JP4827358B2 - Method for producing functional polyalkyleneimine, composition containing said compound and use thereof - Google Patents
Method for producing functional polyalkyleneimine, composition containing said compound and use thereof Download PDFInfo
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- JP4827358B2 JP4827358B2 JP2001560270A JP2001560270A JP4827358B2 JP 4827358 B2 JP4827358 B2 JP 4827358B2 JP 2001560270 A JP2001560270 A JP 2001560270A JP 2001560270 A JP2001560270 A JP 2001560270A JP 4827358 B2 JP4827358 B2 JP 4827358B2
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- polyalkyleneimine
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Description
【０００１】
本発明は細胞にトランスフェクトする核酸を処方するのに有用な機能的ポリアルキレンイミンの製造方法に関する。
【０００２】
バイオテクノロジーの発展に伴い、核酸の効率的な細胞導入は多数のバイオテクノロジー用途で基本技術となっている。例えば組換えタンパク質を生産したり、実験室で遺伝子発現調節の研究、遺伝子クローニング又は他の任意のＤＮＡ操作を行うといった核酸を細胞にｉｎ ｖｉｔｒｏ導入することが含まれる。また、例えばワクチン製造、標識試験又は治療アプローチでは核酸を細胞にｉｎ ｖｉｖｏ導入することが含まれる。更に、例えばトランスジェニック動物の作製には後期再投与の目的で生物から抽出した細胞に遺伝子を導入する。
【０００３】
現在、遺伝子を細胞に導入する手段として最も普及しているのはウイルスベクターの使用である。しかし、ウイルスベクターは全く危険がないとはいえず、合成ベクターの使用に基づく他のいくつかの方法も提案されている。これらの合成ベクターはトランスフェクトしようとする核酸を複合体化して圧縮すること、細胞膜及び場合により２つの核膜の通過を助長するという２つの主機能をもつ。
【０００４】
例えば合成ベクターがいくつか提案されている。それらのうちでは、ポリアルキレンイミン等のカチオンポリマーが特に有用である。カチオンポリマーは特にｉｎ ｖｉｖｏでの核酸トランスフェクションの場合に確かに比較的有効であることが分かっており、毒性も比較的低い。カチオンポリマーが核酸と形成する複合体（「ポリプレクス」とも言う）は注入部位から比較的良好に拡散することも認められている（Ｊ．Ｓ．Ｒｅｍｙら；Ａｄｖａｎｃｅｄ Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ Ｒｅｖｉｅｗｓ，３０，１９９８，ｐｐ．８５−９５）。
【０００５】
他方、トランスフェクトした核酸が生物の他の部分に拡散せずに標的細胞と効果的に相互作用することが重要であるため、適切な核酸を特定臓器、組織、細胞種又は細胞分画を標的とできるベクターを入手することが今日では不可欠であると思われる。その目的は標的細胞以外の細胞と核酸の非特異的作用を完全に避けることである。例えば、ガラクトシル化ポリエチレンイミンはガラクトースに対応する細胞受容体（レクチン）をもつ細胞にプラスミドをｉｎ ｖｉｔｒｏ導入するのに有効なベクターであることが判明した（Ｚａｎｔａら，Ｂｉｏｃｏｎｊ．Ｃｈｅｍ．，８（２），１９９７，ｐ．８３９；Ｔ．Ｂｅｔｔｉｎｇｅｒら，Ｂｉｏｃｏｎｊ．Ｃｈｅｍ．，１９９９）。
【０００６】
現在まで、例えばガラクトシル化ポリエチレンイミン等のポリマーはシアノホウ水素化ナトリウムの存在下にオリゴ糖をポリエチレンイミンと作用させることにより得られていた。しかし、この試薬は高価でしかも毒性が高いという欠点がある。比較的カチオン性である最終生成物にシアン化物イオンが残留する危険があると、医薬用途には全く利用できない。更に、ポリアルキレンイミンは小分子ではなくポリマーであり、多数の溶媒、特に非極性溶媒に溶けないため、代替製法の研究は未だ成果を上げていない。例えば、トリアセトキシホウ水素化ナトリウムを使用する代替法は成功しなかった。硫酸水溶液とピリジン−ボラン混合物中でホウ水素化ナトリウムを使用することもカチオンポリマーには不適合であることが判明した。従って、ポリアルキレンイミン型カチオンポリマーに適合可能であり、医薬的に許容可能な試薬のみを使用する代替法を開発することが必要であると思われた。
【０００７】
今般、チタン（ＩＶ）イソプロポキシドとホウ水素化ナトリウムの存在下にポリアルキレンイミンを機能的ヘミアセタールで処理することにより機能的ポリアルキレンイミンを製造できることが判明した。
【０００８】
このような方法は例えばアルコールのようにポリアルキレンイミンと適合可能な溶媒中で実施できるという利点があり、低廉且つ低毒性の試薬のみで実施できる。
【０００９】
Ｂｈａｔｔａｃｈａｒｙｙａらの各種論文（Ｊ．Ｏｒｇ．Ｃｈｅｍ．，１９９５，６０，ｐｐ．４９２８−４９２９；Ｓｙｎｌｅｔｔ，１９９５，ｐｐ．１０７９−１０８０；Ｊ．Ｃｈｅｍ．Ｓｏｃ．，Ｐｅｒｋｉｎ Ｔｒａｎｓ．１，１９９８，ｐｐ．２５２７−２５３１）にはケトン又はアルデヒドから下記反応：
【００１０】
【化４】
【００１１】
本発明によると、開始ポリアルキレンイミンは一般式：
【００１２】
【化５】
【００１３】
【化６】
【００１４】
当然のことながら、一般式（Ｉ）においてｎの値とＲ基は−ＮＲ−（ＣＨ２）ｎ−から−（ＣＨ２）ｎ−ＮＨ−までの種々のモチーフとなるように選択できる。従って、一般式（Ｉ）は直鎖ポリマーと分枝鎖ポリマーに加え、ホモポリマーとヘテロポリマーも含む。
【００１５】
ｎは２〜５が好ましい。好ましいポリマーは例えばポリエチレンイミン（ＰＥＩ）又はポリプロピレンイミン（ＰＰＩ）である。更に、本発明の実施に好ましく、特にトランスフェクションに有効であることが判明したポリマーは平均分子量１０３〜５×１０６のポリマーである。例えば、平均分子量５００００Ｄａ（５０ＫＰＥＩ）、２５０００Ｄａ（２５ＫＰＥＩ）、２２０００Ｄａ（２２ＫＰＥＩ）のポリエチレンイミン又は８０００００Ｄａ（８００ＫＰＰＩ）のポリプロピレンイミンを挙げることができる。
【００１６】
本発明で使用するポリアルキレンイミンは当業者に公知の種々の方法により得ることができる。例えばアニオン重合（例えばエチレンイミン重合）条件下で対応するモノマーから化学的に合成することもできるし、ジアルデヒドとジアミンの重縮合により得られたイミンの還元により製造することもできる。更に、例えば２５ＫＰＥＩ、２２ＫＰＥＩ又は８００ＫＰＰＩ等の多数のポリアルキレンイミンが市販されている。
【００１７】
