JP6637652B2 - Method for selecting antitumor agent and siRNA contained in the antitumor agent - Google Patents
Method for selecting antitumor agent and siRNA contained in the antitumor agent Download PDFInfo
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Description
本発明は、抗腫瘍剤の選択方法およびその抗腫瘍剤が含有するｓｉＲＮＡに関する。 The present invention relates to a method for selecting an antitumor agent and an siRNA contained in the antitumor agent.
ＭＤＭ２およびＭＤＭ４は癌遺伝子であって、ＴＰ５３がん抑制遺伝子を抑制的に制御する（非特許文献１参照）。従って、ＭＤＭ２およびＭＤＭ４を人為的に制御することによる、腫瘍細胞の治療方法の開発が期待される。 MDM2 and MDM4 are oncogenes and suppressively regulate the TP53 tumor suppressor gene (see Non-Patent Document 1). Therefore, development of a method for treating tumor cells by artificially controlling MDM2 and MDM4 is expected.
しかしながら、どのような場合に、ＭＤＭ２またはＭＤＭ４をどのように制御すれば腫瘍細胞の増殖を制御できるかは明らかではなかった。 However, it was not clear in any case how to control MDM2 or MDM4 to control the growth of tumor cells.
本発明は、効率が良く特異性の高い抗腫瘍剤の選択方法およびその抗腫瘍剤が含有するｓｉＲＮＡを提供することを目的とする。 An object of the present invention is to provide a method for selecting an antitumor agent with high efficiency and high specificity and an siRNA contained in the antitumor agent.
本発明にかかる一実施態様は、腫瘍を治療するための抗腫瘍剤の選択方法であって、採取した腫瘍細胞のＭＤＭ２および／またはＭＤＭ４の発現を調べる工程と、採取した腫瘍細胞においてＭＤＭ４の発現が高い場合、または採取した腫瘍細胞においてＭＤＭ２の発現が低い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ４の発現が低い場合、または採取した腫瘍細胞においてＭＤＭ２の発現が高い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する工程と、を含む抗腫瘍剤の選択方法である。前記腫瘍細胞においてＭＤＭ２の発現が高い場合、ＭＤＭ４の発現を抑制しなくてもよい。また、前記腫瘍細胞が、正常に発現制御される野生型ｐ５３を有していることが好ましい。 One embodiment according to the present invention relates to a method for selecting an antitumor agent for treating a tumor, which comprises examining the expression of MDM2 and / or MDM4 in the collected tumor cells, and expressing the MDM4 in the collected tumor cells. Is high, or when the expression of MDM2 is low in the collected tumor cells, an inhibitor that suppresses the expression of MDM2 and / or MDM4 is selected as an antitumor agent, and when the expression of MDM4 is low in the collected tumor cells, or A step of selecting, as an antitumor agent, an inhibitor that suppresses the expression of MDM2 when the expression of MDM2 is high in the collected tumor cells. When the expression of MDM2 is high in the tumor cells, the expression of MDM4 need not be suppressed. In addition, it is preferable that the tumor cells have wild-type p53 whose expression is regulated normally.
上記実施態様において、前記ＭＤＭ２を抑制する抑制剤が、以下の配列からなる二重鎖部分を有するｓｉＮＡを含有してもよい。 In the above embodiment, the inhibitor for suppressing MDM2 may contain siNA having a double-stranded portion having the following sequence.
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
また、前記ＭＤＭ４を抑制する抑制剤が、以下の配列からなる二重鎖部分を有するｓｉＮＡからなる群:
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４）
GGUCAUGCACUAUUUAGGN （配列番号５）
CCAGUACGUGAUAAAUCCN （配列番号６）
GGUGAAGCAACUUUAUGAN （配列番号７）
CCACUUCGUUGAAAUACUN （配列番号８）
CAACUAUACACCUAGAAGN （配列番号９）
GUUGAUAUGUGGAUCUUCN （配列番号１０）
GUGAUGAUACCGAUGUAGN （配列番号１１）
CACUACUAUGGCUACAUCN （配列番号１２）
より選択されるｓｉＮＡを含有してもよい。。
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
In addition, the inhibitor that suppresses MDM4 is a group consisting of siNA having a double-stranded portion having the following sequence:
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
GGUCAUGCACUAUUUAGGN (SEQ ID NO: 5)
CCAGUACGUGAUAAAUCCN (SEQ ID NO: 6)
GGUGAAGCAACUUUAUGAN (SEQ ID NO: 7)
CCACUUCGUUGAAAUACUN (SEQ ID NO: 8)
CAACUAUACACCUAGAAGN (SEQ ID NO: 9)
GUUGAUAUGUGGAUCUUCN (SEQ ID NO: 10)
GUGAUGAUACCGAUGUAGN (SEQ ID NO: 11)
CACUACUAUGGCUACAUCN (SEQ ID NO: 12)
It may contain more selected siNA. .
本発明にかかる他の一実施態様は、ＭＤＭ２の発現を抑制するためのｓｉＮＡであって、以下の配列からなる二重鎖部分を有するｓｉＮＡである。 Another embodiment according to the present invention is a siNA for suppressing the expression of MDM2, which has a double-stranded portion having the following sequence.
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
本発明にかかるさらなる一実施態様は、ＭＤＭ４の発現を抑制するためのｓｉＮＡであって、以下の配列からなる二重鎖部分を有するｓｉＮＡからなる群:
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４）
GGUCAUGCACUAUUUAGGN （配列番号５）
CCAGUACGUGAUAAAUCCN （配列番号６）
GGUGAAGCAACUUUAUGAN （配列番号７）
CCACUUCGUUGAAAUACUN （配列番号８）
CAACUAUACACCUAGAAGN （配列番号９）
GUUGAUAUGUGGAUCUUCN （配列番号１０）
GUGAUGAUACCGAUGUAGN （配列番号１１）
CACUACUAUGGCUACAUCN （配列番号１２）
より選択されるｓｉＮＡである。
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
A further embodiment according to the present invention is a siNA for inhibiting the expression of MDM4, the group consisting of a siNA having a double-stranded part consisting of the following sequence:
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
GGUCAUGCACUAUUUAGGN (SEQ ID NO: 5)
CCAGUACGUGAUAAAUCCN (SEQ ID NO: 6)
GGUGAAGCAACUUUAUGAN (SEQ ID NO: 7)
CCACUUCGUUGAAAUACUN (SEQ ID NO: 8)
CAACUAUACACCUAGAAGN (SEQ ID NO: 9)
GUUGAUAUGUGGAUCUUCN (SEQ ID NO: 10)
GUGAUGAUACCGAUGUAGN (SEQ ID NO: 11)
CACUACUAUGGCUACAUCN (SEQ ID NO: 12)
This is the siNA that is more selected.
