JP2004532205A - Delivery of nucleic acid molecules to cells and method for evaluating the same - Google Patents

Abstract

Methods for delivering nucleic acid molecules to cells and for measuring nucleic acid delivery and expression to cells are provided. These methods are designed for the introduction of large nucleic acid molecules, including artificial chromosomes, into cells and are practiced in vitro and in vivo.

Description

【０１６４】
配列番号６〜１２に示された配列は、ジェンバンク受託番号第Ｘ８２５６４号の７５５１−１５６７０位に提示された配列とは幾つかの位置が異なっている。上記ダイバージェンスは、ｒＤＮＡの反復単位間におけるランダム突然変異に帰因し得る。
【０１６５】
ここで使用するため、クローンからのｒＤＮＡ挿入体を、コスミドをＮｏｔＩおよびＢｇｌIIで消化することにより調製し、上記要領で精製した。標準的公知方法(例、Sambrook et al.(１９８９)Molecular Cloning: A Laboratory Manual、第２版、コールドスプリングハーバー・ラボラトリー・プレス、参照)を用いて、細菌ストックの培養および維持およびプラスミドの精製を遂行し、そしてミディ‐およびマキシ‐プレップスキット(キアゲン、ミッシサウガ、オンタリオ)を用いてプラスミドを細菌培養物から精製した。
【０１６６】
Ｂ.ＧＦＰ、ネズミＡ９セルラインの調製
細胞培養およびトランスフェクション
ネズミＡ９セルラインをＡＴＣＣから入手し、下記要領で細胞を解凍および維持した。簡単に述べると、細胞を、９０％ＤＭＥＭ(キャナディアン・ライフ・テクノロジーズ・バーリントン、オンタリオ)および１０％ＦＢＳ(キャン・セラ、レキサデール、オンタリオ)を含む成長培地中１５ｃｍ組織培養皿(ファルコン、ベクトン・ディッキンソン・ラブウェア、フランクリンレイクス、ニュージャージー)１枚につき２×１０^６細胞の密度で平板培養し、３７℃、５％ＣＯ_２で維持した。細胞が培養飽和密度の７０〜８０％に達したとき、培養物を常用手順で継代した。継代培養を以下の要領で実施した。培地を吸引除去した。１０ｍｌの１×トリプシン−ＥＤＴＡ(キャナディアン・ライフ・テクノロジーズ・バーリントン、オンタリオ)を細胞単層に分配し、皿を静かに回転させてトリプシン−ＥＤＴＡを分布させた。最後に、トリプシン−ＥＤＴＡの塊を吸引除去し、皿を３７℃で５分間置いた。トリプシン−ＥＤＴＡをクエンチングするため、１０ｍｌの成長培地を皿に加え、単一細胞懸濁液を５０ｍｌ円錐管に移した。細胞計数装置(ベックマン‐コールター、ハイアレア、フロリダ)を用いて細胞計数を実施した。細胞を希釈し、上記要領で再培養した。低温貯蔵のため、トリプシン−ＥＤＴＡ処理により培養物を採取し、計数し、次いで細胞懸濁液を、スイングバケット遠心器中で５分間５００×ｇの遠心分離にかけた。細胞ペレットを、１×１０^７細胞／ｍｌの密度で９０％ＤＭＥＭ、２０％ＦＢＳおよび１０％ＤＭＳＯ(シグマ‐アルドリッチ、オークビル、オンタリオ)を含む凍結培地に再懸濁した。次いで、１ｍｌアリコートの細胞懸濁液を、クライオバイアル(ヌンク、ロチェスター、ニューヨーク)に分配し、イソプロパノール充填容器(ヌンク、ロチェスター、ニューヨーク)中で一晩冷凍し、−７０℃で置き、次いで長期貯蔵のため液体窒素冷凍庫のガス相へ移した。
【０１６７】
Ｃａ_２ＰＯ_４共沈澱法(例、Graham et al.(１９７８)Virology ５２：４５６−４５７；Wigler et al.(１９７９)Proc.Natl.Acad.Sci. U.S.A.７６：１３７３−１３７６；および(１９９０)Current Protocols in Molecular Biology、第１巻、ウィリー・インター‐サイエンス、補遺１４、ユニット９.１.１−９.１.９参照)を用いてＡ９細胞をトランスフェクションした。トランスフェクションの一日前、Ａ９細胞を１０ｃｍ皿１枚当たり２×１０^６細胞の密度で平板培養し、トランスフェクションの３時間前に培地を新鮮な成長培地と取り換えた。１４０μｇの９ｋｂ ｒＤＮＡ、ＮｏｔＩおよび５μｇの２.１ｋＢ ＣＭＶ−ＥＧＦＰ ＸｈｏＩ／ＮｒｕＩフラグメントを混合し、共沈澱させ、それらを用いてＣａ_２ＰＯ_４共沈澱物(リン酸カルシウムトランスフェクションシステム、キャナディアン・ライフ・テクノロジーズ・バーリントン、オンタリオ)を調製し、それをサブコンフルエント(subconfluent)Ａ９細胞の１０ｃｍ皿２枚に分配した。ＤＮＡ−Ｃａ_２ＰＯ_４複合体を１８時間細胞上で放置した後、沈澱物を吸引除去し、細胞を１.５分間グリセリンショックにかけた。グリセリンショック後、細胞単層を２×１０ｍｌのｄＰＢＳ(キャナディアン・ライフ・テクノロジーズ・バーリントン、オンタリオ)で静かに洗浄した後１０ｍｌの予温成長培地を加えた。最後に皿をインキュベーターに戻し、３７℃、５％ＣＯ_２で維持した。３時間回復後、各皿を３×１５ｃｍ組織培養皿で継代した。
