.05% crystal violet for 30 minutes at room temperature, washed with water, and the stained cells were observed. As a result of comparing the tumor killing effect of the virus with the two types of tumor killing viruses, it was shown that the difference from the killing effect by the promoter was not large, and both were confirmed to have excellent tumor selectivity.
図１４は、腫瘍選択的複製可能アデノウイルス（ＣＭＶ ｐｒｏｍｏｔｅｒとＥ１Ｂ ５５ＫＤａが発現されないｄｌ３２４−ＣＭＶ−３４８４とｓｕｒｖｉｖｉｎｇ ｐｒｏｍｏｔｅｒにより選択性を付与し、５５ＫＤａが発現されるｄｌ３２４−ｈＳｕｒｖｉｖｉｎ−３４８４）が正常細胞（ＢＪ細胞）では、複製が起きないのに対し、多様な種類の人間癌細胞では複製が起きて細胞の溶血が起きることを示したものである。
実施例６：癌細胞での効果確認
FIG. 14 shows that tumor-selective replicable adenoviruses (dl324-CMV-3484 in which CMV promoter and E1B 55KDa are not expressed and surviving promoter, and dl324-hSurvivin-3484 in which 55 KDa is expressed are normal cells ( In BJ cells), replication does not occur, whereas in various types of human cancer cells, replication occurs and cell hemolysis occurs.
Example 6: Confirmation of effect in cancer cells
Ａ３７５メルラノ−マ細胞株で人間ｓｈ−ＴＧＦ−β１またはｓｈ−ＴＧＦ−β２を発現する実施例２の複製不能アデノウイルスを１、５、１０、５０、１００ｍｏｉで感染させてから細胞内に存在するＴＧＦ−β１、ＴＧＦ−β２、ＴＧＦ−β３ｍＲＮＡの水準をリアルタイムＰＣＲ方法で行った。 Infected with the non-replicating adenovirus of Example 2 expressing human sh-TGF-β1 or sh-TGF-β2 in the A375 merlanoma cell line at 1, 5, 10, 50, 100 moi and then intracellular The levels of TGF-β1, TGF-β2, and TGF-β3 mRNA were measured by a real-time PCR method.
その結果、ＴＧＦ−β１のｓｈＲＮＡを発現する場合、細胞内ＴＧＦ−β１ ｍＲＮＡを減少させるものの、ＴＧＦ−β２ ｍＲＮＡやＴＧＦ−β３ ｍＲＮＡが増加する傾向を示した。一方、ＴＧＦ−β２のｓｈＲＮＡを発現する場合、細胞内ＴＧＦ−β２ ｍＲＮＡを効果的に減少させると共に、ＴＧＦ−β２ ｍＲＮＡやＴＧＦ−β３ ｍＲＮＡが減少する傾向を示した［図１５］。これは、細胞内の補償効果側面による効能低下の心配が、少なくともＴＧＦ−β２のｓｈＲＮＡを発現する場合には、その現象がなかっただけでなく、他のＴＧＦ−βのｉｓｏｔｙｐｅも抑制させる付随効果も有することができる長所となる。これと同様な結果がＥＬＩＳＡを用いてＴＧＦ−βタンパク質の発現減少効果パターンにもリアルタイムＰＣＲで同様に現れた［図１６］。 As a result, when TGF-β1 shRNA was expressed, intracellular TGF-β1 mRNA was decreased, but TGF-β2 mRNA and TGF-β3 mRNA tended to increase. On the other hand, when TGF-β2 shRNA was expressed, intracellular TGF-β2 mRNA was effectively reduced and TGF-β2 mRNA and TGF-β3 mRNA tended to decrease [FIG. 15]. This is because there is no concern that the effect of reducing the efficacy due to the intracellular compensation effect side is expressed at least when TGF-β2 shRNA is expressed, but also the accompanying effect of suppressing other TGF-β isotypes. It can also be an advantage. A result similar to this appeared in real-time PCR in the expression reduction effect pattern of TGF-β protein using ELISA [FIG. 16].
これは、ＴＧＦ−β２に対するｓｈＲＮＡ発現するアデノウイルスが、ＴＧＦ−β１に対するｓｈＲＮＡ発現するアデノウイルスよりも発現抑制効果が相対的に優れることを確認させたことである。 This is to confirm that the adenovirus expressing shRNA against TGF-β2 has a relatively superior expression suppressing effect than the adenovirus expressing shRNA against TGF-β1.
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Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20160201 |