本発明にかかるさらなる一実施態様は、ＭＤＭ２およびＭＤＭ４の発現を同時に抑制するためのｓｉＮＡペアであって、上記ＭＤＭ２の発現を抑制するためのｓｉＮＡと、上記ＭＤＭ４の発現を抑制するためのｓｉＮＡからなるｓｉＲＮＡペアである。特に、下記配列からなるｓｉＮＡペアであることが好ましい。 A further embodiment according to the present invention relates to a siNA pair for simultaneously suppressing the expression of MDM2 and MDM4, wherein the siNA for suppressing the expression of MDM2 and the siNA for suppressing the expression of MDM4 comprise SiRNA pairs. In particular, a siNA pair having the following sequence is preferable.
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４）
本発明にかかるさらなる一実施態様は、上記ｓｉＮＡペアを含有する抗腫瘍剤である。
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
A further embodiment according to the present invention is an antitumor agent comprising the above siNA pair.
本発明によって、効率が良く特異性の高い抗腫瘍剤の選択方法およびその抗腫瘍剤が含有するｓｉＲＮＡを提供することができるようになった。 According to the present invention, it has become possible to provide a method for selecting an antitumor agent with high efficiency and high specificity, and an siRNA containing the antitumor agent.
以下、上記知見に基づき完成した本発明の実施の形態を、実施例を挙げながら詳細に説明する。 Hereinafter, embodiments of the present invention completed based on the above findings will be described in detail with reference to examples.
実施の形態及び実施例に特に説明がない場合には、M. R. Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.などの標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いる場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。 Unless otherwise described in the embodiments and examples, MR Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012 ); Described in standard protocols such as FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Or a method that is modified or modified. When a commercially available reagent kit or measuring device is used, unless otherwise specified, the protocol attached thereto is used.
なお、本発明の目的、特徴、利点、および、そのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的な実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をこれらに限定するものではない。本明細書で開示されている本発明の意図並びに範囲内で、本明細書の記載に基づき、様々な改変並びに修飾ができることは、当業者にとって明らかである。 The purpose, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art will be able to easily reproduce the present invention from the description in the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description in the present specification within the spirit and scope of the present invention disclosed herein.
（１）ＭＤＭ２および／またはＭＤＭ４高発現腫瘍細胞の治療
本発明の一実施態様において、腫瘍を治療するための薬剤の選択方法は、採取した腫瘍細胞のＭＤＭ２および／またはＭＤＭ４の発現を調べる工程と、採取した腫瘍細胞においてＭＤＭ４の発現が高い場合、または採取した腫瘍細胞においてＭＤＭ２の発現が低い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ４の発現が低い場合、または採取した腫瘍細胞においてＭＤＭ２の発現が高い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する工程とを含む。
(1) Treatment of MDM2 and / or MDM4 Highly Expressing Tumor Cells In one embodiment of the present invention, a method for selecting an agent for treating a tumor comprises a step of examining the expression of MDM2 and / or MDM4 in the collected tumor cells. When the expression of MDM4 is high in the collected tumor cells, or when the expression of MDM2 is low in the collected tumor cells, an inhibitor that suppresses the expression of MDM2 and / or MDM4 is selected as an antitumor agent, and the collected tumor cells Selecting the inhibitor that suppresses the expression of MDM2 as an antitumor agent when the expression of MDM4 is low or the expression of MDM2 is high in the collected tumor cells.
この選択方法には、具体的には少なくとも以下のような場合が含まれる。 This selection method specifically includes at least the following cases.
（１）採取した腫瘍細胞のＭＤＭ２の発現を調べ、採取した腫瘍細胞において採取した腫瘍細胞においてＭＤＭ２の発現が低い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ２の発現が高い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する。 (1) The expression of MDM2 in the collected tumor cells was examined, and when the expression of MDM2 was low in the collected tumor cells, an inhibitor that suppressed the expression of MDM2 and / or MDM4 was selected as an antitumor agent. When the expression of MDM2 is high in the collected tumor cells, an inhibitor that suppresses the expression of MDM2 is selected as an antitumor agent.
（２）採取した腫瘍細胞のＭＤＭ４の発現を調べ、採取した腫瘍細胞においてＭＤＭ４の発現が高い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ４の発現が低い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する。 (2) Examining the expression of MDM4 in the collected tumor cells, and when the expression of MDM4 is high in the collected tumor cells, selecting an inhibitor that suppresses the expression of MDM2 and / or MDM4 as an antitumor agent, and collecting the extracted tumor cells In the case where the expression of MDM4 is low, an inhibitor that suppresses the expression of MDM2 is selected as an antitumor agent.
（３）採取した腫瘍細胞のＭＤＭ２およびＭＤＭ４の発現を調べ、採取した腫瘍細胞においてＭＤＭ４の発現が高い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ２の発現が高い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する。 (3) The expression of MDM2 and MDM4 in the collected tumor cells was examined, and when the expression of MDM4 was high in the collected tumor cells, an inhibitor that suppressed the expression of MDM2 and / or MDM4 was selected as an antitumor agent and collected. When the expression of MDM2 is high in tumor cells, an inhibitor that suppresses the expression of MDM2 is selected as an antitumor agent.
（４）採取した腫瘍細胞のＭＤＭ２およびＭＤＭ４の発現を調べ、採取した腫瘍細胞においてＭＤＭ２の発現が低い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制する抑制剤を抗腫瘍剤として選択し、採取した腫瘍細胞においてＭＤＭ４の発現が低い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する。 (4) The expression of MDM2 and MDM4 in the collected tumor cells was examined, and when the expression of MDM2 was low in the collected tumor cells, an inhibitor that suppressed the expression of MDM2 and / or MDM4 was selected as an antitumor agent and collected. When the expression of MDM4 is low in tumor cells, an inhibitor that suppresses the expression of MDM2 is selected as an antitumor agent.
ここで、高いか低いかは、正常組織における発現を標準レベルとし、標準レベルと比べて判断することとする。この正常組織における発現レベルは、腫瘍細胞が由来する個体とは他の個体の発現レベルであってもよいが、腫瘍細胞と同一個体の発現レベルであることが好ましい。また、腫瘍細胞が由来する組織と異なる組織であってもよいが、腫瘍細胞が由来する組織であることが好ましい。例えば、腫瘍細胞が由来する組織と同じ組織に腫瘍を有しない複数の健常人の当該組織における平均のレベルや、腫瘍細胞が由来する個体において腫瘍細胞が由来する組織のうちの健常な細胞におけるレベルなどが考えられる。 Here, whether the expression is high or low is determined by comparing the expression in a normal tissue with the standard level and comparing the expression with the standard level. The expression level in the normal tissue may be the expression level of an individual other than the individual from which the tumor cells are derived, but is preferably the expression level of the same individual as the tumor cells. The tissue may be different from the tissue from which the tumor cells are derived, but is preferably the tissue from which the tumor cells are derived. For example, the average level in a plurality of healthy individuals having no tumor in the same tissue as the tissue from which the tumor cells are derived, or the level in healthy cells of the tissue from which the tumor cells are derived in the individual from which the tumor cells are derived And so on.