【０１６８】
落射蛍光(epifluorescence)照明を備えた倒立顕微鏡(Ａｘｉｏｖｅｒｔ２５、ツァイス、(ノースヨーク、オンタリオ)および＃４１０１７Ｅｎｄｏｗ ＧＦＰフィルターセット(クロマ・テクノロジーズ、ブラットルボロ、バーモント))を用いて、培養物のＧＦＰ蛍光を培養中視覚的にモニターした。ＧＦＰ発現性集団の濃厚化を下記要領で実施した。
【０１６９】
蛍光活性化細胞選別法によるＧＦＰ発現性細胞集団の濃厚化
ターボ‐ソートオプションおよび２つのイノーヴァ３０６レーザー(コヒーレント、パロアルト、カリフォルニア)を備えたＦＡＣＳヴァンテージフローサイトメーター(ベクトン・ディッキンソン・イムノサイトメトリー・システムズ、サンホセ、カリフォルニア)を用いて細胞選別を実施した。細胞選別のため、７０μｍノズルを使用した。シース緩衝液をＰＢＳに変えた(２０ｐｓｉで維持)。ＧＦＰを４８８ｎｍレーザービームで励起させ、５００ＥＦＬＰフィルターを用いてＦＬ１で励起を検出した。前方および側方への散乱を、生存可能細胞について選別すべく調節した。次いで、生存可能細胞のみ、ＧＦＰ蛍光について分析した。陰性対照として野生型Ａ９細胞および陽性対照としてＧＦＰ ＣＨＯ細胞を用いて、ゲーティングパラメーターを調節した。
【０１７０】
選別の第１ラウンドについては、Ａ９細胞をトランスフェクション４日後に採取し、１０ｍｌの成長培地に再懸濁し、上記パラメーターを用いてＧＦＰ発現性集団について選別した。ＧＦＰ陽性細胞については、１×ペニシリン／ストレプトマイシンを補った５×１０ｍｌ量の成長培地(キャナディアン・ライフ・テクノロジーズ・バーリントン、オンタリオ)中に分配し、非発現性細胞については廃棄の指示をした。同じ培地を用いてさらに発現性細胞を５０ｍｌに希釈し、２×１５ｃｍ皿へ平板培養し、前項の記載と同様に培養した。選別された集団が密集成長に達したとき、ＧＦＰ発現性細胞について濃厚化するためそれらを再選別した。合計４連続選別を実施することにより、最終選別後には８９％ものＧＦＰ発現性細胞の高い濃厚化を達成した。最終ＧＦＰ発現性集団を低温保存および蛍光in situハイブリダイゼーションスクリーニング(下記参照)用に拡大させた。フローサイトメーターを用いてＧＦＰ発現性単細胞を９６ウェルプレートの個々のウェルへ指向させることにより、単細胞クローンを興味の対象である集団から確立した。これらを上記要領で培養した。
【０１７１】
蛍光In−Situハイブリダイゼーション
蛍光Ｉｎ−Ｓituハイブリダイゼーション(ＦＩＳＨ)スクリーニングを、ＧＦＰ濃厚化集団および単細胞クローンについて実施することにより、増幅および／または人工染色体形成を検出した。中期拡散物の調製およびハイブリダイゼーションを遂行した(Telenius et al.(１９９９)Chromosome Res ７：３−７参照)。使用されるプローブには、マウス主要反復を認識するｐＳＡＴ １(例、Wong et al.(１９８８)Nucl.Acids Res.１６：１１６４５−１１６６１)、マウスｒＤＮＡ含有領域とハイブリダイズするｐＦＫ１６１およびマウス小反復に対するＰＣＲ生成プローブが含まれる。
【０１７２】
すなわち、人工染色体、例えばＡＣｅｓの生成について本明細書で提供される一方法では、選択マーカー、例えば蛍光タンパク質またはフローサイトメトリーに基く方法または他の方法、例えば蛍光定量法、細胞イメージングまたは蛍光顕微鏡を用いて容易に検出され得る他のタンパク質をコード化する異種核酸を、細胞へ導入する。例えば、ｒＤＮＡおよび強化緑色蛍光タンパク質(ＥＧＦＰ)をコード化するＤＮＡは、細胞、例えばＡ９細胞へ導入され得る。トランスフェクションされた細胞は、フローサイトメトリーに基く方法または他の方法、例えば蛍光定量法、細胞イメージングまたは蛍光顕微鏡により検出され得る特性、例えば蛍光特性に基いて選別され得る。例えば、蛍光タンパク質を含む細胞は、蛍光活性化細胞選別装置(ＦＡＣＳ)を用いて非トランスフェクション細胞から単離され得る。人工染色体の存在についての細胞の染色体分析前に選別を実施する場合、人工染色体について濃厚化され得るトランスフェクション細胞の集団が提供されるため、後続の細胞染色体分析および人工染色体、例えばＡＣｅｓを含む細胞の同定および選択が容易になる。例えば、細胞は、染色体セグメントの増幅の指標、増幅および新たな人工染色体形成に関連して生じ得る構造の存在および／または人工染色体、例えばＡＣｅｓの存在について分析され得る。細胞分析は、典型的には本明細書記載および／または当業者公知の技術を用いた染色体構造の視覚化方法、例えば、限定されるわけではないが、Ｇ−およびＣ−バンド法およびＦＩＳＨ分析を含む。上記分析は、増幅が行われ得る、特定核酸、例えばサテライトＤＮＡ配列、ヘテロクロマチン、ｒＤＮＡ配列および異種核酸配列の特異的標識を使用し得る。トランスフェクション細胞の分析中における、染色体数の変化および／または、例えば反復単位の増幅から生じる分割増加による特有の染色体構造の様相は、人工染色体含有細胞の同定を助けるものである。