本発明の趣旨では「機能的ポリアルキレンイミン」とは標的要素を共有結合させたポリアルキレンイミン型カチオンポリマーを意味する。これらの標的要素は所望の所定細胞種、所定組織又は所定細胞分画への核酸導入を誘導することができる。標的要素とポリアルキレンイミンの共有結合は機能的ヘミアセタール即ち置換基の１個を前記標的要素としたヘミアセタールと上記反応条件下で反応させることにより得られる。
【００１８】
より詳細には、機能的ヘミアセタールとは本発明の趣旨では一般式：
【００１９】
【化７】
【００２０】
適合可能な基の例としては、ヒドロキシ、炭素原子数１〜４のアルキル（Ｃ１〜４）又はヒドロキシアルキル（Ｃ１〜４）を挙げることができる。
【００２１】
標的要素はヘミアセタールに直接グラフトしてもよいし、二官能性結合分子（「リンカー」とも言う）を介してグラフトしてもよい。前記標的要素をヘミアセタールにグラフトするのは化学的には不可能であるが、「リンカー」はこれを可能にし、及び／又はヘミアセタールから前記標的要素を分離することができる。二官能性結合分子とは、標的要素に共有結合することができる少なくとも１個の官能基とヘミアセタールに共有結合することができる少なくとも１個の官能基を含む任意分子を意味する。
【００２２】
本発明の趣旨では、標的要素とは核酸導入を誘導することが可能な任意分子を意味する。この標的要素は所望の所定細胞種又は所定組織（腫瘍細胞、肝細胞、造血細胞等）へのＤＮＡ導入を誘導することが可能な細胞外標的要素とすることができる。所定の優先細胞区画（例えばミトコンドリア又は核）への核酸導入を誘導することが可能な細胞内標的要素でもよい。
【００２３】
本発明の範囲で利用可能な標的要素としては、糖、ペプチド、タンパク質、オリゴヌクレオチド、脂質、神経伝達物質、ホルモン、ビタミン又はその誘導体が挙げられる。例えば、抗体又は抗体フラグメント、細胞受容体のリガンド又はそのフラグメント、受容体又は受容体フラグメントのような糖、ペプチド又はタンパク質が好ましい。特に、増殖因子受容体リガンド、サイトカイン受容体リガンド、細胞レクチン型受容体のリガンドや、接着タンパク質受容体（例えばインテグリン）に対して親和性をもつＲＧＤ配列を含むリガンドが挙げられる。トランスフェリン受容体、ＨＤＬ及びＬＤＬや、葉酸輸送体も挙げることができる。標的要素はアシアログリコプロテインやシアル酸付加物（例えばシアリルルイスＸ）の受容体等のレクチンを標的とすることが可能な糖や、Ｆａｂ抗体フラグメント又は１本鎖抗体（ＳｃＦｖ）でもよい。例えば、前記糖は単糖、二糖又は三糖から選択することができる。例えばガラクトース、マンノース、フコース、ラムノース、ラクトース又はマルトースが挙げられる。
【００２４】
一般に、アルコール溶媒（例えばメタノール又はエタノール）中で１００℃〜３０℃の温度で反応を実施する。エタノール中で周囲温度即ち１８℃〜２５℃の温度で操作することが好ましい。
【００２５】
更に、一般に導入するポリマー１モル当たり２５〜１００モルのチタン（ＩＶ）イソプロポキシド、より好ましくはポリマー１モル当たり４０〜６０モルのチタン（ＩＶ）イソプロポキシドを使用する。
【００２６】
また、使用するチタン（ＩＶ）イソプロポキシドの量の５０〜８０（モル）％の量のホウ水素化ナトリウムを反応混合物に導入する。
【００２７】
本発明の方法を実施するために使用する機能的ヘミアセタールの量は所望のグラフト率により異なる。ポリマー１モル当たり６〜１００モルの機能的ヘミアセタールを使用すると効果的である。
【００２８】
機能的ヘミアセタールの使用量に応じて１％〜２０％、好ましくは４％〜２０％の割合で機能的ポリアルキレンイミンが得られる。標的要素として糖を使用する場合には、ポリアルキレンイミンにグラフトする糖の百分率はレゾルシノール法を使用して正確に決定することができる。この方法は予め透析しておいた機能的ポリアルキレンイミンをレゾルシノールと硫酸で処理した後、２０分間９０℃に加熱する。冷却後に４９５ｎｍの吸光度を測定し、基準サンプル（標準溶液）と比較する。グラフトしなかった標的要素を透析により除去し、基準サンプルと比較測定することにより、グラフトした標的要素の量を決定することができる。更に、ポリマーは従来小分子で実施されているようなＮＭＲ（核磁気共鳴）や質量測定により特性を決定することができないので、この方法は得られる生成物の特性を決定するのにより確実な手段の１つとなる。
【００２９】
本発明の方法は特定所定組織、所定細胞種又は所定細胞区画を標的とすることが可能な核酸導入ベクターを作製するために廉価で且つ低毒性の代替法を提供するという点で特に効果的である。更に、糖で機能付与したポリアルキレンイミンは非機能的ポリアルキレンイミンよりも細胞毒性が低いことが判明した。
【００３０】
機能的ポリアルキレンイミンは核酸を細胞にトランスフェクトするのに有用である。この目的では、機能的ポリアルキレンイミンを１種以上の核酸と混合し、複合体（「ポリプレクス」とも言い、従って、トランスフェクションでなく「ポリフェクション」と言う場合もある）を形成する。最適トランスフェクション効率を得るためには、形成されるポリプレクスが完全に中性となるか又は低カチオン性となるように機能的ポリアルキレンイミンと核酸の割合を選択することが好ましい。一般に、前記割合は非沈澱性のカチオンポリプレクスを形成するように選択するが、形成されるポリプレクスのカチオン性が増すと毒性も増すのでポリプレクスのカチオン性をさほど高くしてはならない。従って、前記割合はケースバイケースで決定しなければならない。前記割合は一般に機能的ポリアルキレンイミンのアミンと核酸のリン酸のモル比が０．１〜５０、好ましくは０．５〜２０となるように選択する。当然のことながら、この比は使用する機能的ポリアルキレンイミン、核酸、標的細胞、投与方法、又は所期用途（特にトランスフェクトする細胞種）に応じて当業者が容易に調節及び最適化することができる。
【００３１】
上記ポリプレクスにおいて、核酸はデオキシリボ核酸でもリボ核酸でもよい。核酸は天然配列でも人工配列でもよく、特にゲノムＤＮＡ（ｇＤＮＡ）、相補的ＤＮＡ（ｃＤＮＡ）、メッセンジャーＲＮＡ（ｍＲＮＡ）、トランスファーＲＮＡ（ｔＲＮＡ）、リボソームＲＮＡ（ｒＲＮＡ）、ハイブリッド配列、合成又は半合成配列、修飾又は非修飾オリゴヌクレオチドが挙げられる。これらの核酸はヒト、動物、植物、細菌、ウイルス等の起源とすることができる。これらの核酸は当業者に公知の任意方法により得られ、特にライブラリースクリーニング、化学合成、又はライブラリースクリーニングにより得られた配列の化学もしくは酵素修飾を含む混合法により得られる。核酸は化学的に修飾してもよい。
【００３２】
特にデオキシリボ核酸については、１本鎖でも２本鎖でもよいし、短いオリゴヌクレオチドでも長い配列でもよい。特に、核酸は例えばプラスミド、ベクター、エピソーム又は発現カセットから構成すると有利である。これらのデオキシリボ核酸は特に、標的細胞で機能的又は非機能的な複製起点、１個以上のマーカー遺伝子、転写又は複製調節配列、目的治療遺伝子、修飾又は非修飾アンチセンス配列、あるいは他の細胞成分との結合領域等をもつことができる。
【００３３】
核酸は調節配列（例えば標的細胞で活性な１種以上のプロモーターと転写ターミネーター）の制御下におかれた１種以上の目的治療遺伝子を含むことが好ましい。
【００３４】