なお、これらの腫瘍において、発現制御が正常に行われる野生型ｐ５３を有することが好ましい。 It is preferable that these tumors have wild-type p53 whose expression is controlled normally.
腫瘍細胞が由来する腫瘍組織は特に限定されないが、固形腫瘍であることが好ましく、乳がん、子宮がん、胃がん、大腸がん、膵がん、肝がん、肺がん、前立腺がん、骨肉腫、メラノーマ、などが例示できる。 The tumor tissue from which the tumor cells are derived is not particularly limited, but is preferably a solid tumor, such as breast cancer, uterine cancer, stomach cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, osteosarcoma, Melanoma and the like can be exemplified.
ここで、抑制剤は特に限定されないが、アンチセンス核酸、ｓｉＮＡ（すなわち、ｓｉＲＮＡ、ｓｉＤＮＡ，及びｓｉＲＮＡの一部をＤＮＡに置換したキメラ分子）、ｓｈＮＡ（すなわち、ｓｈＲＮＡ、ｓｈＤＮＡ，及びｓｈＲＮＡの一部をＤＮＡに置換したキメラ分子）、ボナック核酸、核酸アプタマー、デコイ核酸などの核酸医薬であることが好ましく、ｓｉＮＡが特に好ましい。これら核酸配列の決定は、当業者には容易であるが、ｓｉＮＡにおいて、腫瘍抑制効果が高く、オフターゲット作用による腫瘍毒性が少ない点で、好ましい配列を以下に述べる。なお、本明細書において、塩基配列はｓｉＲＮＡの場合で代表させて述べるので、特に明記されていない場合、Ｕ（ウラシル）はＴ（チミン）の意味を含むものとする。実際にｓｉＤＮＡやキメラ分子を用いる場合は、Ｕの代わりにＴに用いるものとする。また、ｓｉＮＡを二重らせんの形で表す、すなわち２列の配列で示す場合、上方の鎖をパッセンジャー鎖、下方の鎖をガイド鎖の配列とし、パッセンジャー鎖は左が５‘端、ガイド鎖は右が５’端とする。 Here, although the inhibitor is not particularly limited, antisense nucleic acid, siNA (that is, siRNA, siDNA, and a chimeric molecule in which a part of siRNA is replaced with DNA), shNA (that is, part of shRNA, shDNA, and shRNA) are used. Chimeric molecule in which is replaced by DNA), bonac nucleic acids, nucleic acid aptamers, decoy nucleic acids, and other nucleic acid drugs, and siNA is particularly preferable. Although the determination of these nucleic acid sequences is easy for those skilled in the art, preferred sequences are described below because siNA has a high tumor-suppressing effect and little tumor toxicity due to off-target effects. In the present specification, the base sequence will be described using the case of siRNA as an example. Unless otherwise specified, U (uracil) includes the meaning of T (thymine). When actually using an siRNA or a chimeric molecule, T is used instead of U. When siNA is represented in the form of a double helix, that is, in the case of a two-row sequence, the upper strand is the passenger strand, the lower strand is the guide strand sequence, the passenger strand is the 5 'end on the left, and the guide strand is the guide strand. The right is the 5 'end.
＜腫瘍細胞においてＭＤＭ４の発現が高い場合＞
本発明のｓｉＮＡは、腫瘍細胞においてＭＤＭ４の発現が高い場合における、ＭＤＭ２および／またはＭＤＭ４の発現を抑制するためのｓｉＮＡであって、ＭＤＭ２の発現を抑制するためのｓｉＮＡの二重鎖部分が、
１４８９：
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
であって、
ＭＤＭ４の発現を抑制するためのｓｉＮＡの二重鎖部分が、以下の配列を有するｓｉＮＡからなる群:
４５２：
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４）
３１７：
GGUCAUGCACUAUUUAGGN （配列番号５）
CCAGUACGUGAUAAAUCCN （配列番号６）
３４７：
GGUGAAGCAACUUUAUGAN （配列番号７）
CCACUUCGUUGAAAUACUN （配列番号８）
７８８：
CAACUAUACACCUAGAAGN （配列番号９）
GUUGAUAUGUGGAUCUUCN （配列番号１０）
１０３６：
GUGAUGAUACCGAUGUAGN （配列番号１１）
CACUACUAUGGCUACAUCN （配列番号１２）
より選択される。ＭＤＭ２とＭＤＭ４の発現の一方を抑制する場合、いずれか一方の配列を用いて発現を抑制すればよく、ＭＤＭ２とＭＤＭ４の発現の両方を抑制する場合、両方の配列を用いて発現を抑制すればよい。
<When MDM4 expression is high in tumor cells>
The siNA of the present invention is a siNA for suppressing the expression of MDM2 and / or MDM4 when the expression of MDM4 is high in a tumor cell, wherein the double-stranded portion of the siNA for suppressing the expression of MDM2 is:
1489:
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
And
A group in which the double-stranded portion of siNA for suppressing the expression of MDM4 consists of siNA having the following sequence:
452:
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
317:
GGUCAUGCACUAUUUAGGN (SEQ ID NO: 5)
CCAGUACGUGAUAAAUCCN (SEQ ID NO: 6)
347:
GGUGAAGCAACUUUAUGAN (SEQ ID NO: 7)
CCACUUCGUUGAAAUACUN (SEQ ID NO: 8)
788:
CAACUAUACACCUAGAAGN (SEQ ID NO: 9)
GUUGAUAUGUGGAUCUUCN (SEQ ID NO: 10)
1036:
GUGAUGAUACCGAUGUAGN (SEQ ID NO: 11)
CACUACUAUGGCUACAUCN (SEQ ID NO: 12)
More choice. When suppressing one of the expression of MDM2 and MDM4, the expression may be suppressed by using one of the sequences. When suppressing the expression of both MDM2 and MDM4, the expression may be suppressed by using both sequences. Good.
ここで、パッセンジャー鎖の３‘端の塩基とガイド鎖の５’端の塩基は標的ｍＲＮＡと塩基対を形成しないことが明らかになっており（Betancur et al., Frontiers in Genetics vol.3, p.1-6, 2012; Ma. J-B et al., Nature vol.434, p.666-670, 2005; Frank F. Nature vol.465, p.818-822, 2010 + supplemental information）、どの塩基であってもｓｉＮＡの発現抑制能は低下しないと考えられるので、特に限定されない。 Here, it has been revealed that the base at the 3 ′ end of the passenger strand and the base at the 5 ′ end of the guide strand do not form a base pair with the target mRNA (Betancur et al., Frontiers in Genetics vol. 3, p. .1-6, 2012; Ma.JB et al., Nature vol.434, p.666-670, 2005; Frank F. Nature vol.465, p.818-822, 2010 + supplemental information), which base Even if it is present, the ability to suppress the expression of siNA is not considered to be reduced, and thus is not particularly limited.