【０１７３】
Ｃ．フローサイトメトリーによる人工染色体の精製およびフロー選別染色体からのＤＮＡの調製
人工染色体を、フローサイトメトリーにより宿主細胞から精製した(de Jong(１９９９)Cytometry ３５：１２９−１３３参照)。簡単に述べると、ターボ‐ソート・オプションおよび２つのイノーヴァ３０６レーザー(コヒーレント、パロアルト、カリフォルニア)を備えたＦＡＣＳヴァンテージフローサイトメーター(ベクトン・ディッキンソン・イムノサイトメトリー・システムズ、サンホセ、カリフォルニア)で精製を行った。ターボ‐ソート・オプション調節は、２０ ｌｂ／ｉｎ^２から６０ ｌｂ／ｉｎ^２への最大システム圧の増加、５００００滴／秒から９９０００滴／秒の最大値までの落下駆動頻度および最大６０００Ｖ〜８０００Ｖまでの偏向面電圧の増加を含む。他の調節は、より高圧に適応させるよう器具に加えられる。ヘキスト３５２５８を第一ＵＶレーザービームで励起させ、４２０ｎｍハンドパスフィルターを用いることによりＦＬＩで励起を検出した。クロモマイシンＡ３を４５８ｎｍにセットした第２レーザーにより励起させ、４７５ｎｍロングパスフィルターを用いることにより蛍光をＦＬで検出した。両レーザーとも、出力は２００ｍＷであった。２変量分布(１０２４×１０２４チャンネル)を各選別中に累積した。全染色体選別については、シース圧を３０ ｌｂ／ｉｎ^２に設定し、５０μｍ直径ノズルを取り付けた。落下遅延プロファイルを毎朝実施し、主たるプラグ後に反復した。３.０μｍ直径スフェロレインボービーズ(スフェロテク、リバティービル、イリノイ)を用いることにより、器具のアラインメントを毎日行った。２.０％またはそれより少ないＣＶがＦＬ１およびＦＬ４について達成されたときアラインメントは最適化されたと見なした。
【０１７４】
凝縮剤(へキシレングリコール、スペルミンおよびスペルミジン)を、シース緩衝液に加えることにより、選別後の凝縮染色体を維持した。シース緩衝液は、１５ｎｍのトリスＨＣｌ、０.１ｍＭのＥＤＴＡ、２０ｍＭのＮａＣｌ、１％のへキシレングリコール、１００ｍＭのグリシン、２０μＭのスペルミンおよび５０μＭのスペルミジンを含む。選別された染色体を、４℃、約１×１０^６染色体／ｍｌの濃度で１.５ｍｌネジ蓋式エッペンドルフ管に集め、次いでそれらを４℃で貯蔵した。
【０１７５】
精製ゲノムＤＮＡの調製については、選別された染色体試料を０.５％ＳＤＳ、５０ｍＭのＥＤＴＡおよび１００μｇ／ｍｌプロテイナーゼＫに移し入れ、次いで５０℃で１８時間インキュベーションした。１μｇの２０ｍｇ／ｍｌグリコーゲン溶液(ベーリンガー・マンハイム)を各試料に加えた後、等量のフェノール：クロロホルム：イソアミルアルコール(２５：２４：１)で抽出した。１０分間２１０００×ｇで遠心分離後、水相を新しい微細遠心管に移し、上記と同様に再抽出した。０.２体積の１０Ｍ ＮＨ_４ＯＡＣ、１μｌの２０ｍｇ／ｍｌグリコーゲンおよび１体積のイソプロパノールを、２回抽出水相に加え、次いで渦状に混ぜ、１５分間３００００×ｇ(室温)で遠心分離した。ペレットを２００μｌの７０％エタノールで洗浄し、上記と同様再遠心分離にかけた。洗浄したペレットを風乾し、次に０.５〜２×１０^６染色体等量／μｌで５ｍＭのトリス−Ｃｌ、ｐＨ８.０に再懸濁した。
【０１７６】
本質的には上記と同じ要領(Co et al.(２０００)Chromosome Research ８：１８３−１９１参照)で、ＥＧＦＰおよびＲＡＰＳＹＮに特異的なプライマーセットを用いて選別された染色体試料から調製したＤＮＡにおいてＰＣＲを実施した。簡単に述べると、１０ｍＭのトリス−Ｃｌ、ｐＨ８.３、５０ｍＭのＫＣｌ、２００μＭのｄＮＴＰ、５００ｎＭの前進および逆プライマー、１.５ｍＭのＭｇＣｌ_２、１.２５単位のＴａｑポリメラーゼ(アンプリ‐Ｔａｑ、パーキン‐エルマー・シータス、カリフォルニア)を含む溶液中１００００または１０００染色体に等しいゲノムＤＮＡにおいて、５０μｌのＰＣＲ反応を実施した。各プライマーセットについて別々の反応を実施した。反応条件は以下の通りであった：９５℃で１０分を１サイクル、次いで９４℃で１分、５５℃で１分、７２℃で１分を３５サイクル、および最後に７２℃で１０分を１サイクル。完了後、下記プライマー(各々配列番号１〜４)を用いるアガロースゲル電気泳動による分析まで試料を４℃に保った：
ＥＧＦＰ前進プライマー ５'−cgtccaggagcgcaccatcttctt−３'
ＥＧＦＰ逆プライマー ３'−atcgcgcttctcgttggggtcttt−３'
ＲＡＰＳＹＮ前進プライマー ５'−aggactgggtggcttccaactcccagacac−３'、および
ＲＡＰＳＹＮ逆プライマー ５'−agcttctcattgctgcgcgccaggttcagg−３'。
プライマーは全て、キャナディアン・ライフ・テクノロジーズ(バーリントン、オンタリオ)から入手した。
【０１７７】
実施例２