本発明の趣旨では、目的治療遺伝子とは特に治療効果をもつタンパク性物質をコードする任意遺伝子を意味する。このようにコードされるタンパク性物質としては特にタンパク質又はペプチドが挙げられる。このタンパク性物質は標的細胞に対して同種の外来又は内因物質、即ち標的細胞が疾病をもたないときに標的細胞で正常に発現される物質とすることができる。この場合には、タンパク質が発現されると、例えば細胞での不十分な発現や修飾による不活性又は低活性のタンパク質の発現を補ったり、前記タンパク質を過剰に発現することができる。目的治療遺伝子は例えば安定性を増したり活性を変化させた細胞タンパク質の突然変異体をコードするものでもよい。タンパク性物質は標的細胞に対して異種でもよい。この場合には、発現されるタンパク質は例えば細胞に欠失している活性を補充又は付加し、疾病に対抗できるようにしたり、免疫応答を刺激したりすることができる。
【００３５】
本発明の趣旨で治療物質としては、例えば酵素、血液誘導体、ホルモン、リンホカイン（例えばインターロイキン、インターフェロン又はＴＮＦ：ＦＲ９２／０３１２０）、増殖因子、神経伝達物質又はその前駆物質もしくは合成酵素、栄養因子（例えばＢＤＮＦ、ＣＮＴＦ、ＮＧＦ、ＩＧＦ、ＧＭＦ、ａＦＧＦ、ｂＦＧＦ、ＶＥＧＦ、ＮＴ３、ＮＴ５又はＨＡＲＰ／プレイオトロフィン）、アポリポタンパク質（例えばＡｐｏＡＩ、ＡｐｏＡＩＶ又はＡｐｏＥ：ＦＲ９３／０５１２５）、ジストロフィン又はミニジストロフィン（ＦＲ９１／１１９４７）、膵臓線維症に関連するタンパク質ＣＦＴＲ、腫瘍抑制遺伝子（例えばｐ５３、Ｒｂ、Ｒａｐ１Ａ、ＤＣＣ又はｋ−ｒｅｖ：ＦＲ９３／０４７４５）、凝血に関与する因子（例えばＶＩＩ、ＶＩＩＩ、ＩＸ因子）をコードする遺伝子、ＤＮＡの修復に関与する遺伝子、自殺遺伝子（チミジンキナーゼ、シトシンデアミナーゼ）、ヘモグロビン又は他のタンパク質輸送体の遺伝子、代謝酵素又は同化酵素を挙げることができる。
【００３６】
目的治療核酸は更に、標的細胞で発現されると遺伝子発現又は細胞ｍＲＮＡの転写を調節することが可能なアンチセンス遺伝子又は配列でもよい。このような配列は、例えば特許ＥＰ１４０３０８に記載の技術に従って標的細胞で細胞ｍＲＮＡの相補的ＲＮＡに転写し、そのタンパク質翻訳を阻止することができる。治療遺伝子は更に、標的ＲＮＡを選択的に破壊することが可能なリボソームをコードする配列も含む（ＥＰ３２１２０１）。
【００３７】
上述のように、核酸は更に、ヒト又は動物で免疫応答を発生することが可能な抗原ペプチドをコードする１種以上の遺伝子を含んでいてもよい。従って、この特定態様によると、本発明はヒト又は動物で特に微生物、ウイルス又は癌に対するワクチン又は免疫治療を提供することができる。特に、エプスタイン・バーウイルス、ＨＩＶウイルス、Ｂ型肝炎ウイルス（ＥＰ１８５５７３）、偽狂犬病ウイルス、「シンシチウム形成ウイルス」、他のウイルスの特異的抗原ペプチド又は腫瘍特異的抗原ペプチド（ＥＰ２５９２１２）を挙げることができる。
【００３８】
核酸は更に所望細胞又は臓器で目的治療遺伝子及び／又は抗原ペプチドをコードする遺伝子の発現を可能にする配列も含むことが好ましい。このような配列としては、これらの配列が感染細胞で機能可能なときに該当遺伝子の発現に天然に関与する配列が挙げられる。異なる起源の配列でもよい（他のタンパク質の発現に関与する配列でもよいし、あるいは合成配列でもよい）。特に、真核又はウイルス遺伝子のプロモーター配列が挙げられる。例えば、感染させたい細胞のゲノムに由来するプロモーター配列が挙げられる。また、ウイルスのゲノムに由来するプロモーター配列でもよい。この点では、例えばＥ１Ａ、ＭＬＰ、ＣＭＶ又はＲＳＶ遺伝子のプロモーターを挙げることができる。更に、活性化配列や調節配列等を付加してこれらの発現配列を修飾してもよい。また、誘導プロモーターでも抑制プロモーターでもよい。
【００３９】
こうして形成されるポリプレクスは例えば局所、皮膚、経口、直腸、膣、非経口、鼻腔内、静脈内、筋肉内、皮下、眼内、経皮、気管内又は腹腔内経路で投与するように処方することができる。形成されるポリプレクスは特に所望臓器のレベルに直接注射するための注射用製剤又は局所投与（皮膚及び／又は粘膜）用として医薬的に許容可能なキャリヤーを含有することが好ましい。このようなキャリヤーとしては、特に等張滅菌溶液又は場合に応じて滅菌水もしくは生理的血清を加えると注射可能な溶質を構成することが可能な乾燥組成物、特に凍結乾燥組成物が挙げられる。注射に使用する核酸の用量と投与回数は種々のパラメーター、特に使用する投与方法、該当疾病、発現させようとする遺伝子、又は所望治療期間に応じて選択できる。特に投与方法については、組織（例えば腫瘍レベル）又は循環経路への直接注射や、培養細胞処理後の注射又は移植によるｉｎ ｖｉｖｏ再移植が挙げられる。本発明の範囲に該当する組織は例えば筋肉、皮膚、脳、肺、肝臓、脾臓、骨髄、胸腺、心臓、リンパ、血液、骨、軟骨、膵臓、腎臓、膀胱、胃、腸、精巣、卵巣、直腸、神経系、眼、腺、結合組織である。
【００４０】
上記ポリプレクスを含む組成物は核酸を細胞に導入するために使用することができる。より詳細には、前記組成物は疾病、特にタンパク又は核酸物質の欠損に起因する疾病の治療用医薬の製造に使用することができる。
【００４１】
核酸を細胞に導入する方法の１例は、
（１）上記ポリプレクスを含む組成物を形成する段階と、
（２）（１）で形成された組成物と細胞を接触させる段階を含む。
【００４２】
上記構成に加え、本発明の他の特徴と利点を以下の実施例と図面に記載するが、以下の記載は本発明の例示に過ぎず、発明の範囲を制限するものではない。
【００４３】
図面
図１：電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＥＣＶ３０４細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【００４４】
図２：電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＨｅＬａ細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【００４５】
図３：電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＨｅｐＧ２細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【００４６】
図４：ＤＮＡの細胞導入実験で使用したプラスミドｐＸＬ２７７４の模式図。
【００４７】
実施例
実施例１：ＰＥＩ−４％マルトースの製造［３ａ］
実施した反応は下記模式図で表すことができる。
【００４８】
【化８】
２５ＫＤａＰＥＩ０．０２ｍｍｏｌ（５００ｍｇ，Ａｌｄｒｉｃｈ製品）を無水エタノール２０ｍｌに溶かした。その後、マルトース０．７ｍｍｏｌ（２５２ｍｇ）を加え、こうして得られた溶液を窒素雰囲気下に１５分間混合した。チタンＩＶイソプロポキシド１ｍｍｏｌ（０．３ｍｌ）を反応混合物にゆっくりと加え、撹拌下に一晩維持した。次に、ホウ水素化ナトリウム０．７５ｍｍｏｌ（２８．５ｍｇ）を加え、撹拌を８時間続けた。次に、反応混合物を濾過し、１０ｍｌまで濃縮した。得られた溶液を最後に１２時間透析した（１２０００サイズ排除メンブラン）。収率は８０％であった。