また、これらのｓｉＮＡは、片側の鎖または両側の鎖の３‘末端にオーバーハングが付加されていてもよく、その配列及びヌクレオチドの個数は限定されないが、配列はｍＲＮＡに相補的な配列、あるいはポリＴであることが好ましく、個数は２個であることが好ましい。 In addition, these siNAs may have an overhang added to the 3 ′ end of one strand or both strands, and the sequence and the number of nucleotides are not limited, but the sequence is a sequence complementary to mRNA, or It is preferably poly T, and the number is preferably two.
＜腫瘍細胞においてＭＤＭ２の発現が高い場合＞
本発明のｓｉＮＡは、腫瘍細胞においてＭＤＭ２の発現が高い場合における、ＭＤＭ２の発現を抑制するためのｓｉＮＡであって、ＭＤＭ２の発現を抑制するためのｓｉＲＮＡが、
１４８９：
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
である。
<When MDM2 expression is high in tumor cells>
The siNA of the present invention is a siNA for suppressing the expression of MDM2 when the expression of MDM2 is high in a tumor cell, wherein the siRNA for suppressing the expression of MDM2 is:
1489:
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
It is.
この時、ＭＤＭ４の発現を抑制してもよいが、抑制しなくてもかまわない。発現を抑制する場合は、腫瘍細胞においてＭＤＭ４の発現が高い場合に用いられる上記５つの配列から選択してもよい。 At this time, expression of MDM4 may be suppressed, but may not be suppressed. When the expression is suppressed, it may be selected from the above five sequences used when the expression of MDM4 is high in tumor cells.
（２）医薬への応用
本発明に係る抗腫瘍剤はまた、有効成分である核酸の他、必要に応じて、一般に用いられる各種成分をさらに含み得るものであり、例えば、1種以上の医薬的に許容され得る賦形剤、崩壊剤、希釈剤、滑沢剤、着香剤、着色剤、甘味剤、矯味剤、懸濁化剤、湿潤剤、乳化剤、分散剤、補助剤、防腐剤、緩衝剤、結合剤、安定剤、コーティング剤などを含み得る。
(2) Application to medicine The antitumor agent according to the present invention may further contain, if necessary, various commonly used components in addition to the nucleic acid as an active ingredient. Excipients, disintegrants, diluents, lubricants, flavors, colorants, sweeteners, flavoring agents, suspending agents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives , Buffers, binders, stabilizers, coatings and the like.
投与経路は、全身投与または局所投与のいずれも選択することができる。この場合、疾患、症状などに応じた適当な投与経路を選択する。本発明に係る抗腫瘍剤は、経口経路、非経口経路のいずれによっても投与できる。非経口経路としては、通常の静脈内投与、動脈内投与の他、皮下、皮内、筋肉内などへの投与を挙げることができる。さらに、経粘膜投与または経皮投与を実施することができる。 The administration route can be selected from either systemic administration or local administration. In this case, an appropriate administration route is selected according to the disease, symptom and the like. The antitumor agent according to the present invention can be administered by either the oral route or the parenteral route. Parenteral routes include subcutaneous, intradermal, intramuscular, etc., in addition to normal intravenous and intraarterial administration. Further, transmucosal or transdermal administration can be performed.
剤形は、特に限定されず、種々の剤形、例えば、経口投与のためには、錠剤、カプセル剤、散剤、顆粒剤、丸剤、液剤、乳剤、懸濁液、溶液剤、酒精剤、シロップ剤、エキス剤、エリキシル剤とすることができる。非経口剤としては、例えば、皮下注射剤、静脈内注射剤、筋肉内注射剤、腹腔内注射剤などの注射剤；経皮投与または貼付剤、軟膏またはローション；口腔内投与のための舌下剤、口腔貼付剤；ならびに経鼻投与のためのエアゾール剤；坐剤とすることができるが、これらには限定されない。これらの製剤は、製剤工程において通常用いられる公知の方法により製造することができる。また本発明に係る薬剤は、持続性または徐放性剤形であってもよい。 The dosage form is not particularly limited, and various dosage forms, for example, for oral administration, tablets, capsules, powders, granules, pills, solutions, emulsions, suspensions, solutions, spirits, Syrups, extracts and elixirs can be prepared. Parenteral preparations include, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections; transdermal or patch, ointment or lotion; sublinguals for oral administration Oral patches; and aerosols for nasal administration; suppositories, but are not limited to these. These preparations can be manufactured by a known method usually used in a preparation process. The medicament according to the present invention may be in a sustained or sustained release form.
注射剤を調製する場合は、有効成分にpH調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤などを添加し、常法により皮下、筋肉内及び静脈内用注射剤を製造することができる。この場合のpH調節剤及び緩衝剤としてはクエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどを挙げることができる。安定化剤としてはピロ亜硫酸ナトリウム、 エチレンジアミン四酢酸（EDTA）、チオグリコール酸、チオ乳酸などを挙げることができる。局所麻酔剤としては塩酸プロカイン、塩酸リドカインなどを挙げることができる。等張化剤としては、塩化ナトリウム、ブドウ糖などが例示できる。 When preparing an injection, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the active ingredient, and a subcutaneous, intramuscular and intravenous injection is prepared by a conventional method. can do. In this case, examples of the pH adjusting agent and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the tonicity agent include sodium chloride and glucose.
軟膏剤を調製する場合は、有効成分に通常使用される基剤、安定剤、湿潤剤、保存剤などが必要に応じて配合され、常法により混合、製剤化される。基剤としては、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィンなどを挙げることができる。保存剤としては、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピルなどを挙げることができる。 When preparing an ointment, a base, a stabilizer, a wetting agent, a preservative, and the like, which are usually used for an active ingredient, are blended as necessary, and mixed and formulated according to a conventional method. Examples of the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.
抗腫瘍剤に含有される有効成分の量は、該有効成分の用量範囲や投薬の回数などにより適宜決定できる。 The amount of the active ingredient contained in the antitumor agent can be appropriately determined depending on the dose range of the active ingredient, the number of times of administration, and the like.
用量範囲は特に限定されず、含有される成分の有効性、投与形態、投与経路、疾患の種類、対象の性質（体重、年齢、病状および他の医薬の使用の有無など）、および担当医師の判断など応じて適宜選択される。一般的には適当な用量は、例えば対象の体重1kgあたり約0．01μg〜100mg程度、好ましくは約0．1μg1mg程度の範囲であることが好ましい。しかしながら、当該分野においてよく知られた最適化のための一般的な常套的実験を用いてこれらの用量の変更を行うことができる。上記投与量は1日1回乃至数回に分けて投与することができる。 The dose range is not particularly limited, and the efficacy of the ingredients contained, dosage form, administration route, type of disease, nature of the subject (body weight, age, medical condition and whether or not other medicines are used, etc.), and the It is appropriately selected according to the judgment. In general, a suitable dose is, for example, in the range of about 0.01 μg to 100 mg, preferably about 0.1 μg 1 mg / kg body weight of the subject. However, variations in these dosages can be made using routine experimentation for optimization well known in the art. The above dose can be administered once or several times a day.
