JP7481694B2 - 核酸送達複合体 - Google Patents
核酸送達複合体 Download PDFInfo
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Description
[1] 核酸を含むナノ粒子と、非エンベロープウイルスの中空粒子を含む複合体、
[2] 核酸が、ホスホロジアミデート化モルフォリノオリゴマー(PMO)、ペプチド付加PMO(P-PMO)、トリシクロDNA(tcDNA)及び2’Oメチルオリゴマー(2’OMe)からなる群より選択される核酸誘導体である、[1]記載の複合体、
[3] 核酸誘導体が、配列番号1又は配列番号2に記載の配列を有するペプチドを含むP-PMOである、[1]又は[2]記載の複合体、
[4] 最も長い部分が50~1000nmである、[1]~[3]のいずれか1項に記載の複合体、
[5] 中空粒子がアデノ随伴ウイルスの中空粒子である、[1]~[4]のいずれか1項に記載の複合体、
[6] 核酸が、ジストロフィン遺伝子のアンチセンス核酸である、[1]~[5]のいずれか1項に記載の複合体、
[7] ナノ粒子が、核酸が集合して形成されている、[1]~[6]のいずれか1項記載の複合体、
[8] 以下の工程を含む、核酸を含むナノ粒子とカプシドウイルスを含む複合体の製造方法;
(i)核酸を含むナノ粒子を製造する工程、
(ii)非エンベロープウイルスの中空粒子を製造する工程、及び
(iii)(i)のナノ粒子と(ii)の非エンベロープウイルスの中空粒子を混合する工程、
[9] (iii)の工程において、核酸と中空粒子を、中空粒子1に対して核酸が150~1500のモル比で混合する、[8]記載の製造方法。
[10] [1]~[7]いずれか1項に記載の複合体を含む医薬組成物、
[11] デュシェンヌ型筋ジストロフィー(DMD)治療用である、[10]記載の医薬組成物、
[12] 経静脈全身投与用である、[10]又は[11]に記載の医薬組成物、
[13] 全身投与用である、[10]又は[11]に記載の医薬組成物、
[14] [1]~[7]いずれか1項に記載の複合体を対象に投与することを含む、疾患の予防又は治療方法、
[15] デュシェンヌ型筋ジストロフィー(DMD)を治療するための、[13]記載の方法、
[16] 複合体が対象に全身投与される、[14]又は[15]記載の方法、
[17] 複合体が対象に経静脈全身投与される、[16]記載の方法、
[18] [1]~[7]いずれか1項に記載の複合体のDDSとしての使用、
[19] DDSが全身投与用である、[18]記載の使用、
[20] DDSが経静脈全身投与用である、[19]記載の使用、
[21] 核酸医薬の担体としての、非エンベロープウイルスの中空粒子の使用、
[22] 核酸医薬が、ホスホロジアミデート化モルフォリノオリゴマー(PMO)、ペプチド付加PMO(P-PMO)、トリシクロDNA(tcDNA)及び2’Oメチルオリゴマー(2’OMe)からなる群より選択される核酸誘導体を含む、[21]記載の使用、
[23] 核酸誘導体が、配列番号1又は配列番号2に記載の配列を有するペプチドを含むP-PMOである、[22]記載の使用、
[24] 核酸医薬と中空粒子が複合体を形成し、該複合体の最も長い部分が50~1000nmである、[21]~[23]のいずれか1項に記載の使用、
[25] 中空粒子がアデノ随伴ウイルスの中空粒子である、[21]~[24]のいずれか1項に記載の使用、
[26] 核酸医薬が、ジストロフィン遺伝子のアンチセンス核酸を含む、[21]~[25]のいずれか1項に記載の使用、
[27] 核酸医薬が、核酸が集合して形成されたナノ粒子を含む、[21]~[26]のいずれか1項記載の使用、
[28] 核酸医薬が全身投与される、[21]~[27]のいずれか1項記載の使用、及び
[29] 核酸医薬が経静脈全身投与される、[28]記載の使用
に関する。
本発明の複合体は、核酸を含むナノ粒子と、非エンベロープウイルスの中空粒子を含む複合体である。
本発明は、前記(1)に記載する本発明の複合体の製造方法を提供し、以下の工程を含む。
(i)核酸を含むナノ粒子を製造する工程、
(ii)非エンベロープウイルスの中空粒子を製造する工程、及び
(iii)(i)のナノ粒子と(ii)の非エンベロープウイルスの中空粒子を混合する工程。
本発明の医薬組成物は、前記(2)の複合体を有効成分として含む。医薬組成物の投与形態は、医薬的に許容可能な投与形態であれば特に制限されず、治療方法に応じて選択することができる。例えば、静脈内投与、動脈内投与などの全身投与、筋肉内投与、皮下投与、経口投与、組織内投与、経皮投与などの局所投与が好ましい。また、本発明の組成物が取り得る剤型としては、特に制限されないが、例えば、各種の注射剤、経口剤、点滴剤、吸入剤、軟膏剤、ローション剤等を挙げることができる。
スキッピング効率(%)=A/(A+B)×100