【００５０】
グラフトしたマルトース残基の百分率を上述したレゾルシノール法により決定した処、マルトース残基２３個が２５ＫＤａＰＥＩにグラフトしており、アミノ基グラフト率４％であった。
【００５１】
実施例２：ＰＥＩ−１２％マルトースの製造［３ｂ］
２５ＫＤａＰＥＩ０．０２ｍｍｏｌ（５００ｍｇ，Ａｌｄｒｉｃｈ製品）を無水エタノール２０ｍｌに溶かした。その後、マルトース２ｍｍｏｌ（７２０ｍｇ）を加え、こうして得られた溶液を窒素雰囲気下に１５分間混合した。チタンＩＶイソプロポキシド２．７ｍｍｏｌ（０．８ｍｌ）を反応混合物にゆっくりと加え、撹拌下に一晩維持した。次に、ホウ水素化ナトリウム２ｍｍｏｌ（７６ｍｇ）を加え、撹拌を８時間続けた。次に、反応混合物を濾過し、１０ｍｌまで濃縮した。得られた溶液を最後に１２時間透析した（１２０００サイズ排除メンブラン）。収率は８０％であった。
【００５２】
グラフトしたマルトース残基の百分率を上述したレゾルシノール法により決定した処、マルトース残基７０個が２５ＫＤａＰＥＩにグラフトしており、アミノ基グラフト率１２％であった。
【００５３】
実施例３：ＰＥＩ−６％ラクトースの製造［４ａ］
実施した反応は下記模式図で表すことができる。
【００５４】
【化９】
２５ＫＤａＰＥＩ０．０２ｍｍｏｌ（５００ｍｇ，Ａｌｄｒｉｃｈ製品）を無水エタノール２０ｍｌに溶かした。その後、ラクトース１ｍｍｏｌ（３６０ｍｇ）を加え、こうして得られた溶液を窒素雰囲気下に１５分間混合した。チタンＩＶイソプロポキシド１．３５ｍｍｏｌ（０．４ｍｌ）を反応混合物にゆっくりと加え、撹拌下に一晩維持した。次に、ホウ水素化ナトリウム１ｍｍｏｌ（３８ｍｇ）を加え、撹拌を８時間続けた。次に、反応混合物を濾過し、１０ｍｌまで濃縮した。得られた溶液を最後に１２時間透析した（１２０００サイズ排除メンブラン）。収率は８０％であった。
【００５６】
グラフトしたラクトース残基の百分率を上述したレゾルシノール法により決定した処、ラクトース残基３５個が２５ＫＤａＰＥＩにグラフトしており、アミノ基グラフト率６％であった。
【００５７】
実施例４：ＰＥＩ−１７％ラクトースの製造［４ｂ］。
【００５８】
２５ＫＤａＰＥＩ０．０２ｍｍｏｌ（５００ｍｇ，Ａｌｄｒｉｃｈ製品）を無水エタノール２０ｍｌに溶かした。その後、ラクトース３ｍｍｏｌ（１０８０ｍｇ）を加え、こうして得られた溶液を窒素雰囲気下に１５分間混合した。チタンＩＶイソプロポキシド４ｍｍｏｌ（１．２ｍｌ）を反応混合物にゆっくりと加え、撹拌下に一晩維持した。次に、ホウ水素化ナトリウム３ｍｍｏｌ（１１４ｍｇ）を加え、撹拌を８時間続けた。次に、反応混合物を濾過し、１０ｍｌまで濃縮した。得られた溶液を最後に１２時間透析した（１２０００サイズ排除メンブラン）。収率は８０％であった。
【００５９】
グラフトしたラクトース残基の百分率を上述したレゾルシノール法により決定した処、ラクトース残基９９個が２５ＫＤａＰＥＩにグラフトしており、アミノ基グラフト率１７％であった。
【００６０】
使用例：ＰＥＩ−４％及び１２％マルトース又はＰＥＩ−６％及び７％ラクトースに結合したプラスミドＤＮＡの各種細胞種へのｉｎ ｖｉｔｒｏトランスフェクション
本実施例は上記のように合成した機能的ポリアルキレンイミンがＤＮＡを細胞にトランスフェクトする能力について検討する。
【００６１】
使用したＤＮＡはプラスミドｐＸＬ２７７４であり、１５０ｍＭ塩化ナトリウム混合物に濃度８０μｇ／ｍｌで溶かした。このプラスミドはサイトメガロウイルスのＣＭＶプロモーターの制御下にルシフェラーゼをコードするｌｕｃ遺伝子を含む。その寸法は４５００ｂｐである。このプラスミドの模式図を図４に示す。プラスミドｐＸＬ２７７４は特許出願ＷＯ９７／３５００２に記載の方法により精製した。
【００６２】
ポリプレクスはプラスミドｐＸＬ２７７４溶液と所望電荷比に応じて可変濃度に水で希釈した機能的ＰＥＩ溶液を等容量混合することにより調製した。
【００６３】
２４ウェルマイクロプレートにＨｅＬａ細胞（ＡＴＣＣ）６００００個／ウェルを播き、２４時間後にトランスフェクトした。ポリプレクス溶液５０μｌを各ウェルに滴下した。血清の不在下でトランスフェクションよりも２時間前に培地に予めＦＣＳ（ウシ胎仔血清）を加えた。次に細胞を３７℃で４時間培養した。次に複合体を含む培地を取出し、ＤＭＥＭと１０％ウシ胎仔血清の混合物に交換した。次に、細胞を２４時間再培養した。最後に、細胞を溶解させ、ルシフェラーゼテストキット（Ｐｒｏｍｅｇａ）とＤｙｎｅｘ ＭＬＸルミノメーターを使用して試験した。更に、細胞溶解液の濃度を測定することによりポリプレクスの毒性を調べ、溶解液タンパク質１μｇ当たりの酵素活性で表した。
【００６４】
図１、２及び３に示す結果はＤＮＡとの電荷比を変えて上記のように合成した各種機能的ＰＥＩを３種の異なる細胞系で使用した場合のトランスフェクション効率を表す。
【００６５】
全般にグラフト率の高いＰＥＩから形成したポリプレクスを使用した場合を除き、導入効率は比較的高いことが判明した（ＰＥＩ−１２％マルトースの効率はＰＥＩ−４％マルトースよりも劣り、ＰＥＩ−１７％ラクトースの効率はＰＥＩ−６％ラクトースよりも劣る）。従って、グラフト率は所期用途に応じて最適化することが重要なパラメーターである。
【００６６】
更に、アミノ基を機能的ヘミアセタールで置換すると、特にＤＮＡに対する電荷比が高い場合にはＰＥＩにより誘導される細胞毒性が有意に低下することが判明した。即ち、細胞生存率百分率（図１、２及び３）はＤＮＡをトランスフェクトするために非機能的ＰＥＩを使用した場合に得られる細胞生存率（結果は示さず）よりも著しく高く維持される。
【００６７】
従って、本実施例から明らかなように、確実で廉価な本発明の方法により得られる機能的ポリアルキレンイミンはＤＮＡの細胞トランスフェクションに有用な候補である。
【００６８】
【図面の簡単な説明】
【図１】 電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＥＣＶ３０４細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【図２】 電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＨｅＬａ細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【図３】 電荷比（＋／−）３〜２８の種々のグリコシル化ポリアルキレンイミン／ＤＮＡ処方のＨｅｐＧ２細胞におけるルシフェラーゼ活性をＲＬＵ／μｇタンパク質（ＲＬＵ：Ｒｅｌａｔｉｖｅ Ｌｉｇｈｔ Ｕｎｉｔ即ち相対発光量）で表したヒストグラム。実線の曲線は使用した各処方［３ａ］、［３ｂ］、［４ａ］及び［４ｂ］のグリコシル化ポリアルキレンイミンとＤＮＡの電荷比（＋／−）に伴う細胞生存率百分率を示す。
【図４】 ＤＮＡの細胞導入実験で使用したプラスミドｐＸＬ２７７４の模式図。[0001]
The present invention relates to a process for the production of functional polyalkyleneimines useful for formulating nucleic acids that transfect cells.