カチオン小胞の調製
以下の要領に従い７００ナノモル／ｍｌ脂質の脂質濃度(カチオン脂質／ＤＯＰＥ１：１)で小胞を調製した。ガラス管(１０ｍｌ)中で、３５０ｎｍｏｌのカチオン脂質(ＳＡＩＮＴ−２)を、３５０ｎｍｏｌジオレオイルホスファチジルエタノールアミン(ＤＯＰＥ)と混合し、両方を有機溶媒(クロロホルム、メタノールまたはクロロホルム／メタノール１：１、ｖ／ｖ)中で可溶化した。ジオレオイルホスファチジルエタノールアミン(ＤＯＰＥ；アヴァンティ極性脂質、アラバスター、アラバマ)は、膜に逆六辺形相を形成し、膜を弱める。使用され得る他のエフェクターは、シス不飽和ホスファチジルエタノールアミン、シス不飽和脂肪酸およびコレステロールである。シス不飽和ホスファチジルコリンは有効性が劣る。
【０１７８】
溶媒を窒素気流下で蒸発させた(室温で１５分／２５０μｌ溶媒)。真空ポンプからの高度真空下デシケーター中で１５分間脂質を乾燥することにより、残存する溶媒を完全除去した。乾燥混合物に、１ｍｌの超高純度水を加えた。これを約５分間激しく渦状に攪拌した。生成した溶液を、澄明溶液が得られるまで超音波浴(ラボラトリー・サプライズ、インコーポレイテッド、ニューヨーク)中で音波処理にかけた。生成した懸濁液は、サイズ分布が５０〜１００ｎｍの間である単ラメラ小胞の集団を含んでいた。
【０１７９】
実施例３
アルコール注入によるカチオン小胞の調製
ガラス管(１０ｍｌ)中、３５０ｎｍカチオン脂質(セイント−２)を、３５０ｎｍｏｌのＤＯＰＥと混合し、両方を有機溶媒(クロロホルム、メタノールまたはクロロホルム／メタノール１／１)中で可溶化した。溶媒を窒素気流下で蒸発させた(室温で１５分／２５０μｌ溶媒)。高度真空下で１５分間脂質を乾燥することにより、残存する溶媒を完全除去した。次いで、これを１００μｌ純エタノール中で再構成した。
【０１８０】
実施例４
Ｖ７９−４セルラインへのベータＡＣｅｓのトランスフェクション
様々なトランスフェクション剤についてのトランスフェクション手順
全化合物をチャイニーズハムスター肺線維芽細胞ライン(Ｖ７９−４、ＡＴＣＣ番号ＣＣＬ−３９)で試験した。トランスフェクションの約１７時間前(２細胞倍加)、指数的成長中の細胞をトリプシン処理し、ダルベッコ修飾イーグル培地(ライフ・テクノロジーズ、バーリントン、オンタリオ)を含み、１０％ＦＢＳ(キャン・セラ、レキサデール、オンタリオ)を補った６ウェルペトリ皿に１ウェル当たり２５００００細胞で平板培養した。トランスフェクション時点で、１ウェル当たりの細胞数を評価したところ約１００万であった。トランスフェクションするため、裸のＤＮＡへの複合体形成に関する個々の製造業者の各プロトコルに従ったが、例外として使用するトランスフェクション剤の量を変えることにより、存在するＤＮＡの異なる量およびタイプ、並びに複合体形成の異なるイオン強度を反映させた。典型的には１００万のＡＣｅｓ(８００μｌの体積で)を、広範な濃度(製造業者が示唆した最低濃度の５倍および１００倍の間)でのトランスフェクション剤と合わせた。ＡＣｅｓ／トランスフェクション混合物を、製造業者が推奨する時間、０.８ｍｌ〜１.９ｍｌの範囲の容量で複合させた。複合体形成反応に培地を加えることを推奨する製造業者もある。次いで、複合混合物を受容細胞に適用し、製造業者のプロトコルにしたがってトランスフェクションを続行させた。異なる薬剤で使用される様々な条件に関する詳細は、表１に示されている。
【０１８１】
スーパーフェクト剤についてのトランスフェクション手順
スーパーフェクトを、チャイニーズハムスター肺線維芽細胞ライン(Ｖ７９−４、ＡＴＣＣ番号ＣＣＬ−３９)で試験した。トランスフェクションの約１７時間前(２細胞倍加)、指数的成長中の細胞をトリプシン処理し、ダルベッコ修飾イーグル培地(ライフ・テクノロジーズ、バーリントン、オンタリオ)を含み、１０％ＦＢＳ(キャン・セラ、レキサデール、オンタリオ)を補った６ウェルペトリ皿に１ウェル当たり２５００００細胞で平板培養した。選別バファー８００μｌ中１００万のＡＣｅｓが１０μｌのスーパーフェクト試薬と複合体形成した。複合体を室温で１０分間インキュベーションした。トランスフェクション時点で、１ウェル当たりの細胞数を評価したところ約１００万であった。培地をウェルから除去し、６００μｌのＤＭＥＭおよび１０％ＦＢＳを加えた。スーパーフェクト：ＡＣｅｓ複合体をウェルへ滴下し、３７℃で３時間インキュベーションした。インキュベーション後、トランスフェクション細胞をトリプシン処理し、２５ｍｌのＤＭＥＭおよび１０％ＦＢＳを含む１５ｃｍ皿に移し、２４時間結合させた。２４時間後、０.７ｍｇ／ｍｌのハイグロマイシンＢを含む選択培地を各ウェルに加えた。選択培地を２〜３日ごとに変えた。１０〜１２日後、コロニーを、無傷の染色体検出のためにベータ‐ガラクトシダーゼ発現および／またはＦＩＳＨについてのスクリーニングにかけた。
【０１８２】
クロモス指数の測定の適用例
約１×１０^６のＶ７９−４細胞を、送達因子(すなわち、リポフェクタミン・プラスおよびリポフェクタミンまたはスーパーフェクト)と複合した１×１０^６のＩｄＵｒｄ標識ＡＣｅｓによりトランスフェクションした。次いで、トランスフェクション細胞をエタノールに固定した。固定された細胞を変性させ、ＢｒｄＵ／ＩｄＵｒｄ標識核酸に特異的に結合するＦＩＴＣコンジュゲート抗体に暴露した。