(1)8型及び9型AAV中空粒子の製造
国際公開パンフレット WO2012/144446記載の293EB細胞 4×108個を、10%FBS+/DMEM/F12培地を含む225cm2フラスコ28本(又は6320cm2の10段フラスコ1個)に接種し、5%CO2下37℃にて培養した。培養48時間後に回収した細胞に、AAVのITRとその間に挿入されたeGFP遺伝子を保持するAAVベクタープラスミド、8型AAV(AAV8)又は9型AAV(AAV9)のrep及びcap遺伝子を保持するAAVヘルパープラスミド、並びに2型アデノウイルスのE2A、E4、VA RNA遺伝子を保持するアデノウイルスヘルパープラスミドをそれぞれ650μgずつリン酸カルシウム法にて導入した。そのまま培養を継続し、AAV粒子及びAAV中空粒子を該細胞内で複製させた。プラスミド導入の72時間後に細胞を遠心にて回収し、細胞ペレットを30mLのトリス緩衝生理食塩水(TBS)にて懸濁した。この懸濁液の凍結融解を4~6回繰り返し、融解時に毎回ボルテックス操作にて懸濁液を十分に混合した。
遠心チューブに入れた1.50g/mL塩化セシウム溶液上に1.25g/mL塩化セシウム溶液を重層し、さらにこの上に前記(1)で回収した熱処理後の上清を重層した。チューブを16℃で25,000×g、3時間超遠心後、内容液を遠心チューブ下方から0.5mLずつ回収した。各画分の屈折率(RI:refractive index:)を測定し、RIが1.365~1.368の画分を集め、その容量の約100倍量のMHNバッファー(3.33mM MES、3.33mM HEPES(pH6.5)、3.33mM NaOAc)に対し、30分間透析した。得られた透析物をその容量の約5倍量のMHNバッファーで希釈した。
イオン交換担体として基材表面にスルホン酸基を保持する陽イオン交換膜ムスタングSアクロディスク(ポール コーポレーション社製)を使用した。FPLCシステムとして、AKTA explorer 100(GEヘルスケア社製)を用い、以下に示す精製操作を行った。MHNバッファーでムスタングSアクロディスクを平衡化した後、流速3mL/分で、前記(2)で得た透析後の希釈液をムスタングSアクロディスクにロードし、中空粒子を膜に吸着させるとともにAAV粒子を分離、除去した。10CV(ディスクの容量の10倍量)のMHNバッファーでムスタングSアクロディスクを洗浄し、0~2M NaCl/50CVの濃度勾配条件にて中空粒子を溶出し、画分量1mLで回収した。280nmにおける吸光度のピークの画分に含まれる中空粒子を集め、電子顕微鏡により、中心に黒い部分がある、ウイルスゲノムが入っていない中空粒子であることを確認した。
(1)~(3)で調製したAAV8及びAAV9の中空粒子を、それぞれPBS(-)(リン酸緩衝生理食塩水)で4×1013 v.p.相当/mLの濃度に調製し、Viscotek 802(Malvern社製)を使用して動的光散乱(DLS)法により粒子径を測定した。その結果、AAV8及びAAV9の中空粒子の純度は、それぞれ96.8%、98.3%であり、粒子径はいずれも28nm程度であった。さらに、Zetasizer nano(Malvern社製)を用いてゼータ電位を測定した。AAV8及びAAV9の中空粒子の電気伝導率は15mS/cm、等電点はpH8.5付近であることを確認した。
(1)P-PMOの製造
ペプチド修飾型PMO(peptide-conjugated phosphorodiamidate morpholino oligomer:P-PMO)をEzzat K, et al., 2015, NANO LETTERS,vol.15,pp4364記載の方法に従って調製した。本実施例で調製したP-PMOはPip6aペプチド(配列番号1)と、そのC末端にアミドリンカーにより連結されたマウスジストロフィン遺伝子のエクソン51の配列番号3に記載される配列を標的とするPMOアンチセンスオリゴヌクレオチドからなる。P-PMO溶液をPBS(-)で50μMの濃度に調製した。
実施例2(1)で調製したP-PMOを、実施例1で調製したAAV8中空粒子と、PBS(-)またはOpti-MEM培地(Thermo Fisher Scientific社製)中で、1500:1、750:1、150:1のモル比で混合し、室温で15分静置した。
(1)H2K-mdx52筋芽細胞
