JP2006518996A - 心筋収縮性をinvivoで調節するための方法 - Google Patents
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Abstract
Description
本発明は、1995年11月4日出願の現在も係属中の米国特許出願第08/420,306号の一部継続出願である。
本発明は、心筋収縮性を調節するための方法に関する。より詳細には、本発明は、SERCA2蛋白質を作動的にコードする遺伝子をin vivoで送達することによって、心臓の筋小胞体(SR)カルシウム2++ATPアーゼ(SERCA2)のin vivoのレベルを上昇させる方法に関する。
うっ血性心不全は、米国において成人の主な死亡原因のうちの1つである。心虚血(心臓への血液供給が妨害され又は失われることにより生じる急性の事象)と比べて、うっ血性心不全は、心筋の収縮性及びストレスに対する心臓の適応性の漸進的な消失を伴う比較的潜行性の事象である。結局、有効な治療法はなく、CHF心臓は、身体の代謝要求を満たすのに十分な速度で血液をポンプ輸送する能力を失う。
以下の定義は、本発明の考察を簡単にするために提供される。しかし、これらの定義が本発明の正当な範囲又は趣旨から逸脱しない等価物を含むように拡張できることを、当業者なら理解するであろう。この理由から、これらの定義は本発明を限定するものと解釈されるべきではない。
1.「SERCA2ポリヌクレオチド」とは、DNA又はRNAを意味し、本発明に従って行う治療の目的に適したセンス鎖及びアンチセンス鎖を含むことができる。本明細書では、「ポリヌクレオチド」とは、分離した断片の形又は大きな構築体の成分としての、デオキシリボヌクレオチド又はリボヌクレオチドの重合体を意味する。ポリヌクレオチドの配列は、遺伝コードから推測できるが、コードの縮重を考慮しなければならない。本発明のポリヌクレオチドは、遺伝コードの結果として縮重した配列を含み、その配列は当業者によって容易に決定することができる。
2.「作動的にコードする」とは、所望の翻訳産物、例えばペプチド又は蛋白質の発現及び望むならその分泌に必要なプロモーター及び他の配列を含むように改変されたポリヌクレオチドを意味する。本発明の全ての実施形態は、公知の組換え発現ベクターを使用して実施することができる。これらのベクターは、所望の翻訳産物をコードするcDNAを含むことが好ましい。従って、文脈上別の意味で解釈する必要がない限り、「ポリヌクレオチド」とは、適切な組換え発現ベクター中に含まれ作動的にコードする配列を意味すると想定され、その例が本明細書で提供される。
3.「合成」とは、ポリヌクレオチド配列及びポリペプチド配列を合成する公知の手段を意味し、天然のポリヌクレオチド及び蛋白質の単離及び精製を含むことができる。
4.「ペプチド」とは、in vivoで所望の生体効果を有する小型ペプチド、ポリペプチド、オリゴペプチド、及び蛋白質を意味する。
5.「送達」とは、作動的にコードするポリヌクレオチドを宿主に導入する既知の方法を意味する。当業者なら、これらの送達手段を熟知しており、或いは容易に特定することができる。しかし、特に有用な送達手段に関しては、「新規の薬物送達系(Novel Drug Delivery Systems)」(Marcel Dekker、1992年)を参照することができ、その関連する開示内容を、薬物送達技術に関する当技術分野の知識状態を例示するために参照により本明細書に組み込む。
6.「宿主」とは、本発明に従って行われる治療の受容者を意味する。宿主はどんな脊椎動物でもよいが、好ましくは哺乳動物である。哺乳動物の場合は、宿主はヒトが好ましいが、飼育された家畜でもペット動物でもよい。
7.「標的組織」とは、作動的にコードするポリヌクレオチドの発現を得ようとする、宿主の組織を意味する。
8.「抗体」とは、任意のクラスの免疫グロブリン全体、キメラ抗体、2つ又は複数の抗原特異性を有するハイブリッド抗体、及びハイブリッド断片を含む断片を意味する。また、このような断片の結合体、例えば米国特許第4,704,692号に記載されているいわゆる抗原結合性蛋白質(単鎖抗体)、及び、例えば米国特許第4,699,880号に記載されている抗イディオタイプ抗体(他の抗体に結合する抗体)も、「抗体」の意味の範囲内に含まれる。