（１）腫瘍細胞株におけるＭＤＭ２及びＭＤＭ４の発現とｓｉＲＮＡによる発現抑制
各腫瘍細胞株（表１参照）の細胞抽出液を用いて、ウエスタンブロッティングを行った。
(1) Expression of MDM2 and MDM4 in tumor cell lines and suppression of expression by siRNA Western blotting was performed using a cell extract of each tumor cell line (see Table 1).
１次抗体として、抗ＭＤＭ２マウスモノクローナル抗体（２Ａ１０）（Abcam社）、抗ＭＤＭ４ウサギポリクローナル抗体（Bethyl Laboratories社）、抗ｐ５３マウスモノクローナル抗体（ＢＰ５３−１２）（Cell Sciences社）、抗ｐ２１マウスモノクローナル抗体（ＤＣＳ６０）（Cell Signaling Technology社）、抗βアクチンマウスモノクローナル抗体（８Ｈ１０Ｄ１０）（Cell Signaling Technology社）を用い、２次抗体は、ＨＲＰ結合抗マウスＩｇＧヒツジ抗体及びＨＲＰ結合抗ウサギＩｇＧロバ抗体（GE Healthcare社）を用いた。ＨＲＰの基質としてECL Prime Western Blotting Detection Reagent (GE Healthare社)を用い、Ez-Capture II Imaging System (アトー株式会社)によって化学発光を検出した。結果を図１に示す。 As primary antibodies, anti-MDM2 mouse monoclonal antibody (2A10) (Abcam), anti-MDM4 rabbit polyclonal antibody (Bethyl Laboratories), anti-p53 mouse monoclonal antibody (BP53-12) (Cell Sciences), anti-p21 mouse monoclonal antibody (DCS60) (Cell Signaling Technology) and anti-β-actin mouse monoclonal antibody (8H10D10) (Cell Signaling Technology), and the secondary antibodies were HRP-conjugated anti-mouse IgG sheep antibody and HRP-conjugated anti-rabbit IgG donkey antibody (GE Healthcare). Using ECL Prime Western Blotting Detection Reagent (GE Healthare) as a substrate for HRP, chemiluminescence was detected by an Ez-Capture II Imaging System (Ato Corporation). The results are shown in FIG.
wtTP53と示したＭＣＦ−７からＡ５４９まで細胞株は野生型のｐ５３遺伝子を有しており、ｐ５３の発現は抑制されている。これらの細胞では、ＭＤＭ２かＭＤＭ４のいずれかが高レベルで発現している。また、mtTP52と示したＫａｔｏＩＩＩからＤＬＤ−１までの細胞株は変異型の不活性なｐ５３遺伝子を有しているため、その発現が検出される細胞株もあるが、いずれもＭＤＭ４が中〜高レベルで発現していた。 Cell lines from MCF-7 to A549 indicated as wtTP53 have a wild-type p53 gene, and the expression of p53 is suppressed. In these cells, either MDM2 or MDM4 is expressed at high levels. Further, since the cell lines from KatoIII to DLD-1 indicated as mtTP52 have a mutated inactive p53 gene, there are cell lines whose expression is detected. Was expressed at the level.
これらの細胞でＭＤＭ２またはＭＤＭ４の発現を抑制するため、表２（ＭＤＭ２）及び表３（ＭＤＭ４）に示す配列を有するｓｉＲＮＡ及キメラｓｉＮＡを、Lipofectamine RNAiMAX （Invitrogen社）を用いて細胞に導入（６穴ディッシュの場合、導入前日に０．６〜１．５x１０５個播種した培養に対し、１ｎＭのｓｉＲＮＡを用い、９６穴ディッシュの場合、導入前日に０．５〜１．５x１０３個播種した培養に対し、０．２、１．０、５．０ｎＭのｓｉＲＮＡを用いた）し、ＭＤＭ２またはＭＤＭ４の発現及びｐ５３の発現を同様にウエスタンブロッティングで調べた。その結果を図２及び表４（ＭＤＭ２）、図３及び表５（ＭＤＭ４）に示す。図３では、ｐ５３の活性化の指標として、ｐ２１の発現も同時に調べた。なお、表２においては、３４７から１７１３までは公知の配列であり、４８０から７４１４までは新規に設計した配列であり、表３においては、２３５から１６０３までは公知の配列であり、３１７から２２６３までは新規に設計した配列である。コントロールとしては、ｓｉＲＮＡ不添加（Mock）、無関係な配列を有するｓｉＲＮＡ（Naito-1）、ランダムな配列を有するｓｉＲＮＡ（Naito random）を用いた。これらの配列を表６に示す。 In order to suppress the expression of MDM2 or MDM4 in these cells, siRNA and chimeric siNA having the sequences shown in Table 2 (MDM2) and Table 3 (MDM4) were introduced into the cells using Lipofectamine RNAiMAX (Invitrogen) (6). In the case of a well dish, 0.6-1.5 × 10 5 cells were seeded on the day before the introduction, and 1 nM siRNA was used. In the case of a 96-well dish, 0.5-1.5 × 10 3 cells were seeded on the day before the introduction. Again, 0.2, 1.0, 5.0 nM siRNA was used), and the expression of MDM2 or MDM4 and the expression of p53 were similarly examined by Western blotting. The results are shown in FIG. 2 and Table 4 (MDM2), and FIG. 3 and Table 5 (MDM4). In FIG. 3, p21 expression was also examined simultaneously as an indicator of p53 activation. In Table 2, 347 to 1713 are known sequences, 480 to 7414 are newly designed sequences, and in Table 3, 235 to 1603 are known sequences, and 317 to 2263 are known sequences. Up to this is a newly designed sequence. As controls, siRNA-free (Mock), siRNA having an unrelated sequence (Naito-1), and siRNA having a random sequence (Naito random) were used. These sequences are shown in Table 6.
また、いくつかの配列に対しては、キメラｓｉＮＡでも同様に実験を行い、表にその結果を示した。キメラｓｉＮＡ（dsRDC）の構造（パッセンジャー鎖は１６から２３番目、ガイド差は１から６番目がＤＮＡで、残りの塩基はＲＮＡ）を図４に示す。 In addition, for some sequences, the same experiment was performed with chimeric siNA, and the results are shown in the table. The structure of the chimeric siNA (dsRDC) (passenger strand 16th to 23rd, guide difference 1st to 6th is DNA, and the remaining bases are RNA) is shown in FIG.
このように、新規配列として、６４３、６９１、８３０、１４８９、２３８１、６０６８は、十分なＭＤＭ２抑制活性を有し、非特異的なｐ５３発現亢進活性は有していない。また、新規配列として、３１７、３４７、４５２、７８８、１０３６は、十分なＭＤＭ４抑制活性を有し、非特異的なｐ５３発現亢進活性は有していない。 Thus, 643, 691, 830, 1489, 2381, and 6068 as new sequences have sufficient MDM2 inhibitory activity and do not have nonspecific p53 expression-enhancing activity. In addition, 317, 347, 452, 788, and 1036 as the novel sequences have sufficient MDM4 inhibitory activity and do not have nonspecific p53 expression-enhancing activity.