[0002]
With the development of biotechnology, efficient cell introduction of nucleic acids has become a basic technology for many biotechnology applications. For example, in vitro introduction of a nucleic acid into a cell, such as producing a recombinant protein or performing gene expression regulation studies, gene cloning or any other DNA manipulation in the laboratory. Also, for example, vaccine production, labeling tests or therapeutic approaches include introducing nucleic acids into cells in vivo. Furthermore, for example, in the production of transgenic animals, genes are introduced into cells extracted from organisms for the purpose of late re-administration.
[0003]
Currently, the most popular means for introducing genes into cells is the use of viral vectors. However, viral vectors are not at all dangerous and several other methods based on the use of synthetic vectors have also been proposed. These synthetic vectors have two main functions: complexing and compressing the nucleic acid to be transfected and facilitating passage through the cell membrane and possibly two nuclear membranes.
[0004]
For example, several synthetic vectors have been proposed. Of these, cationic polymers such as polyalkyleneimines are particularly useful. Cationic polymers have proven to be relatively effective, especially in the case of in vivo nucleic acid transfection, and have a relatively low toxicity. Complexes formed by cationic polymers with nucleic acids (also referred to as “polyplexes”) have also been found to diffuse relatively well from the injection site (JS Remy et al; Advanced Drug Delivery Reviews, 30, 1998, pp. .85-95).
[0005]
On the other hand, it is important that the transfected nucleic acid interacts effectively with the target cell without diffusing to other parts of the organism, so the appropriate nucleic acid is targeted to a specific organ, tissue, cell type or cell fraction It seems to be indispensable today to obtain a vector that can be used. Its purpose is to completely avoid non-specific actions of cells and nucleic acids other than the target cells. For example, galactosylated polyethyleneimine has been found to be an effective vector for in vitro introduction of plasmids into cells with cell receptors (lectins) corresponding to galactose (Zanta et al., Bioconj. Chem., 8 (2 ), 1997, p. 839; T. Bettinger et al., Bioconj. Chem., 1999).
[0006]
To date, polymers such as galactosylated polyethyleneimine have been obtained by reacting oligosaccharides with polyethyleneimine in the presence of sodium cyanoborohydride. However, this reagent has the disadvantage of being expensive and highly toxic. The danger of residual cyanide ions in the end product, which is relatively cationic, is completely unusable for pharmaceutical use. In addition, polyalkylenimines are polymers rather than small molecules and are insoluble in many solvents, especially nonpolar solvents, so research on alternative processes has not yet yielded results. For example, alternative methods using sodium triacetoxyborohydride have not been successful. The use of sodium borohydride in aqueous sulfuric acid and pyridine-borane mixtures has also proved incompatible with cationic polymers. It therefore appeared necessary to develop an alternative method that is compatible with polyalkyleneimine type cationic polymers and uses only pharmaceutically acceptable reagents.
[0007]
It has now been found that functional polyalkyleneimines can be prepared by treating polyalkyleneimines with functional hemiacetals in the presence of titanium (IV) isopropoxide and sodium borohydride.
[0008]
Such a method has an advantage that it can be carried out in a solvent compatible with polyalkyleneimine such as alcohol, and can be carried out only with a low cost and low toxicity reagent.
[0009]
Bhattacharya et al. (J. Org. Chem., 1995, 60, pp. 4928-4929; Synlett, 1995, pp. 1079-1080; J. Chem. Soc., Perkin Trans. 1, 1998, pp. 2527). -2531) includes the following reaction from ketone or aldehyde:
[0010]
[Formula 4]
[0011]
According to the present invention, the starting polyalkylenimine has the general formula:
[0012]
[Chemical formula 5]
[0013]
[Chemical 6]
[0014]
Of course, in general formula (I), the value of n and the R group are -NR- (CH 2 ) n -To-(CH 2 ) n It can be selected to be various motifs up to -NH-. Accordingly, general formula (I) includes homopolymers and heteropolymers in addition to linear and branched polymers.
[0015]
n is preferably 2 to 5. Preferred polymers are for example polyethyleneimine (PEI) or polypropyleneimine (PPI). Furthermore, polymers that have been found to be preferred for the practice of the invention and particularly effective for transfection have an average molecular weight of 10 3 ~ 5x10 6 The polymer. Examples thereof include polyethyleneimine having an average molecular weight of 50000 Da (50 KPEI), 25000 Da (25 KPEI), 22000 Da (22 KPEI), or polypropyleneimine having 80000 Da (800 KPPI).
[0016]
The polyalkylenimine used in the present invention can be obtained by various methods known to those skilled in the art. For example, it can be chemically synthesized from the corresponding monomer under anionic polymerization (for example, ethyleneimine polymerization) conditions, or can be produced by reduction of imine obtained by polycondensation of dialdehyde and diamine. In addition, many polyalkylenimines are commercially available, such as 25KPEI, 22KPEI or 800KPPI.
[0017]
In the meaning of the present invention, “functional polyalkyleneimine” means a polyalkyleneimine-type cationic polymer having a target element covalently bonded thereto. These target elements can induce nucleic acid introduction into a desired predetermined cell type, predetermined tissue or predetermined cell fraction. The covalent bond between the target element and the polyalkylenimine is obtained by reacting a functional hemiacetal, that is, a hemiacetal having one of the substituents as the target element under the above reaction conditions.
