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Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion

Shibata, Saiko 京都大学 DOI:10.14989/doctor.k23065

2021.03.23

概要

嚢胞性線維症(CF)はcystic fibrosis transmembrane conductance regulator (CFTR)遺伝子の変異を原因とする遺伝性疾患である。塩化物イオンチャネルである CFTR は細胞内外の水分量を調節することから、CFTR が不活性化すると全身の分泌液・粘液が粘稠となり管腔が閉塞することで多臓器障害が引き起こされる。現在までにCF を発症させる CFTR 遺伝子変異は 2,000 種類以上報告されており、CFTR の機能異常に基づいて 1-6 型に分類されている。近年 2, 3, 及び 4 型の CFTR 機能不全を直接改善する CFTR 調節薬が承認された一方で、スプライシング異常を原因とする5 型に対しては未だ対症療法しかないことから新規治療薬の開発が求められている。そこで本研究では5型の中でも最も患者数の多い遺伝子変異c.3849+10kb C>T の低分子治療薬の探索を行った。

全CF 患者の約2%が保有するc.3849+10kb C>T は、CFTR の第22 番イントロンの 12,191 番目のシトシンがチミンに置き換わった遺伝子変異である。この変異により第 22 番イントロンの 84 塩基が偽エクソンとして mRNA に取り込まれることで正常な CFTR 遺伝子の発現が阻害される。低分子治療薬の探索にあたり、はじめに遺伝子変異によるスプライシング異常のメカニズム解明に取り組んだ。遺伝子変異周辺の塩基配列の解析より、偽エクソン中の RNA 配列にスプライシング反応を促進する RNA 結合タンパク質 serine/arginine-rich splicing factors (SRSFs)が直接結合すること、また SRSFs の結合が偽エクソンの認識に必須であることを明らかにした。さらにSRSFs の機能発現に必要なリン酸化を担うCDC-like kinase (CLK)の阻害剤であるTG003 処理を行うと正常なスプライシングの回復が認められた。よって CLK よりリン酸化を受けた SRSFs が偽エクソン内の RNA 配列に結合することでスプライシング異常が引き起こされること、またCLK 阻害によりスプライシング異常を改善できることを解明した。そこで次に、より高活性な CLK 阻害剤取得を目指し、京都大学大学院医学研究科形態形成機構学で開発されたレポーターベクターthe splicing reporter assay for disease gene with dual color (SPREADD)を用いて約800 化合物からなるTG003 類縁化合物ライブラリーのスクリーニングを行った。その結果、偽エクソン認識抑制の EC50 値が TG003 よりも20.4 倍低い高活性なCaNDY を取得した(CaNDY: EC50 = 1.2 μM)。さらに CaNDY による偽エクソン認識抑制作用により、患者由来 B 細胞において正常な CFTR mRNA 発現が回復すること、CF モデル細胞において機能的なCFTR タンパク質の発現も回復することを確認した。よって遺伝子変異c.3849+10kb C>T を原因とするCF に対する新規低分子治療薬候補としてCaNDY を見出すことができた。近年のトランスクリプトーム解析の進歩により CF 以外にも偽エクソンが生じる遺伝性疾患が数多く報告されていることから、偽エクソンが生じるメカニズムを明らかにすることにより、CaNDY が治療薬となり得る遺伝性疾患が他にも見出される可能性がある。個々の変異によるスプライシング異常の分子機構を解明し是正する治療法を確立していくことは、多様な遺伝性疾患に対しての個別化医療の実現に繋がると期待される。

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