【０１８３】
ＩｄＵｒｄ標識ＡＣｅｓを含むトランスフェクション細胞のパーセントを、フローサイトメトリーを用い、ＦＩＴＣ蛍光を集めて測定した。データを蓄積すると、前方散乱対緑色蛍光(ＩｄＵｒｄ−ＦＩＴＣ)を示す２変量チャンネル分布が形成された。１％が陽性領域に現れるように陰性細胞についてのゲートを設定した形で、陰性対照細胞のヒストグラムの視覚的検査により、細胞が陽性であると測定された蛍光レベルを確立した。
【０１８４】
トランスフェクションの２４時間後に回収された細胞数を、コールターカウンターを用いてアリコートを計数することにより測定した。受容セルラインの対照平板効率を測定するため、未処理細胞を、成長培地中１０ｃｍペトリ皿１枚につき６００〜１０００細胞で平板培養し、５細胞周期間または５０細胞から成る平均コロニーが形成されるまで３７℃で、５％ＣＯ_２インキュベーター中で静置した。この時点で、生存し得るコロニーの数を測定した。ＣＰＥが０.１〜０.２を越える場合、処理細胞を１０００細胞で播種した。ＣＰＥが低い場合、播種密度を皿１枚当たり５０００〜５００００細胞に増加させた。
【０１８５】
実施例５
リポフェクタミンによるＬＭＴＫ(−)細胞の超音波伝達トランスフェクション
ＬＭ(ｔｋ−)細胞を、４５００ｍｇ／ＬのＤ−グルコース、Ｌ−グルタミン、ピリドキシン塩酸塩および１０％胎児ウシ血清を含むＤＭＥＭ中、３７℃、５％ＣＯ_２で培養した。使用２４時間前１２ウェル皿のコーナーウェルに皿１枚当たり２０００００細胞を播種した(これは、他のウェルからの超音波からの干渉が無いことを確実にするためである)。
【０１８６】
ＧＦＰ染色体を計数することにより、１ｍｌにつき約１×１０^６のＡＣｅｓが実証された。染色体を迅速に動かすことにより管中で再懸濁した。１０μｌの染色体懸濁液を除去し、等量の３０ｍｇ／ｍｌＰＩ(よう化プロピジウム)染料と混合した。染色された染色体８μｌをペトロフ・ハウサー計数チャンバーへローディングし、染色体を計数した。
【０１８７】
培地を細胞から除去し、細胞を３７℃に温めておいたＨＢＳＳ(フェノールレッド不含有、ギブコＢＲＬ)で２回洗浄した。温めておいたＨＢＳＳ５００μｌを細胞(１μｌ)の各ウェルに加え、リポフェクタミン(ギブコＢＲＬ)を各ウェルに加えた。次いで、プレートをパラフィルムテープで密閉し、室温で３０分間、２０ｒｐｍで静かに振とうした(スタッガープレート−操作の容易さのため１０分)。
【０１８８】
インキュベーション後、ウルトラサウンドゲル(アザー‐ソニック・ジェネリック超音波透過ゲル、ファーマシューティカル・イノヴェーションズ、インコーポレイテッド、ニューアーク、ニュージャージー)を、２.５ｃｍソノポレーター(sonoporator)ヘッドに適用した。超音波を、プレートの底部を通して、６０秒間、ＩｍａＲＸソノポレーター１００により２.０ワット／ｃｍ^２の出力エネルギーで細胞に適用した。ウェルの超音波適用後、播種細胞(２×１０^５)当たり一染色体またはシース緩衝液(１５ｎＭのトリスＨＣｌ、０.１ｍＭのＥＤＴＡ、２０ｍＭのＮａＣｌ、１％へキシレングリコール、１００ｍＭのグリシン、２０μＭのスペルミンおよび５０μＭのスペルミジン)中の２００μｌのＧＦＰ ＡＣｅｓを直ちにウェルに加えた。(超音波を必要とするプレート上の試料が全て処理されるまで反復)。次いで、プレートをパラフィルムテープで再度密閉し、室温で１時間静かに(２０ｒｐｍ)振とうした。
【０１８９】
インキュベーション後、１ｍｌ(ＤＭＥＭ、４５００ｍｇ／ＬのＤ−グルコース、Ｌ−グルタミンおよびピリドキシン塩酸塩、１０％胎児ウシ血清、および１００００単位／ｍｌのペニシリンおよび１００００ｍｇ／ｍｌのストレプトマイシンからのペニシリンおよびストレプトマイシンの１×溶液、１００×ストック溶液含有)を各ウェルに加え、細胞を３７℃で１８〜２４時間インキュベーションした。
【０１９０】
次いで、プレート中の細胞を抗生物質含有培地で洗浄し、２ｍｌの培地を各ウェルに入れた。細胞をトランスフェクション／音響穿孔の４８時間後まで５％ＣＯ_２、３７℃でインキュベーションし続けた。次いで、細胞をトリプシン処理し、ＤＭＥＭ中１×１０^６の濃度で再懸濁し、フローサイトメトリーにより分析した。
【０１９１】
結果：ターボ‐ソートオプションおよび２つのイノーヴァ３０５レーザー(コヒーレント、パロアルト、カリフォルニア)を備えたＦＡＣＳヴァンテージ(ＢＤＩＳ、サンホセ、カリフォルニア)においてフロー分析を遂行した。ＧＦＰシグナル励起は４８８ｎｍであり、５００ｎｍロングパスフィルターを用いてＦＬ１で放射を検出した。トランスフェクション細胞の分析により、１３〜２７％の範囲に及ぶＧＦＰ陽性細胞の集団が生成された。非音響穿孔対照値は５％であった。
【０１９２】
実施例６
セイント−２による超音波伝達トランスフェクション
Ａ．セイント−２によるＣＨＯ−ＫＩ細胞の超音波伝達トランスフェクション