[0018]
More particularly, functional hemiacetal is a compound of the general formula:
[0019]
[Chemical 7]
[0020]
Examples of compatible groups include hydroxy, alkyl having 1 to 4 carbon atoms (C1-4) or hydroxyalkyl (C1-4).
[0021]
The targeting element may be grafted directly to the hemiacetal or may be grafted via a bifunctional binding molecule (also referred to as a “linker”). Although it is chemically impossible to graft the target element onto a hemiacetal, a “linker” allows this and / or separates the target element from the hemiacetal. By bifunctional binding molecule is meant any molecule that contains at least one functional group that can be covalently bound to a target element and at least one functional group that can be covalently bound to a hemiacetal.
[0022]
For the purposes of the present invention, target element means any molecule capable of inducing nucleic acid introduction. This target element can be an extracellular target element capable of inducing DNA introduction into a desired predetermined cell type or predetermined tissue (tumor cell, hepatocyte, hematopoietic cell, etc.). It may be an intracellular targeting element capable of inducing nucleic acid introduction into a given preferred cell compartment (eg mitochondrion or nucleus).
[0023]
Target elements that can be used within the scope of the present invention include sugars, peptides, proteins, oligonucleotides, lipids, neurotransmitters, hormones, vitamins or derivatives thereof. For example, sugars, peptides or proteins such as antibodies or antibody fragments, cell receptor ligands or fragments thereof, receptors or receptor fragments are preferred. In particular, growth factor receptor ligands, cytokine receptor ligands, cell lectin-type receptor ligands, and ligands containing RGD sequences that have an affinity for adhesion protein receptors (for example, integrins). Transferrin receptors, HDL and LDL, and folate transporters can also be mentioned. The target element may be a sugar capable of targeting a lectin such as a receptor for asialoglycoprotein or a sialic acid adduct (eg, sialyl Lewis X), a Fab antibody fragment or a single chain antibody (ScFv). For example, the sugar can be selected from monosaccharides, disaccharides or trisaccharides. Examples include galactose, mannose, fucose, rhamnose, lactose, or maltose.
[0024]
In general, the reaction is carried out in an alcohol solvent (for example methanol or ethanol) at a temperature of from 100 ° C to 30 ° C. It is preferred to operate in ethanol at ambient temperature, i.e. a temperature between 18C and 25C.
[0025]
Furthermore, generally from 25 to 100 moles of titanium (IV) isopropoxide are used per mole of polymer introduced, more preferably from 40 to 60 moles of titanium (IV) isopropoxide per mole of polymer.
[0026]
Also, sodium borohydride in an amount of 50-80 (mol)% of the amount of titanium (IV) isopropoxide used is introduced into the reaction mixture.
[0027]
The amount of functional hemiacetal used to carry out the method of the present invention depends on the desired grafting rate. It is advantageous to use 6 to 100 moles of functional hemiacetal per mole of polymer.
[0028]
Depending on the amount of functional hemiacetal used, functional polyalkylenimine is obtained in a proportion of 1% to 20%, preferably 4% to 20%. When sugar is used as the target element, the percentage of sugar grafted to the polyalkyleneimine can be accurately determined using the resorcinol method. In this method, a previously dialyzed functional polyalkyleneimine is treated with resorcinol and sulfuric acid and then heated to 90 ° C. for 20 minutes. After cooling, the absorbance at 495 nm is measured and compared with a reference sample (standard solution). The amount of grafted target element can be determined by removing ungrafted target element by dialysis and measuring it relative to a reference sample. In addition, since the polymer cannot be characterized by NMR (nuclear magnetic resonance) or mass measurements as is conventionally done with small molecules, this method is a more reliable means of determining the properties of the resulting product. It becomes one of.
[0029]
The method of the present invention is particularly effective in that it provides an inexpensive and low toxicity alternative method for producing a nucleic acid transfer vector capable of targeting a specific predetermined tissue, a predetermined cell type or a predetermined cell compartment. is there. Furthermore, it has been found that polyalkyleneimines functionalized with sugars are less cytotoxic than non-functional polyalkyleneimines.
[0030]
Functional polyalkyleneimines are useful for transfecting cells with nucleic acids. For this purpose, a functional polyalkyleneimine is mixed with one or more nucleic acids to form a complex (also referred to as “polyplex” and therefore sometimes referred to as “polyfection” rather than transfection). In order to obtain optimal transfection efficiency, it is preferable to select the ratio of functional polyalkylenimine to nucleic acid so that the formed plexes are completely neutral or low cationic. In general, the ratio is selected to form a non-precipitating cationic plex, but the cationicity of the plex should not be so high because the increased cationicity of the formed plex increases the toxicity. Therefore, the ratio must be determined on a case-by-case basis. The ratio is generally selected such that the molar ratio of functional polyalkylenimine amine to nucleic acid phosphoric acid is 0.1 to 50, preferably 0.5 to 20. Of course, this ratio is easily adjusted and optimized by one skilled in the art depending on the functional polyalkylenimine, nucleic acid, target cell, method of administration, or intended use (especially the cell type to be transfected) used. Can do.
[0031]
In the above plex, the nucleic acid may be deoxyribonucleic acid or ribonucleic acid. Nucleic acids may be natural or artificial sequences, especially genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences, synthetic or semi-synthetic sequences , Modified or unmodified oligonucleotides. These nucleic acids can originate from humans, animals, plants, bacteria, viruses and the like. These nucleic acids are obtained by any method known to those skilled in the art, and in particular by library screening, chemical synthesis, or mixed methods involving chemical or enzymatic modification of sequences obtained by library screening. The nucleic acid may be chemically modified.
[0032]
In particular, deoxyribonucleic acid may be single-stranded or double-stranded, and may be a short oligonucleotide or a long sequence. In particular, the nucleic acid is advantageously composed of, for example, a plasmid, vector, episome or expression cassette. These deoxyribonucleic acids are particularly functional or non-functional origins of replication in target cells, one or more marker genes, transcriptional or replication regulatory sequences, target therapeutic genes, modified or unmodified antisense sequences, or other cellular components And can have a coupling region.
[0033]
The nucleic acid preferably includes one or more target therapeutic genes placed under the control of regulatory sequences (eg, one or more promoters and transcription terminators active in the target cell).
[0034]
For the purposes of the present invention, the target therapeutic gene means any gene that encodes a proteinaceous substance having a therapeutic effect in particular. Examples of proteinaceous substances encoded in this way include proteins or peptides. This proteinaceous substance can be a foreign or endogenous substance of the same type as the target cell, that is, a substance that is normally expressed in the target cell when the target cell has no disease. In this case, when the protein is expressed, it is possible to compensate for the expression of an inactive or low activity protein due to insufficient expression or modification in cells, or to overexpress the protein. The target therapeutic gene may, for example, encode a mutant of a cellular protein with increased stability or altered activity. The proteinaceous material may be heterologous to the target cell. In this case, the expressed protein can, for example, supplement or add to the activity that is lacking in the cell, to combat the disease, or to stimulate an immune response.