9.「組換え発現ベクター」は、真核生物又は原核生物で発現可能なポリペプチドを作動的にコードするポリヌクレオチドの系を意味する。真核生物又はウイルスの配列を有するDNA配列を原核生物で発現する方法は当技術分野で公知である。宿主中で発現及び複製できる、生物的に機能的なウイルス及びプラスミドDNAベクターも当技術分野で公知である。宿主としては、微生物、酵母、昆虫、及び哺乳類生物を挙げることができる。
ラットSERCA2のゲノムクローンのヌクレオチド配列(Rohrer他、J.Biol.Chem.、263:6941〜6944、1988年(ラットSERCA2 mRNA)も参照のこと。この論文で報告された配列を、参照のために本明細書に組み込む)。このクローンは、Rohrer他、J.Biol.Chem.、266:8638〜8646、1991年(その開示内容を参照のために本明細書に組み込む)に記載されているような従来のハイブリダイゼーション技術によって得られた(実施例1を参照のこと)。この論文は、クローンを転写するための開始コドン及び終止コドンも記載している。
SERCA2ポリヌクレオチドの組成物及びSERCA2ポリヌクレオチドの混合物は、製薬上許容される懸濁液、溶液、又は乳濁液中に入れてよい。適切な媒体は生理食塩水を含み、SERCA2ポリヌクレオチドを標的組織に送達するために抗原提示細胞を使用しない実施形態の場合は、リポソーム調製物を含んでよい。
本発明では、SERCA2ポリヌクレオチドが、それがコードする生物的に活性なペプチドを発現させるのに十分な用量で供給されれば十分である。実現される発現レベルが、「通常の」内在性SERCA2の活性を実質的に補充するのに十分であることが好ましい。SERCA2ポリヌクレオチドが、組換え発現ベクター、好ましくはAAVベクターに含まれ、(セクションCで前述したように)製薬上許容される組成物に配合されると有利である。
ラットが、その心臓には側副循環がないが、ヒトCHFの状態を満足できる程度に予測できる、うっ血性心不全の再現性のある実験モデルであることが明らかになった。特に、冠動脈の外科的結紮を受けたラットは、ヒトの心筋梗塞後のCHFの特に良いモデルである。CHF動物モデルとしてのラットを作製及び使用するための実験プロトコールは、当技術分野で十分に記載されており、参照文献については、当業者なら、Pfieffer他、Am.J.Med.、76:99〜103、1984年;Johns及びOlsen、Ann.Surg.、140:675〜682、1954年;並びに、Selye他、Angiology、11:398〜407、1960年(その開示内容を、CHF動物モデルの開発及び使用に関する当技術分野の知識を例示するために参照により本明細書に組み込む)を参照できよう。さらに、甲状腺の状態が減退したマウス、又は心臓の収縮性が低減しカルシウムトランジェントが遅くなっている甲状腺機能低下マウスも、再現性のある実験モデルとして使用することができる。
SERCA2の発現をモニターするために、本発明に従って宿主に導入されるSERCA2ポリヌクレオチドを、既知のレポーター遺伝子を含むように改変してよい。例えば、Norton他、Mol.Cell.Biol.、5:281、1985年に記載のpRSV lac−Z DNAベクターは、蛋白質発現とともにβ−ガラクシターゼを産生する。ルシフェラーゼ及びクロラムフェニコールアセチル基転移酵素(「CAT」;pRSV−CATプラスミドの構築については、例えば前述のGorman他を参照のこと)も使用できる。別の有用なレポーター分子は、免疫測定法で容易に検出することができるフラッグ抗原性ペプチドである。フラッグ抗原性ペプチドの8個のアミノ酸配列及びそれをコードする領域は、当技術分野で既知である。この点に関して参照するためには、当業者なら、Chiang他、Peptide Res.、6:62〜64、1993年を参照できよう。(例えば、開始コドンから約15塩基対の位置に遺伝子を挿入することにより)コード用のレポーター遺伝子をSERCA2蛋白質のC末端に挿入しても、SERCA2触媒活性は妨害されない。このようなレポーター遺伝子の発現を検出する手段は当技術分野で公知であり、詳細には記載しないが、以下に要約する。