（２）腫瘍細胞株に対するＭＤＭ２抑制配列の増殖抑制活性
ＭＤＭ２抑制配列として（１）で選択された配列（６４３、６９１、８３０、１４８９、２３８１、６０６８）を有するｓｉＲＮＡ及びキメラｓｉＮＡについて、ＭＤＭ２抑制によるｐ５３の発現抑制解除を通じた増殖抑制活性を調べた。
(2) Growth inhibitory activity of MDM2 inhibitory sequence against tumor cell line siRNA and chimeric siNA having the sequence (643, 691, 830, 1489, 2381, 6068) selected in (1) as an MDM2 inhibitory sequence were subjected to MDM2 inhibition. The growth inhibitory activity through the release of p53 expression suppression was examined.
具体的には、ＳＪＳＡ−１細胞に対し、１ｎＭの各ｓｉＲＮＡをトランスフェクトして５日間培養し、その後ＷＳＴ−８細胞増殖アッセイを行い、ｓｉＲＮＡを導入していないコントロール細胞の細胞数に対する、ｓｉＲＮＡ導入細胞の細胞数の割合（ここでは細胞残存率と呼ぶ）を調べた。ＭＤＭ２抑制による細胞増殖阻害効果が大きいほど、細胞残存率が小さくなる。表６にその結果を示したが、ここでは、細胞残存率が６０％以下であるものを合格とした。なお、いくつかの配列については、キメラｓｉＮＡ分子を用いて同様に細胞残存率を調べ、増殖阻害率＝１００−細胞残存率とし、表７にその結果を示したが、ここでは、その比が４０％以上であるものを合格とした。 Specifically, 1 nM of each siRNA was transfected into SJSA-1 cells and cultured for 5 days, and then a WST-8 cell proliferation assay was performed. The ratio of the number of the introduced cells (herein referred to as cell survival rate) was examined. The greater the cell growth inhibitory effect of MDM2 suppression, the lower the cell survival rate. The results are shown in Table 6. Here, those having a cell residual rate of 60% or less were accepted. For some sequences, the cell survival rate was similarly examined using chimeric siNA molecules, and the growth inhibition rate = 100−cell survival rate. Table 7 shows the results. Those that were 40% or more were judged to be acceptable.
次に、増殖阻害効果がｐ５３の発現抑制解除によるかどうかを調べた。 Next, it was examined whether the growth inhibitory effect was caused by the release of p53 expression suppression.
まず、ＳＪＳＡ-１細胞（ＭＤＭ２高発現骨肉種）を用いて変異型ｐ５３の安定発現細胞を作製した。ヒト変異型ｐ５３株（Ａ１３８Ｖ）は、野生型ヒトｐ５３のコドン１３８のアラニンをバリンに変換したドミナントネガティブ変異体で、野生型ｐ５３の機能を有さず、野生型ｐ５３を拮抗抑制する。（Yamato, K et al., A human temperature-sensitive p53 mutant p53Val-138: modulation of the cell cycle, viability and expression of p53-responsive genesOncogene 11:1-6, 1995）。そのヒトTP53(A138V) cDNAをpCMVneo（Tsutsumi-Ishii Y, et al, Response of heat shock element within the human HSP70 promoter to mutated p53 genes. Cell Growth Differ 6:1-8, 1955）のＢａｍＨＩサイトに導入し、ＴＰ５３（Ａ１３８Ｖ）／ｐＣＭＶｎｅｏを作製した。一方、ＳＪＳＡ-１細胞を１ウエル当り２ｍｌの培地（１０％ＦＣＳ加ＲＰＭＩ１６４０）に１×１０５細胞で播種した。翌日、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ＬＴＸ（Ｉｎｖｉｔｒｏｇｅｎ社）６μｌとＰｌａｓｍｉｄ ＤＮＡ ２μｇをＯｐｔｉ-ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）で希釈してそれぞれ２００μｌとした後、両者を混和し、室温で３０分間静置し、その後全量を培養に添加して、トランスフェクションを行った。２４時間後に、細胞をトリプシン処理し、０．９ｍｇ／ｍｌのＧ４１８添加培地に再懸濁し、１ウエル当り培地量１００μｌ、細胞数１００細胞で播種した。その後、３−４日毎に培地を交換し、十分に細胞が増えた所で、残存した複数のクローンについて変異型ｐ５３の発現を調べ、最も高い発現を示したクローン８を選択し、変異型ｐ５３の安定発現細胞株とした。正常のＳＪＳＡ−１細胞と同様にキメラｓｉＮＡ分子を用いて同様に細胞残存率を調べ、増殖阻害率＝１００−細胞残存率とし、表７にその結果を示したが、その比が４０％以上であるものを合格とした。 First, stable expression cells of mutant p53 were prepared using SJSA-1 cells (MDM2 highly expressing bone meat species). The human mutant p53 strain (A138V) is a dominant negative mutant in which alanine at codon 138 of wild-type human p53 is converted to valine, has no function of wild-type p53, and antagonizes wild-type p53. (Yamato, K et al., A human temperature-sensitive p53 mutant p53Val-138: modulation of the cell cycle, viability and expression of p53-responsive genes Oncogene 11: 1-6, 1995). The human TP53 (A138V) cDNA was introduced into the BamHI site of pCMVneo (Tsutsumi-Ishii Y, et al, Response of heat shock element within the human HSP70 promoter to mutated p53 genes. Cell Growth Differ 6: 1-8, 1955). And TP53 (A138V) / pCMVneo. On the other hand, SJSA-1 cells were seeded at 1 × 10 5 cells per well in 2 ml of medium (RPMI1640 with 10% FCS). The next day, 6 μl of Lipofectamine LTX (Invitrogen) and 2 μg of Plasmid DNA were diluted with Opti-MEM (Invitrogen) to 200 μl each, mixed, allowed to stand at room temperature for 30 minutes, and then the entire amount was added to the culture. Then, transfection was performed. Twenty-four hours later, the cells were trypsinized, resuspended in G418-supplemented medium at 0.9 mg / ml, and seeded with a medium volume of 100 μl per well and a cell number of 100 cells. Thereafter, the medium was changed every 3-4 days, and when the number of cells was sufficiently increased, the expression of mutant p53 was examined for the remaining clones, and clone 8 showing the highest expression was selected. Was used as the stable expression cell line. Similar to normal SJSA-1 cells, the cell survival rate was similarly examined using the chimeric siNA molecule, and the growth inhibition rate = 100−cell survival rate. The results are shown in Table 7, and the ratio was 40% or more. Was passed.