[0035]
In the meaning of the present invention, therapeutic substances include, for example, enzymes, blood derivatives, hormones, lymphokines (for example, interleukin, interferon or TNF: FR92 / 03120), growth factors, neurotransmitters or precursors or synthetic enzymes thereof, nutrient factors ( For example, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, NT5 or HARP / pleiotrophin), apolipoprotein (eg ApoAI, ApoAIV or ApoE: FR93 / 05125), dystrophin or minidystrophin (FR91 / 11947), protein CFTR associated with pancreatic fibrosis, tumor suppressor genes (eg p53, Rb, Rap1A, DCC or k-rev: FR93 / 04745), factors involved in coagulation (eg VI , The gene encoding VIII, factor IX), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), genes for hemoglobin or other protein transporters include a metabolic enzyme or assimilation enzyme.
[0036]
The therapeutic nucleic acid of interest may further be an antisense gene or sequence capable of regulating gene expression or transcription of cellular mRNA when expressed in the target cell. Such a sequence can be transcribed into the complementary RNA of cellular mRNA in the target cell according to the technique described in patent EP140308, for example, to block its protein translation. The therapeutic gene further includes a sequence encoding a ribosome capable of selectively destroying the target RNA (EP321201).
[0037]
As described above, the nucleic acid may further comprise one or more genes that encode antigenic peptides capable of generating an immune response in humans or animals. Thus, according to this particular embodiment, the present invention can provide a vaccine or immunotherapy against humans or animals, especially against microorganisms, viruses or cancers. In particular, Epstein-Barr virus, HIV virus, hepatitis B virus (EP185573), pseudorabies virus, “syncytium-forming virus”, specific antigen peptides of other viruses or tumor-specific antigen peptides (EP259212) can be mentioned. .
[0038]
The nucleic acid preferably further includes a sequence that allows expression of the gene encoding the target therapeutic gene and / or antigenic peptide in the desired cell or organ. Such sequences include sequences that are naturally involved in the expression of the gene of interest when these sequences are capable of functioning in infected cells. It may be a sequence of different origin (a sequence involved in the expression of another protein or a synthetic sequence). In particular, eukaryotic or viral gene promoter sequences. For example, a promoter sequence derived from the genome of a cell to be infected can be mentioned. It may also be a promoter sequence derived from the viral genome. In this respect, for example, promoters of E1A, MLP, CMV or RSV genes can be mentioned. Furthermore, these expression sequences may be modified by adding an activation sequence or a regulatory sequence. Moreover, an inducible promoter or a repressible promoter may be used.
[0039]
The polyplex thus formed is formulated to be administered, for example, by topical, dermal, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal route. be able to. The formed plexes preferably contain an injectable preparation for direct injection at the level of the desired organ or a pharmaceutically acceptable carrier for topical administration (skin and / or mucosa). Such carriers include, in particular, isotonic sterile solutions or dry compositions, especially freeze-dried compositions, which can constitute injectable solutes when added with sterile water or physiological serum as the case may be. The dose of nucleic acid used for injection and the number of administrations can be selected according to various parameters, particularly the administration method used, the disease concerned, the gene to be expressed, or the desired treatment period. Particularly, the administration method includes direct injection into a tissue (for example, tumor level) or a circulatory route, and in vivo re-transplantation by injection or transplantation after treatment of cultured cells. Tissues falling within the scope of the present invention include, for example, muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testis, ovary, The rectum, nervous system, eye, gland, connective tissue.
[0040]
The composition containing the above plex can be used to introduce nucleic acids into cells. More specifically, the composition can be used for the manufacture of a medicament for the treatment of diseases, particularly diseases caused by deficiencies in proteins or nucleic acid substances.
[0041]
One example of a method for introducing a nucleic acid into a cell is:
(1) forming a composition comprising the above-described plex,
(2) including a step of contacting the cell formed with the composition formed in (1).
[0042]
In addition to the above configuration, other features and advantages of the present invention are described in the following examples and drawings. However, the following description is merely illustrative of the present invention and does not limit the scope of the invention.
[0043]
Drawing
FIG. 1: Histogram of luciferase activity in ECV304 cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit or relative luminescence). . The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
[0044]
FIG. 2: Histogram of luciferase activity in HeLa cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit or relative luminescence). . The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
[0045]
FIG. 3: Histogram of luciferase activity in HepG2 cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit or relative luminescence). . The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
[0046]
FIG. 4: Schematic diagram of plasmid pXL2774 used in the DNA cell introduction experiment.
[0047]
Example
Example 1: Production of PEI-4% maltose [3a]
The implemented reaction can be represented by the following schematic diagram.
[0048]
[Chemical 8]
25 KDaPEI 0.02 mmol (500 mg, Aldrich product) was dissolved in 20 ml of absolute ethanol. Thereafter, 0.7 mmol (252 mg) of maltose was added, and the solution thus obtained was mixed for 15 minutes under a nitrogen atmosphere. 1 mmol (0.3 ml) of titanium IV isopropoxide was slowly added to the reaction mixture and kept under stirring overnight. Next, 0.75 mmol (28.5 mg) of sodium borohydride was added and stirring was continued for 8 hours. The reaction mixture was then filtered and concentrated to 10 ml. The resulting solution was finally dialyzed for 12 hours (12000 size exclusion membrane). The yield was 80%.
[0050]
When the percentage of the grafted maltose residues was determined by the resorcinol method described above, 23 maltose residues were grafted on 25 KDaPEI, and the amino group grafting rate was 4%.
[0051]
Example 2: Production of PEI-12% maltose [3b]
25 KDaPEI 0.02 mmol (500 mg, Aldrich product) was dissolved in 20 ml of absolute ethanol. Thereafter, 2 mmol (720 mg) of maltose was added, and the solution thus obtained was mixed for 15 minutes under a nitrogen atmosphere. 2.7 mmol (0.8 ml) of titanium IV isopropoxide was slowly added to the reaction mixture and kept under stirring overnight. Next, 2 mmol (76 mg) of sodium borohydride was added and stirring was continued for 8 hours. The reaction mixture was then filtered and concentrated to 10 ml. The resulting solution was finally dialyzed for 12 hours (12000 size exclusion membrane). The yield was 80%.
[0052]
When the percentage of the grafted maltose residues was determined by the resorcinol method described above, 70 maltose residues were grafted on 25 KDaPEI, and the amino group graft ratio was 12%.
[0053]
Example 3: Production of PEI-6% lactose [4a]
The implemented reaction can be represented by the following schematic diagram.
[0054]
[Chemical 9]
25 KDaPEI 0.02 mmol (500 mg, Aldrich product) was dissolved in 20 ml of absolute ethanol. Thereafter, 1 mmol (360 mg) of lactose was added and the solution thus obtained was mixed for 15 minutes under a nitrogen atmosphere. Titanium IV isopropoxide 1.35 mmol (0.4 ml) was slowly added to the reaction mixture and kept under stirring overnight. Next, 1 mmol (38 mg) of sodium borohydride was added and stirring was continued for 8 hours. The reaction mixture was then filtered and concentrated to 10 ml. The resulting solution was finally dialyzed for 12 hours (12000 size exclusion membrane). The yield was 80%.
[0056]
When the percentage of the grafted lactose residues was determined by the resorcinol method described above, 35 lactose residues were grafted on 25 KDaPEI, and the amino group graft ratio was 6%.
[0057]
Example 4: Production of PEI-17% lactose [4b].