SERCA2 cDNAを従来のクローニング技術を使用してアデノ随伴ウイルスベクター中にクローニングした。SERCA2を発現するアデノ随伴ウイルス又はGFPレポーター配列の構築は、基本的に以下のように実施した。
CHF心臓の条件を模倣した条件下で導入遺伝子を含む動物におけるSERCA2の追加発現の効果を試験するために、性別、年齢、体重が同程度の動物群を処理して、心臓狭窄を誘発させ、腹大動脈狭窄を発症させ、食塩負荷させた。CHF心臓で顕著なこのような条件下で、これらの動物は心肥大(CH)を発症し、心臓への圧力過負荷(PO)に伴う心臓異常を起こす。導入遺伝子発現を心臓組織に対して特異的にするために、SERCA2 CMV/β−アクチントランスジェニック動物を使用した。
AAV/SERCA2構築体を使用して開発したトランスジェニック動物は、手術の6週間後に試験すると(SERCA2のmRNAレベルを検出して判定された)SERCA2遺伝子転写が有意に減少し、通常、10〜12週で心不全を発症し始める。SERCA2 mRNAレベル及び蛋白質レベルを、前述のノーザンブロット法及びウェスタンブロット法又は他の分析技術(例えば、やはり前述したPCR)によって決定した。トランスジェニック動物及び対照動物の(SRカルシウムトランジェントに関係している)酵素活性レベル及び左心室機能を下記に記載するように測定した。他のCHF動物モデル及び本発明の方法による治療を受けているヒトにおいて、以下に記載するプロトコールに従って、心臓性能に関する同じパラメータを測定することができる。
A.心室の機能性を判定するための心エコー検査のプロトコール(in vivo)
心エコー図の撮像は、7Mhzで作動する2周波フェーズドアレイトランスデューサー、及び積算ドプラー出力機能を備えた「ACCUSON(登録商標)」128超音波コンソールを用いて、適切な動物モデルで実施する。心エコー事象のタイミングは、皮下電極から得られる同時の心電図記録と相関がある。撮像の深さは2cm、セクタ角度はフレームレートが50/秒になる60にセットする。パワー設定は75dBとする。2次元イメージを使用して、Mモード記録(1000ライン/秒で得られる)に適したカーソル位置を選択することができる。
マウスを例にすると、体重20〜30gの成体マウス(PO有り又は無しの対象及びトランスジェニック)をケタミン(100mg/kg、腹腔内投与(IP))及びキシラジン(5mg/kg、腹腔内投与(IP))の混合物で麻酔する。解剖顕微鏡下で、動物をうつ伏せにし、正中線頚部切開を行って気管及び頚動脈を露出させる。次に、20ゲージの鈍針を、総(tital)容量0.2ml、呼吸数100/分のボリュームサイクル方式げっ歯類用人工呼吸器(ハーバードアパレイタス(Harvard Apparatus)社製)に連結されている気管カニューレを補助するように気管に通す。換気の妥当性を、胸部の膨張を目視検査して判定する。挿管後、大動脈圧を測定するために、1本の頚動脈に、液体入りカテーテル(管状になるよう火炎で延伸したポリエチレン(PE))付きカニューレを挿入する。次に、胸部を開き、2F高忠実度のマイクロマノメータ(ミラー(Millar)社)を僧帽弁から挿入して、LV中に取り付ける。
このプロトコールをラットで例示すると、切除による損傷を最小限にし拘縮を防ぐために25mM BDMを含む酸素化タイロード液中で左心室乳頭筋を切除し、受動的伸張を避ける。その端を短い5−0シルク縫合糸で結びつけて調製物の心室壁及び弁の端に付着させ、過剰伸張を防ぐ。筋肉の一方の端はCambridge400 Isometric force transducer(等尺性力トランスデューサー)(フルスケール5g、分解能100μg)のガラスロッドに付着し、もう一方の端はトランスデューサー台(Newport423、±1μm)上の小型のステンレススチールフックに付着するようにする。都合よく見られるように、1mlの試験浴をOlympus SZ45ステレオ顕微鏡の視野内におく。浴の温度を33℃に維持する。
心筋細胞は、コラゲナーゼ灌流法を使用して成体ラットの心臓から調製することができる。この筋細胞は、4%ウシ胎児血清の不在下又は存在下で少なくとも4日間維持し、成体の心筋細胞の形態及び代謝的特徴を維持することができる。1匹の成体ラットの心臓から、9〜10x106個の生存可能な筋細胞を得ることができる。