ＣＨＯ−ＫＩ細胞を、ＣＨＯ−Ｓ−ＳＦＭ２培地(ギブコＢＲＬ、ペーズリー、イギリス国)中、３７℃、５％ＣＯ_２で培養した。２×１０^５および５×１０^５間の細胞を、使用の２４時間前１２ウェルプレートにおいて滅菌ガラススライドへ平板培養した。
【０１９３】
細胞のトランスフェクションを以下の要領で遂行した。培地を細胞から除去し、細胞を３７℃のＨＢＳＳ(フェノールレッド不含有ハンクス平衡塩溶液(ギブコＢＲＬ、イギリス国))により２回洗浄した。次いで、１ウェルにつき３７℃の５００μｌのＨＢＳＳを加えた後、１０μｌの新たに調製した小胞溶液(実施例２で調製)を加え、２３.３ｎｍｏｌ／ｍｌの最終濃度とした。
【０１９４】
別法として、培地を細胞から除去し、細胞をＨＢＳＳで２回洗浄した。３７℃のＨＢＳＳ／脂質溶液５００μｌを各ウェルに加えた。１μｌのエタノール性脂質溶液(上記要領で調製)を５００μｌのＨＢＳＳに激しく渦状に攪拌しながら加えることにより、ＨＢＳＳ／脂質溶液を調製した。次いで、プレートをパラフィルムテープで密閉し、室温で３０分間静かに振とうした。インキュベーション後、超音波を０.５ワット／ｃｍ^２の出力エネルギーで６０秒間プレート底部を通して細胞に適用した。超音波は、変換器およびプレート間において超音波ゲル(アクアソニック１００、パーカー、ニュージャージー)により伝達された。超音波をＩｍａＲＸソノポレーター１００で適用した。超音波適用直後、播種細胞(２×１０^５〜５×１０^５)(実施例１で調製)につき一ＧＦＰ染色体を加えた。次いでプレートを再度密閉し、室温で１時間静かに振とうした。インキュベーション後、１ｍｌの培地(ＣＨＯ−Ｓ−ＳＦＭ２、１０％胎児ウシ血清、１００００μｇ／ｍｌペニシリンおよび１００００μｇ／ｍｌストレプトマイシン含有(ギブコＢＲＬ、ペーズリー、イギリス国))を各ウェルに加え、細胞を３７℃で２４時間インキュベーションした。次いで、細胞を培地で洗浄し、１ｍｌの培地を加え、細胞をさらに２４時間３７℃でインキュベーションした。次いで、発現された遺伝子の検出を顕微鏡により評価するかまたは導入された染色体の検出をＦＩＳＨ分析法により評価した。陰性対照試験は、染色体を細胞に加えないこと以外、同じ方法で遂行した。
【０１９５】
結果
トランスフェクション後、視覚的検査を用いると、細胞の３０％がガラススライドに残存し、そのうち１０％が４８時間後に緑色蛍光タンパク質発現について陽性であった(最初の集団の３％)。２週間培養後、ＦＩＳＨを細胞において遂行したところ、細胞の１.４％が無傷の人工染色体を含んでいた。
【０１９６】
Ｂ．セイント−２によるＨｅｐ−Ｇ２細胞の超音波伝達トランスフェクション
Ｈｅｐ−Ｇ２細胞を、４５００ｍｇ／ｌのグルコース、ピリドキシン／ＨＣｌ、１０％胎児ウシ血清、１００００μｇ／ｍｌのストレプトマイシンおよび１０００μｇ／ｍｌのペニシリンを加えたＤＭＥＭ中において、３７℃、５％ＣＯ_２で培養した。２×１０^５および５×１０^５間の細胞を、使用の２４時間前１２ウェルプレートにおいて滅菌ガラススライドへ平板培養した。
【０１９７】
ＣＨＯ−ＫＩ培地をＨｅｐ−Ｇ２培地と置き換えること以外は実施例６Ａの手順を用いて細胞をＧＦＰ染色体でトランスフェクションした。
【０１９８】
結果
トランスフェクション後、細胞の３０％がガラススライド上に残存していた。これらの細胞の８０％は、緑色蛍光タンパク質発現について陽性であった。
【０１９９】
Ｃ．セイント−２によるＡ９細胞の超音波伝達トランスフェクション
Ａ９細胞を、４５００ｍｇ／ｌのグルコース、ピリドキシン／ＨＣｌ、１０％胎児ウシ血清、１００００μｇ／ｍｌのストレプトマイシンおよび１００００μｇ／ｍｌのペニシリンを加えたＤＭＥＭ(ギブコＢＲＬ、ペーズリー、イギリス国)中において、３７℃、５％ＣＯ_２で培養した。２×１０^５および５×１０^５間の細胞を、使用の２４時間前１２ウェルプレートにおいて滅菌ガラススライドへ平板培養した。
【０２００】
ＣＨＯ−ＫＩ培地をＡ９培地と置き換えること以外は実施例６Ａの手順を用いて細胞をＧＦＰ染色体でトランスフェクションした。
【０２０１】
結果
トランスフェクション後、細胞の３０％がガラススライド上に残存しており、そのうち５０％は、緑色蛍光タンパク質発現について陽性であった。
【０２０２】
実施例７
滑膜細胞、骨格筋線維芽細胞および皮膚線維芽細胞へのＡＣｅｓの送達
米国特許第６０２５１５５および６０７７６９７号およびＰＣＴ出願公開第ＷＯ／９７４０１８３号の記載にしたがって調製された、一次ネズミ中心周囲ヘテロクロマチンを含み、リポーター遺伝子(ｌａｃＺ)およびハイグロマイシンＢ選択可能マーカー遺伝子を含む哺乳類(ネズミ)ＡＣｅｓ人工染色体(〜６０Ｍｂ)を、一次ラット線維芽細胞様滑膜細胞、ラット皮膚線維芽細胞およびラット骨格筋線維芽細胞ライン(Ｌ８細胞；ＡＴＣＣ受託番号ＣＲＬ−１７６９)へ送達した。送達前、本明細書記載の要領でＡＣｅｓをヨードデオキシウリジン(ＩｄＵｒｄ)により標識した。
【０２０３】
細胞の調製