[0058]
25 KDaPEI 0.02 mmol (500 mg, Aldrich product) was dissolved in 20 ml of absolute ethanol. Thereafter, 3 mmol (1080 mg) of lactose was added and the solution thus obtained was mixed for 15 minutes under a nitrogen atmosphere. 4 mmol (1.2 ml) of titanium IV isopropoxide was slowly added to the reaction mixture and kept under stirring overnight. Next, 3 mmol (114 mg) of sodium borohydride was added and stirring was continued for 8 hours. The reaction mixture was then filtered and concentrated to 10 ml. The resulting solution was finally dialyzed for 12 hours (12000 size exclusion membrane). The yield was 80%.
[0059]
When the percentage of the grafted lactose residues was determined by the resorcinol method described above, 99 lactose residues were grafted on 25 KDaPEI, and the amino group graft ratio was 17%.
[0060]
Example of use: In vitro transfection of plasmid DNA conjugated to PEI-4% and 12% maltose or PEI-6% and 7% lactose into various cell types
This example examines the ability of functional polyalkylenimines synthesized as described above to transfect DNA into cells.
[0061]
The DNA used was plasmid pXL2774 and was dissolved in a 150 mM sodium chloride mixture at a concentration of 80 μg / ml. This plasmid contains the luc gene encoding luciferase under the control of the cytomegalovirus CMV promoter. Its size is 4500 bp. A schematic diagram of this plasmid is shown in FIG. Plasmid pXL2774 was purified by the method described in patent application WO 97/3502.
[0062]
Polyplexes were prepared by mixing equal volumes of plasmid pXL2774 solution and functional PEI solution diluted with water to a variable concentration depending on the desired charge ratio.
[0063]
24-well microplates were seeded with 60000 HeLa cells (ATCC) / well and transfected 24 hours later. 50 μl of the polyplex solution was dropped into each well. In the absence of serum, FCS (fetal calf serum) was added to the medium in
[0064]
The results shown in FIGS. 1, 2 and 3 show the transfection efficiency when various functional PEIs synthesized as described above with different charge ratios with DNA are used in three different cell lines.
[0065]
In general, the introduction efficiency was found to be relatively high except when using plexes formed from PEI with a high graft rate (PEI-12% maltose was inferior to PEI-4% maltose, PEI-17% The efficiency of lactose is inferior to PEI-6% lactose). Therefore, it is an important parameter to optimize the graft ratio according to the intended use.
[0066]
Furthermore, it has been found that substitution of amino groups with functional hemiacetal significantly reduces the cytotoxicity induced by PEI, especially when the charge ratio to DNA is high. That is, the cell viability percentage (FIGS. 1, 2 and 3) remains significantly higher than the cell viability obtained when using non-functional PEI to transfect DNA (results not shown).
[0067]
Thus, as is apparent from this example, functional polyalkyleneimines obtained by the reliable and inexpensive method of the present invention are useful candidates for cell transfection of DNA.
[0068]
[Brief description of the drawings]
FIG. 1 Luciferase activity in ECV304 cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit or relative luminescence). histogram. The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
FIG. 2 represents luciferase activity in HeLa cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit or relative luminescence). histogram. The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
FIG. 3 shows luciferase activity in HepG2 cells of various glycosylated polyalkyleneimine / DNA formulations with charge ratios (+/−) 3 to 28 expressed as RLU / μg protein (RLU: Relative Light Unit). histogram. The solid curve shows the percent cell viability with the charge ratio (+/−) of the glycosylated polyalkyleneimine to DNA of each formulation [3a], [3b], [4a] and [4b] used.
FIG. 4 is a schematic diagram of plasmid pXL2774 used in DNA cell introduction experiments.
Claims (13)
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0002059A FR2805271B1 (en) | 2000-02-18 | 2000-02-18 | PROCESS FOR THE PREPARATION OF FUNCTIONALIZED POLYALKYLENIMINES, COMPOSITIONS CONTAINING THEM AND USES THEREOF |
| FR00/02059 | 2000-02-18 | ||
| US20390700P | 2000-05-12 | 2000-05-12 | |
| US60/203,907 | 2000-05-12 | ||
| PCT/FR2001/000460 WO2001060890A2 (en) | 2000-02-18 | 2001-02-15 | Method for preparing functionalised polyalkylenimines, composition containing same and uses thereof |
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| JP2003529633A JP2003529633A (en) | 2003-10-07 |
| JP4827358B2 true JP4827358B2 (en) | 2011-11-30 |
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| JP2001560270A Expired - Fee Related JP4827358B2 (en) | 2000-02-18 | 2001-02-15 | Method for producing functional polyalkyleneimine, composition containing said compound and use thereof |
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| FR (1) | FR2805271B1 (en) |
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| JP4786052B2 (en) * | 2001-04-04 | 2011-10-05 | 花王株式会社 | Sugar graft polymer |
| US6878374B2 (en) * | 2003-02-25 | 2005-04-12 | Nitto Denko Corporation | Biodegradable polyacetals |
| JP2008510829A (en) * | 2004-08-25 | 2008-04-10 | ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン | Compositions based on dendrimers and their use |
| US8703197B2 (en) * | 2012-09-13 | 2014-04-22 | International Business Machines Corporation | Branched polyamines for delivery of biologically active materials |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10502918A (en) * | 1994-07-13 | 1998-03-17 | ローン−プーラン・ロレ・ソシエテ・アノニム | Compositions containing nucleic acids, their production and use |
| JPH11187874A (en) * | 1997-09-30 | 1999-07-13 | Hoechst Marion Roussel Deutsche Gmbh | Biologically acceptable low-molecular weight polyethyleneimine |
| JP2002506441A (en) * | 1997-06-20 | 2002-02-26 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Complexes for transporting nucleic acids to higher eukaryotic cells |
| JP2002531468A (en) * | 1998-12-03 | 2002-09-24 | アバンテイス・フアルマ・エス・アー | Novel nucleic acid transfer agent, composition containing the nucleic acid transfer agent, and use thereof |
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| CN1162528C (en) * | 1996-05-03 | 2004-08-18 | 普罗格特-甘布尔公司 | Cotton soil release polymers |
| US5962400A (en) * | 1998-12-22 | 1999-10-05 | National Starch And Chemical Investment Holding Corporation | Amino acid copolymers having pendent polysaccharide moieties and uses thereof |
-
2000
- 2000-02-18 FR FR0002059A patent/FR2805271B1/en not_active Expired - Fee Related
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10502918A (en) * | 1994-07-13 | 1998-03-17 | ローン−プーラン・ロレ・ソシエテ・アノニム | Compositions containing nucleic acids, their production and use |
| JP2002506441A (en) * | 1997-06-20 | 2002-02-26 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Complexes for transporting nucleic acids to higher eukaryotic cells |
| JPH11187874A (en) * | 1997-09-30 | 1999-07-13 | Hoechst Marion Roussel Deutsche Gmbh | Biologically acceptable low-molecular weight polyethyleneimine |
| JP2002531468A (en) * | 1998-12-03 | 2002-09-24 | アバンテイス・フアルマ・エス・アー | Novel nucleic acid transfer agent, composition containing the nucleic acid transfer agent, and use thereof |
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| JP2003529633A (en) | 2003-10-07 |
| FR2805271A1 (en) | 2001-08-24 |
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