簡単に言うと、新生児の心筋細胞をSERCA2アデノウイルスベクターでトランスフェクトし、実施例3で記載したようにカルシウムトランジェントを測定し、対照(トランスフェクトされていない)細胞と比べる。図3において、細胞#42は対照の新生児心筋細胞の挙動の平均を表し、細胞#27は、トランスフェクトした新生児心筋細胞の挙動の平均を表す(ADV)。図3で示されるように、カルシウムトランジェントの最大減少の半分までの時間は、対照細胞と比べてトランスフェクトした細胞で33±19%短縮された(n=4、p≦0.01)。これらのデータは、カルシウムトランジェントがSERCA2 ADVでトランスフェクトした筋細胞で増大され、その結果、in vivoで測定すれば、心筋でより速い拡張期弛緩が促進されるはずであるという原理の証拠を提供する。
Claims (4)
- SERCA2をコードする導入遺伝子を含むアデノ随伴ウイルス(AAV)ベクターを宿主の心筋に送達することを含む、宿主の心筋細胞の筋小胞体へのSERCA2媒介カルシウムイオン輸送を増大させるための方法であって、前記導入遺伝子の発現が、処理されていない筋細胞と比べて処理された筋細胞で、SERCA2媒介カルシウムイオン輸送の増大を誘起する方法。
- 前記AAVベクターがコロイド分散系に含まれている請求項1記載の方法。
- 前記宿主がうっ血性心不全と診断されたことがある請求項1記載の方法。
- 前記宿主がヒトである請求項3記載の方法。
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US10/342,120 US7745416B2 (en) | 1995-04-11 | 2003-01-13 | Method for in vivo regulation of cardiac muscle contractility |
PCT/US2004/000719 WO2004062618A2 (en) | 2003-01-13 | 2004-01-12 | Method for in vivo regulation of cardiac muscle contractility |
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AU (2) | AU2004204815B2 (ja) |
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DE (1) | DE602004018269D1 (ja) |
ES (1) | ES2318264T3 (ja) |
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Also Published As
Publication number | Publication date |
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IL169663A (en) | 2011-04-28 |
AU2010202432A1 (en) | 2010-07-01 |
WO2004062618A2 (en) | 2004-07-29 |
ATE416792T1 (de) | 2008-12-15 |
EP1590001B1 (en) | 2008-12-10 |
US20030211080A1 (en) | 2003-11-13 |
AU2004204815A1 (en) | 2004-07-29 |
ES2318264T3 (es) | 2009-05-01 |
WO2004062618A3 (en) | 2004-12-09 |
AU2004204815B2 (en) | 2010-03-11 |
US7745416B2 (en) | 2010-06-29 |
EP1590001A4 (en) | 2006-02-01 |
HK1083065A1 (en) | 2006-06-23 |
CA2513249A1 (en) | 2004-07-29 |
DE602004018269D1 (de) | 2009-01-22 |
EP1590001A2 (en) | 2005-11-02 |
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