次に、この変異型ｐ５３導入細胞の細胞残存率に対するＳＪＳＡ−１細胞の細胞残存率の比を調べた。すなわち、ＭＤＭ２に対するｓｉＲＮＡによってＭＤＭ２発現が抑制され、ｐ５３の発現抑制解除が生じても、変異型ｐ５３導入細胞では、変異型ｐ５３がｐ５３に拮抗し、ｓｉＲＮＡの効果が相殺される。従って、ＳＪＳＡ−１細胞において、ＭＤＭ２発現の抑制によって特異的にｐ５３発現抑制が解除され、それによって増殖抑制が生じる場合、変異型ｐ５３導入細胞では増殖抑制が生じないことになる。そこで、変異型ｐ５３導入細胞の細胞残存率に対するＳＪＳＡ−１細胞の細胞残存率の比が、ＳＪＳＡ−１細胞の細胞残存率より大きいと、ｐ５３によるのではない非特異的増殖が生じていることがわかる。こうすることで、オフターゲット効果の低い配列を選択することができた。 Next, the ratio of the cell survival rate of the SJSA-1 cells to the cell survival rate of the mutant p53-introduced cells was examined. That is, even if MDM2 expression is suppressed by siRNA against MDM2 and the suppression of p53 expression is released, mutant p53 antagonizes p53 in mutant p53-introduced cells, and the effect of siRNA is offset. Therefore, in SJSA-1 cells, when suppression of p53 expression is specifically released by suppression of MDM2 expression, thereby suppressing growth, growth-suppressing does not occur in mutant p53-introduced cells. Therefore, if the ratio of the residual cell rate of the SJSA-1 cells to the residual cell rate of the mutant p53-introduced cells is greater than the residual cell rate of the SJSA-1 cells, non-specific proliferation not due to p53 occurs. I understand. In this way, a sequence having a low off-target effect could be selected.
このように、配列１４８９が、最も効果的に且つ特異的に、ｐ５３の発現抑制解除を通じて細胞増殖を阻害する。 Thus, sequence 1489 most effectively and specifically inhibits cell growth through derepression of p53 expression.
（３）腫瘍細胞株に対するＭＤＭ４抑制配列の増殖抑制活性
ＭＤＭ４発現抑制配列として（１）で選択された配列（３１７、３４７、４５２、７８８、１０３６）を有するキメラｓｉＮＡについて、ＭＤＭ４発現抑制によるｐ５３の発現抑制解除を通じた増殖抑制活性を調べた。
(3) Proliferation inhibitory activity of MDM4 inhibitory sequence against tumor cell line For chimeric siNA having the sequence (317, 347, 452, 788, 1036) selected in (1) as an MDM4 expression inhibitory sequence, p53 was inhibited by inhibiting MDM4 expression. The growth inhibitory activity through expression suppression release was examined.
まず、ＭＣＦ７細胞を用い、キメラｓｉＮＡを導入したときのＭＤＭ４ｍＲＮＡの発現を、以下のようにして、リアルタイムＲＴ−ＰＣＲによって調べた。９６穴プレート１ウエル当りＭＣＦ−７細胞を４０００個播種し、翌日キメラｓｉＮＡをＬｉｐｏｆｅｃｔａｍｉｎｅ ＲＮＡｉＭＡＸを用いて導入した。４８時間後、RealTime ready Cell Lysis Kit（Ｒｏｃｈｅ社）とTranscriptor universal cDNA Master（Ｒｏｃｈｅ社）を用いて、ＲＮＡ抽出とｃＤＮＡ合成を行なった。１ウエルから４０μｌのＲＮＡ抽出液を得て、そのうち２μｌのＲＮＡを用いて２０μｌのスケールで逆転写反応を行なった。反応後の逆転反応液から０．８μｌのｃＤＮＡと、１μｌの２０Ｘ ＭＤＭ４ プライマー・ＴａｑＭａｎ プローブ （アッセイＩＤ： Ｈｓ００９６７２３８、ＡＢＩ社）と０．３μｌ２０Ｘ １８Ｓ ｒＲＮＡプライマー・ＴａｑＭａｎ プローブ（アッセイＩＤ： Ｈｓ０３９２８９９０、ＡＢＩ社）、１０μｌ 2x FastStart Universal Probe Master （ＲＯＸ）（Ｒｏｃｈｅ社）、７．９μｌ精製水 （合計２０μｌ）を用いて９６穴プレートで、Applied Biosystems 7500 Fast Real-Time PCR Systemを用いてＭＤＭ４ｃＤＮＡを増幅した。表７に、１８ＳｒＲＡを内部コントロールとして、Naito-I導入細胞に対する相対的ＭＤＭ４ ｍＲＮＡ量を示した。 First, using MCF7 cells, the expression of MDM4 mRNA when chimeric siNA was introduced was examined by real-time RT-PCR as follows. 4000 MCF-7 cells were seeded per well of a 96-well plate, and the next day, chimeric siNA was introduced using Lipofectamine RNAiMAX. After 48 hours, RNA extraction and cDNA synthesis were performed using RealTime ready Cell Lysis Kit (Roche) and Transcriptor universal cDNA Master (Roche). A 40 μl RNA extract was obtained from one well, and a reverse transcription reaction was performed on a 20 μl scale using 2 μl of the RNA extract. 0.8 μl of cDNA, 1 μl of 20 × MDM4 primer and TaqMan probe (Assay ID: Hs0097238, ABI) and 0.3 μl of 20 × 18S rRNA primer and TaqMan probe (Assay ID: Hs03928990, ABI) from the reverse reaction solution after the reaction MDM4 cDNA was amplified using a Applied Biosystems 7500 Fast Real-Time PCR System in a 96-well plate using 10 μl 2 × FastStart Universal Probe Master (ROX) (Roche) and 7.9 μl purified water (20 μl in total). Table 7 shows the relative amount of MDM4 mRNA relative to Naito-I-transfected cells using 18SrRA as an internal control.
次に、（２）と同様にしてＷＳＴ８による細胞残存率の解析を行い、Naito-Iを用いたときの細胞残存数を１００％としたときの細胞残存率を算出した。そして、本実施例では、得られた細胞残存率をＭＤＭ４発現抑制効果と比較した。ＭＤＭ４発現抑制効果に対して、細胞残存率があまりに小さいものは非特異的細胞致死が生じていると考えられるが、細胞残存率が６０％以下で、且つ、細胞残存率／ＭＤＭ４ｍＲＮＡ発現が６０％以上を合格としたとき、これら５つの配列については、すべて合格になった。このように、これら５つの配列は、オフターゲット効果が低いと考えられる。比較例（配列８６１及び１６０３）とともに、その結果を表９に示す。 Next, the cell remaining ratio was analyzed by WST8 in the same manner as in (2), and the cell remaining ratio was calculated when the number of cells remaining when Naito-I was used was set to 100%. Then, in this example, the obtained cell remaining rate was compared with the MDM4 expression suppression effect. If the cell survival rate is too small for the MDM4 expression inhibitory effect, non-specific cell killing is considered to have occurred. However, the cell survival rate is 60% or less, and the cell survival rate / MDM4 mRNA expression is 60%. When the above was regarded as a pass, all of these five sequences passed. Thus, these five sequences are considered to have low off-target effects. The results are shown in Table 9 together with the comparative examples (sequences 861 and 1603).