筋ジストロフィーのモデル動物であるmdxマウス由来のH2K細胞株(H2K-mdx52)(Proc Natl Acad Sci U S A. 2012 Aug 21; 109(34): 13763-13768.)の細胞50,000個を0.5mLの分化培地(5%ウマ血清を添加したDMEM培地)で4日間培養し筋管細胞に分化させた。その後、培地を1500:1、750:1または150:1のモル比のP-PMO/AAV中空粒子複合体を(P-PMOとして)50μMの濃度で含むOpti-MEM培地(サーモフィッシャーサイエンティフィック社製)に交換し、トランスフェクション試薬を添加せずに48時間培養した。コントロールとして、複合体の代わりにP-PMOのみを同濃度で含む培地を添加した細胞を使用した。培養した細胞からTRIzol (Invitrogen社製)を用いてTotal RNAを抽出した。このRNAを鋳型とし、配列番号4記載のプライマーEx50F及び配列番号5記載のプライマーEx53Rを使用してRT-PCRによりジストロフィン遺伝子のエクソン50から53を増幅させた。増幅産物の塩基長をMultiNA(Shimadzu社製)により解析して、エクソン51スキップの効率を測定した。その結果を図3に示す。また、培養した細胞に0.1%Triton-X100を15μL添加して30分間静置し、ピペッティングで細胞を懸濁させ、遠心分離した上清中の乳酸脱水素酵素(LDH)をCytoSelect(商標) LDH細胞毒性アッセイキット(CBL社製)を使用して細胞膜障害マーカーとして測定した。
国立研究開発法人国立精神・神経医療研究センター(NCNP)で飼育・繁殖中の5週齢のmdx52マウス(Biochem Biophys Res Commun. 1997;238:492-497.)の両側前脛骨筋に、1500:1又は150:1のモル比のP-PMO/AAV中空粒子複合体を、P-PMOの量として10μg局所筋肉内投与し、2週間後に投与部位の骨格筋を回収した。コントロールとして複合体の代わりにP-PMOのみ投与して得た骨格筋を使用した。回収した骨格筋に対して抗ジストロフィン抗体NCL-DYS1(Leica Microsystems社製)を使用してジストロフィン免疫組織化学染色を行い、筋肉の横断面積あたりのジストロフィン陽性筋線維数及び蛍光強度を測定した。その結果を図4に示す。さらに、回収した骨格筋からComplete Mini Protease Inhibitor Cocktail(Roche社製)を添加したRIPAバッファーによりタンパク質を抽出してウエスタンブロット解析を行い、ハウスキーピングタンパク質GAPDHに対するジストロフィン量の割合を測定した。その結果を図5に示す。
5週齢のmdx52マウスに、1500:1又は750:1のモル比のP-PMO/AAV中空粒子複合体を3mg/kg又は6mg/kgで経静脈全身投与し、2週間後に各部位の骨格筋を回収した。コントロールとしてP-PMOを投与しないマウスの(blank)及び複合体の代わりにP-PMOのみ投与したマウスを使用した。回収した骨格筋からタンパク質を抽出し、実施例3(2)同様にウエスタンブロット解析を行った。その結果を図6に示す。
SEQ ID NO: 2: B peptide
SEQ ID NO: 3: mouse dystrophin gene exon 51 partial sequence
SEQ ID NO: 4: Ex50F primer
SEQ ID NO: 5: Ex53R primer
Claims (12)
- 核酸を含むナノ粒子と、非エンベロープウイルスの中空粒子を含む複合体であって、該核酸を含むナノ粒子と、該非エンベロープウイルスの中空粒子とが静電気的又は化学的に結合した、複合体。
- 核酸が、ホスホロジアミデート化モルフォリノオリゴマー(PMO)、ペプチド付加PMO(P-PMO)、トリシクロDNA(tcDNA)及び2’Oメチルオリゴマー(2’OMe)からなる群より選択される核酸誘導体である、請求項1記載の複合体。
- 核酸誘導体が、配列番号1又は配列番号2に記載の配列を有するペプチドを含むP-PMOである、請求項2記載の複合体。
- 透過型電子顕微鏡(TEM)で測定した場合の最も長い部分の長さが50~1000nmである、請求項1~3のいずれか1項に記載の複合体。
- 中空粒子がアデノ随伴ウイルスの中空粒子である、請求項1~4のいずれか1項に記載の複合体。
- 核酸が、ジストロフィン遺伝子の配列に相補的なアンチセンス核酸である、請求項1~5のいずれか1項に記載の複合体。
- ナノ粒子が、核酸が集合して形成されている、請求項1~6のいずれか1項記載の複合体。
- 以下の工程を含む、核酸を含むナノ粒子と非エンベロープウイルスの中空粒子を含む複合体の製造方法;
(i)核酸を含むナノ粒子を製造する工程、
(ii)非エンベロープウイルスの中空粒子を製造する工程、及び
(iii)(i)のナノ粒子と(ii)の非エンベロープウイルスの中空粒子を混合する工程。 - (iii)の工程において、核酸と中空粒子を、中空粒子1に対して核酸が150~1500のモル比で混合する、請求項8記載の製造方法。
- 請求項1~7いずれか1項に記載の複合体を含む医薬組成物。
- デュシェンヌ型筋ジストロフィー(DMD)治療用である、請求項10記載の医薬組成物。
- 経静脈全身投与用である、請求項10又は11に記載の医薬組成物。
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