一次線維芽細胞様滑膜細胞およびラット皮膚線維芽細胞を、標準的方法を用いてラットから入手した[例、Aupperle et al.(１９９９)J.Immunol. １６３：４２７−４３３およびAlvaro-Garcia et al.(１９９０)J.Clin.Invest.８６：１７９０]。上記方法には、げっし動物の膝から一般的には皮膚および筋肉を除去後次いで膝関節組織を細かく刻むことによる滑膜細胞の単離が含まれる。次いで、細かく刻んだ組織をコラゲナーゼとインキュベーションし、ナイロンメッシュにより濾過し、十分に洗浄する。細胞は一晩培養され得、その後の時点で、非接着細胞を除去する。接着細胞は、培養され、培養物が密集成長に達したときに一定希釈度で再平板培養することにより継代され得る。低グルコースＤＭＥＭ、１−グルタミン、ペニシリン／ストレプトマイシンおよび２０％ＦＢＳを含む培地中６ウェル皿１枚につき５００００〜７５０００細胞の割合で細胞を平板培養する。約８０％密集成長または１ウェル当たり５０００００細胞になるまで、細胞を３〜５日間３７℃の５％ＣＯ_２インキュベーター中で培養した。
【０２０４】
ＡＣｅｓによる細胞のトランスフェクション
１００万のＩｄＵｒｄ標識ＡＣｅｓを、以下の要領で２、５または１０μｌのスーパーフェクト(キアゲン)またはリポフェクタミン・プラス(ライフ・テクノロジーズ；ギブコ)と複合体形成させた。スーパーフェクトとの複合体形成を室温で１０分間実施した。リポフェクタミン・プラスとの複合体形成については、指示量のプラス試薬を１００万のＡＣｅｓに加え、室温で１５分間複合体形成させた。次に、指示量のリポフェクタミンを２００μｌの低グルコースＤＭＥＭ(ＦＢＳ不含有)に加え、室温で１５分間ＡＣｅｓ／プラス試薬複合体と合わせた。次いで、複合体化ＡＣｅｓを、６００μｌの培地中の細胞に滴下した(約１.４ｍｌの最終容量)。５％ＣＯ_２インキュベーター中３７℃で３時間後、総量３ｍｌの培養培地(低グルコースＤＭＥＭ、ｌ−グルタミン、ペニシリン／ストレプトマイシンおよび２０％ＦＢＳ)を加えた。２４〜４８時間後、細胞をトリプシン処理して単細胞懸濁液を形成し、遠心分離にかけることにより、上清を除去し、次いで最小限１時間冷７０％エタノールに固定した。固定細胞のアリコートを顕微鏡分析用に取っておいた。
【０２０５】
ＡＣｅｓのＦＩＴＣ−コンジュゲート抗体標識
トランスフェクション後、ＡＣｅｓを、ＢｒｄＵ−またはＩｄＵｒｄ−標識核酸に特異結合するＦＩＴＣコンジュゲート抗体で標識し、細胞をＦＩＴＣ蛍光および顕微鏡染色についてＦＡＣｓにより分析した。固定細胞を室温で３０分間２ＮのＨＣｌおよび０.５％トリトン−Ｘ中で変性させた。変性誘発後、細胞を４℃での一連の洗浄工程により中和した。バックグラウンド染色を最小にするため、試料を、最小限１５分間ＰＢＳおよび４％ＦＢＳまたはＢＳＡおよび０.１％トリトン−Ｘ(遮断緩衝液)に再懸濁した。次いで、細胞を沈澱させ、室温で２時間ＦＩＴＣコンジュゲート抗体に暴露した。細胞を遮断緩衝液で洗浄後、試料をフローサイトメトリー分析用に準備した。顕微鏡分析用試料をスライド上で乾燥し、上記染色プロトコルに従ったが、例外としてＢｒｄＵ／ＩｄＵｒｄ抗体を１／５希釈し、２４時間細胞に暴露した。
【０２０６】
結果
無傷ＡＣｅｓの送達を、トランスフェクション後２４〜４８時間以内に検出した。トランスフェクション２４時間後に回収された細胞数を、コールターカウンターを用いてアリコートを計数することにより測定した。受容セルラインの対照平板効率またはトランスフェクション細胞の平板効率を測定するため、細胞を成長培地中１０ｃｍペトリ皿１枚につき１０００〜１００００細胞の割合で平板培養し、１０日間３７℃の５％ＣＯ_２インキュベーター中で静置した。その時点で、生存し得るコロニーの数を測定した。正規化平板効率を本明細書の記載にしたがって計算した。
【０２０７】
スーパーフェクトを送達因子として使用したとき、フローサイトメトリーにより測定された線維芽細胞様滑膜細胞への送達パーセントは、〜２４％から〜６６.３％の範囲であった。２μｌのスーパーフェクトを使用したとき正規化平板効率％は〜３６％であり、５μｌのスーパーフェクトを使用したとき〜１６％であった。高用量のスーパーフェクトは、低用量の場合と比べて毒性および１細胞につき多数のＡＣｅｓを伴う。リポフェクタミン・プラスを送達因子として使用したとき、フローサイトメトリーにより測定された線維芽細胞様滑膜細胞への送達パーセントは、〜１１％から〜２７％の範囲であり、因子の用量を増やすと送達パーセントは増加した。
【０２０８】
ＡＣｅｓでトランスフェクションされたＬ８およびラット皮膚線維芽細胞(ＲＳＦ)を、ハイグロマイシンＢ選択下で培養し、ｌａｃＺ発現について分析した。この実施例では、ハイグロマイシン選択遺伝子がＡＣｅｓに含まれていたが、多様な他の選択マーカー遺伝子もあり、それらは、上記遺伝子を含むことが望ましい場合に細胞への異種核酸導入に関連して使用され得る。上記選択系は、当業者には公知である。選択マーカー遺伝子の選択は、例えば、トランスフェクションについての宿主細胞に対する選択剤の毒性レベルを考慮し得る。適切な選択マーカー遺伝子の同定は、本明細書で適用されているガイダンスを用いて常用手順で行われる。
【０２０９】
ｌａｃＺを発現するＬ８細胞およびＲＳＦ細胞のクローンを同定した。これらの結果は、ＩｄＵｒｄ−標識ＡＣｅｓが一次細胞およびセルラインへ有効に送達され得ること、およびＡＣｅｓに含まれる導入遺伝子がトランスフェクションされた細胞で発現されることを立証している。