（４）腫瘍細胞に対するＭＤＭ２またはＭＤＭ４発現抑制の細胞増殖に対する効果
表１に示した細胞のうち、野生型のｐ５３遺伝子を有しているＭＣＦ−７からＣ３２ＴＧまでの細胞株について、ＭＤＭ２またはＭＤＭ４の発現を抑制し、それに伴う細胞残存率を測定した。具体的な実験方法は（２）（３）と同様にし、コントロールの配列（siNaito-1）の細胞残存率を１００％としたときの相対的な細胞残存率を算出した。なお、発現抑制にはキメラｓｉＮＡ分子を用い、ＭＤＭ２については配列１０６８及び１４８９、ＭＤＭ４については配列４５２及び１０３６を用いた。その結果を図５に示す。
(4) Effect of MDM2 or MDM4 Expression Suppression on Tumor Cells on Cell Proliferation Among the cells shown in Table 1, MDM2 or MDM4 was inhibited in cell lines from MCF-7 to C32TG having a wild-type p53 gene. Expression was suppressed, and the cell retention rate associated with the expression was measured. The specific experimental method was the same as in (2) and (3), and the relative cell survival rate was calculated assuming the cell survival rate of the control sequence (siNaito-1) to be 100%. In addition, a chimeric siNA molecule was used for expression suppression, sequences 1068 and 1489 were used for MDM2, and sequences 452 and 1036 were used for MDM4. The result is shown in FIG.
ＭＤＭ４の発現が高いＭＣＦ−７からＡ５４９までの腫瘍細胞において、ＭＤＭ２およびＭＤＭ４のいずれの発現を抑制した場合も効率よく細胞増殖が抑制された。一方、ＭＤＭ２の発現が高いＳＪＳＡ−１からＣ３２ＴＧまでの腫瘍細胞においては、ＭＤＭ２の発現を抑制した場合に効率よく細胞増殖が抑制されたが、ＭＤＭ４の発現を抑制した場合には、それほど細胞増殖が抑制されなかった。このことから、腫瘍細胞が発現している遺伝子によって、抑制する遺伝子を決定するのが、治療法として効果であることがわかる。すなわち採取した腫瘍細胞のＭＤＭ２およびＭＤＭ４の発現を調べ、腫瘍細胞においてＭＤＭ４の発現が高い場合、ＭＤＭ２および／またはＭＤＭ４の発現を抑制し、ＭＤＭ２の発現が高い場合、ＭＤＭ２の発現を抑制するのが好ましく、後者の場合、ＭＤＭ４の発現を抑制しても、配列によっては全く効果がない訳ではないが、それほど効果的ではない。 In the tumor cells from MCF-7 to A549 in which the expression of MDM4 was high, even when the expression of both MDM2 and MDM4 was suppressed, the cell proliferation was efficiently suppressed. On the other hand, in the tumor cells from SJSA-1 to C32TG in which MDM2 expression was high, cell growth was efficiently suppressed when MDM2 expression was suppressed, but not so much when MDM4 expression was suppressed. Was not suppressed. This indicates that determining a gene to be suppressed based on the gene expressed by the tumor cell is effective as a therapeutic method. That is, the expression of MDM2 and MDM4 in the collected tumor cells is examined. When the expression of MDM4 is high in the tumor cells, the expression of MDM2 and / or MDM4 is suppressed, and when the expression of MDM2 is high, the expression of MDM2 is suppressed. Preferably, in the latter case, suppressing the expression of MDM4 is not completely ineffective depending on the sequence, but is not so effective.
Claims (10)
前記患者から採取した腫瘍細胞のＭＤＭ４の発現を調べる工程と、
前記採取した腫瘍細胞においてＭＤＭ４の発現が高い場合、ＭＤＭ２の発現を抑制する抑制剤を抗腫瘍剤として選択する工程と、
を含む抗腫瘍剤の選択方法。 A method of selecting an antitumor agent for treating a patient having a tumor,
Examining the expression of MDM4 in tumor cells collected from the patient;
When the expression of MDM4 is high in the collected tumor cells, selecting an inhibitor that suppresses the expression of MDM2 as an antitumor agent;
A method for selecting an antitumor agent comprising:
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２） The method for selecting an antitumor agent according to claim 1 or 2, wherein the inhibitor that suppresses MDM2 contains siNA having a double-stranded portion having the following sequence.
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２） A siNA for suppressing the expression of MDM2, which has a double-stranded portion having the following sequence.
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４）
GGUCAUGCACUAUUUAGGN （配列番号５）
CCAGUACGUGAUAAAUCCN （配列番号６）
GGUGAAGCAACUUUAUGAN （配列番号７）
CCACUUCGUUGAAAUACUN （配列番号８）
CAACUAUACACCUAGAAGN （配列番号９）
GUUGAUAUGUGGAUCUUCN （配列番号１０）
GUGAUGAUACCGAUGUAGN （配列番号１１）
CACUACUAUGGCUACAUCN （配列番号１２）
より選択されるｓｉＮＡ。 A group of siNAs for suppressing the expression of MDM4, the siNAs having a double-stranded portion having the following sequence:
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
GGUCAUGCACUAUUUAGGN (SEQ ID NO: 5)
CCAGUACGUGAUAAAUCCN (SEQ ID NO: 6)
GGUGAAGCAACUUUAUGAN (SEQ ID NO: 7)
CCACUUCGUUGAAAUACUN (SEQ ID NO: 8)
CAACUAUACACCUAGAAGN (SEQ ID NO: 9)
GUUGAUAUGUGGAUCUUCN (SEQ ID NO: 10)
GUGAUGAUACCGAUGUAGN (SEQ ID NO: 11)
CACUACUAUGGCUACAUCN (SEQ ID NO: 12)
SiNA selected from
ＭＤＭ２の発現を抑制するためのｓｉＮＡが、請求項４に記載のｓｉＮＡであって、
ＭＤＭ４の発現を抑制するためのｓｉＮＡが、請求項５に記載のｓｉＮＡであるｓｉＲＮＡペア。 A siNA pair for simultaneously suppressing the expression of MDM2 and MDM4,
The siNA for suppressing the expression of MDM2 is the siNA according to claim 4 , wherein
An siRNA pair, wherein the siNA for suppressing the expression of MDM4 is the siNA according to claim 5 .
CAGCCAUCAACUUCUAGUN （配列番号１）
GUCGGUAGUUGAAGAUCAN （配列番号２）
CCCUCUCUAUGAUAUGCUN （配列番号３）
GGGAGAGAUACUAUACGAN （配列番号４） The siNA pair according to claim 6, consisting of the following sequence.
CAGCCAUCAACUUCUAGUN (SEQ ID NO: 1)
GUCGGUAGUUGAAGAUCAN (SEQ ID NO: 2)
CCCUCUCUAUGAUAUGCUN (SEQ ID NO: 3)
GGGAGAGAUACUAUACGAN (SEQ ID NO: 4)
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