JP6818358B2 - 神経系血管バリアーの機能回復剤及び神経系疾患治療剤 - Google Patents
神経系血管バリアーの機能回復剤及び神経系疾患治療剤 Download PDFInfo
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Description
[1]以下の(a)〜(c)のいずれかを有効成分とする神経系血管バリアーの破綻抑制剤。
(a)ベイシジン(basigin)遺伝子の発現を抑制する機能性核酸;
(b)ベイシジンの活性を消失又は減退する機能性核酸;
(c)上記(a)又は(b)の機能性核酸をコードするDNAを含む該機能性核酸発現ベクター;
[2]ベイシジン遺伝子の発現を抑制する機能性核酸がsiRNAであることを特徴とする上記[1]記載の神経系血管バリアーの破綻抑制剤。
[3]ベイシジン遺伝子の発現を抑制する機能性核酸が、配列番号1に示すヌクレオチドのセンス鎖配列と配列番号2に示すその相補的なアンチセンス鎖配列から構成されるsiRNA、又は、配列番号3に示すヌクレオチドのセンス鎖配列と配列番号4に示すその相補的なアンチセンス鎖配列から構成されるsiRNAであることを特徴とする上記[2]記載の神経系血管バリアーの破綻抑制剤。
[4]以下の(a)〜(c)のいずれかを有効成分とする神経系血管バリアーの破綻に起因する神経系疾患治療剤。
(a)ベイシジン(basigin)遺伝子の発現を抑制する機能性核酸;
(b)ベイシジンの活性を消失又は減退する機能性核酸;
(c)上記(a)又は(b)の機能性核酸をコードするDNAを含む該機能性核酸発現ベクター;
[5]神経系疾患が、脳神経系疾患又は網膜神経系疾患であることを特徴とする上記[4]記載の神経系疾患治療剤。
[6]神経系疾患が、脳浮腫又は網膜浮腫であることを特徴とする上記[5]記載の神経系疾患治療剤。
[7]ベイシジン遺伝子の発現を抑制する機能性核酸がsiRNAであることを特徴とする上記[4]〜[6]のいずれか記載の神経系疾患治療剤。
[8]ベイシジン遺伝子の発現を抑制する機能性核酸が、配列番号1に示すヌクレオチドのセンス鎖配列と配列番号2に示すその相補的なアンチセンス鎖配列から構成されるsiRNA、又は、配列番号3に示すヌクレオチドのセンス鎖配列と配列番号4に示すその相補的なアンチセンス鎖配列から構成されるsiRNAであることを特徴とする上記[7]記載の神経系疾患治療剤。
本発明者らはこれまでに、上述のように1)難治性神経系疾患の血管内皮細胞の細胞膜からのクローディン−5の消失、2)血管バリアー機能の破綻、3)病態悪化、というカスケードを明らかにして報告した。そこで、1次スクリーニングとして、対象因子の発現を特異的に抑制するsiRNAを作製し、種々の刺激(低酸素、サイトカインなど)によるマウス脳血管内皮細胞株(bEND.3細胞)の細胞膜からのクローディン−5消失に共通した阻害効果を示すsiRNAを、血管バリアー破綻に対する治療薬候補とした。そのなかでベイシジンに対するsiRNAを候補とし、かかる候補siRNAの血管バリアー破綻に対する治療薬としての有用性について、以下に示す方法でin vitro系を用いた機能的解析を行った。
図1、2に示すように、siRNA無し若しくはNon−silence siRNAを導入した場合には、VEGF添加、TNF−α添加、低酸素状態の全ての病的刺激によってbEND.3細胞の細胞膜からのクローディン−5消失現象が観察された。一方、BSGsiRNA#1又は2をbEND.3細胞に導入してベイシジンの発現を阻害したところ、VEGF添加、TNF−α添加、低酸素状態のいずれもbEND.3細胞の細胞膜からのクローディン−5消失現象が抑制された。さらに、図4に示すように、VEGF添加、TNF−α添加におけるbEND.3細胞単層のTEERを経時的に測定したところ、BSGsiRNA#1又は2は、VEGF添加、TNF−α添加によるTEER低下を抑制することが明らかとなった。したがって、ベイシジン遺伝子の発現を抑制する機能性核酸は、低酸素状態のみならずVEGFやTNF−αなどの様々な誘因による血管バリアー破綻に対して作用することが明らかとなった。
上記BSGsiRNA#1又は2の代わりに以下の表2に示すADAM12siRNA(センス鎖配列:配列番号9、アンチセンス鎖配列:配列番号10)、ADAM17siRNA(センス鎖配列:配列番号11、アンチセンス鎖配列:配列番号12)の2種類を用いて、上記と同様にbEND.3細胞の細胞膜からのクローディン−5消失現象を調べた。結果を図5に示す。
VEGFやTNF−αなどの病的刺激は異なる細胞内シグナル伝達系を介して働くことから、ベイシジンは、異なる細胞内シグナル伝達系に共通した下流において血管バリアー機能を調節する因子(バリアーを開く因子)であると解釈される。したがって、ベイシジン遺伝子の発現を抑制する機能性核酸は、種々の誘因が混在して血管バリアー破綻が起こる神経系疾患に対する有用な治療標的をなることが示唆される。そこで、ベイシジンの治療標的としての有用性について、以下に示す方法でマウスを用いたin vivo系を用いた機能的解析を行った。なお、網膜は、個体発生の過程で中枢神経系が出芽する形で形成される組織であり、脳と同様に中枢神経系の一部である。網膜の血管系は、長軸方向に全長に渡って2次元的に観察・評価することが可能であるため、本研究では、中枢神経系の代表として網膜を解析材料として用いた。
Claims (3)
- 以下の(a)〜(c)のいずれかを有効成分とする神経系血管バリアーの機能回復剤。
(a)ベイシジン(basigin)遺伝子の発現を抑制する機能性核酸;
(b)ベイシジンの活性を消失又は減退する機能性核酸;
(c)上記(a)又は(b)の機能性核酸をコードするDNAを含む該機能性核酸発現ベクター; - ベイシジン遺伝子の発現を抑制する機能性核酸がsiRNAであることを特徴とする請求項1記載の神経系血管バリアーの機能回復剤。
- ベイシジン遺伝子の発現を抑制する機能性核酸が、配列番号1に示すヌクレオチドのセンス鎖配列と配列番号2に示すその相補的なアンチセンス鎖配列から構成されるsiRNA、又は、配列番号3に示すヌクレオチドのセンス鎖配列と配列番号4に示すその相補的なアンチセンス鎖配列から構成されるsiRNAであることを特徴とする請求項2記載の神経系血管バリアーの機能回復剤。
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