【０２１０】
実施例８
ラット関節へのリポーター遺伝子のエクスビボ導入
インビボ環境への異種遺伝子の導入および遺伝子のインビボ発現を調べるため、上記のＡＣｅｓでトランスフェクションしたＬ８細胞を、アジュバント誘発関節炎を患うラットの足関節に注射した。０日目、関節炎のアジュバント誘発をルイスラットにおいて行った。動物モデルにおけるアジュバント誘発方法は、当業界では公知である[例、Kong et al.(１９９９)Nature ４０２３：３０４−３０９]。関節炎のアジュバント誘発についてのプロトコルの一例では、ルイスラットを、０日目に０.１ｍｌ鉱油中の１ｍｇのマイコバクテリウム・ツベルクロシス(Mycobacterium tuberculosis)Ｈ３７ＲＡ(ディフコ、デトロイト、ミシガン)により尾の付け根で免疫化する。前足腫脹は、典型的には１０日目前後に始まる。
【０２１１】
１２日目、右足関節へのトランスフェクションＬ８細胞(〜０.７×１０^６細胞)または非トランスフェクション対照細胞の関節内注射を実施した。１４日目、移植されたトランスフェクションＬ８細胞の存在について関節を分析するためラットを殺した。
【０２１２】
殺したラットの異なる組織を、ｌａｃＺｍＲＮＡの存在についてＲＴ−ＰＣＲ分析により調べた。全ｍＲＮＡを組織から抽出し、ｌａｃＺ遺伝子に特異的なプライマーを用いてＲＴ−ＰＣＲを遂行した。増幅産物は、他の組織(肝臓、腎臓、心臓、脾臓および肺)ではなく滑膜からのみ検出された。また、殺したラットからの滑膜を、in situ酵素的染色法、β−ｇａｌ活性に関するＸ−ｇａｌ染色法により分析した。滑膜の急速冷凍後、８μｍおよび２０μｍ断片を切断し、メイヤーのヘマトキシリンで対比染色し、細胞の青色染色について分析した。染色は、非トランスフェクション細胞を注射した滑膜ではなく、ＡＣｅｓによりトランスフェクションされたＬ８細胞を注射した滑膜から検出された。これらの結果は、マーカー遺伝子含有ＡＣｅｓを用いたラットアジュバント関節炎モデルにおける有効なエクスビボ遺伝子導入、すなわち遺伝子治療用の非ウイルスベクターとしてＡＣｅｓを用いた関節炎および他の結合組織疾患処置の可能性を立証している。
【０２１３】
実施例９
人工染色体の送達測定についてのフローサイトメトリー技術
生産セルライン(実施例１参照)を、０.１６８μｇ／ｍｌのハイグロマイシンＢ(カルビオケム、サンディエゴ、カリフォルニア)と共に１０％胎児ウシ血清(キャン・セラ、レキサデール、オンタリオ)を含むＭＥＭ培地(ギブコＢＲＬ)中で培養した。ヨードデオキシウリジンまたはブロモデオキシウリジンを、指数的成長相における生産セルライン(ＣＨＯ Ｅ４２０１９)の培養培地に直接加えた。ストックのヨードデオキシウリジンをトリス基剤ｐＨ１０中で調製し、ブロモデオキシウリジンストックをＰＢＳ中で調製した。１５分パルスで５〜５０μＭの２０〜２４時間の連続標識用に０.０５〜１μＭの最終濃度とした。２４時間後、指数的成長中の細胞を、採取前の７時間コルチシン(１.０μｇ／ｍｌ)により有糸分裂で遮断した。次いで、染色体を単離し、ヘキスト３３２５８(２.５μｇ／ｍｌ)およびクロモマイシンＡ３(５０μｇ／ｍｌ)で染色した。ＦＡＣＳヴァンテージフローサイトメーター(ベクトン・ディッキンソン・イムノサイトメトリーシステム、サンホセ、カリフォルニア)を用いて人工染色体の精製を行った。クロモマイシンＡ３を４５７ｎｍにセットした第一レーザーで励起し、４７５ｎｍロングパスフィルターを用いて放射を検出した。ヘキストを第二のＵＶレーザーにより励起し、４２０／４４ｎｍバンドパスフィルターを用いて放射を検出した。両レーザー共、出力は１５０ｍＷであった。細胞核型を示す２変量分布を各ソートから蓄積した。ＡＣｅｓを他の染色体からゲートし、選別した。凝縮剤(へキシレングリコール、スペルミンおよびスペルミジン)をシース緩衝液に加えて、選別後の凝縮した無傷染色体を維持した。選別された染色体のＩｄＵ標識指数を顕微鏡により測定した。選別した染色体のアリコート(２〜１０μｌ)を５分間０.２％ホルムアルデヒド溶液に固定した後、汚染されていない顕微鏡スライド上で乾燥した。顕微鏡試料を７０％エタノールにより固定した。風乾したスライドを室温で３０分間２ＮのＨＣｌによりコプリンジャー中で変性させ、２〜３回ＰＢＳで洗浄した。非特異的結合を、ＰＢＳおよび４％ＢＳＡまたは血清で最小限の１０分間遮断した。最終体積が６０〜１００μｌのＦＩＴＣコンジュゲートＩｄＵ／ＢｒｄＵ抗体(ベクトン・ディッキンソン)の１／５希釈物を、スライドに適用した。プラスチックストリップ、Durraシール(ダイヴァーシファイド・バイオテック、ボストン、マサチューセッツ)をスライドにかぶせ、そしてスライドを８〜２４時間加湿遮蔽箱中で４％Ｃの暗所に保った。ベクターシールド中のＤＡＰＩ(シグマ)１μｇ／ｍｌを対比染色として用いた。落射蛍光用に備えられたツァイスのアクシオプラン２顕微鏡を用いて蛍光を検出した。最小限の１００染色体を、標識％を測定するため評点した。非標識染色体を陰性対照として使用した。
【０２１４】
トランスフェクション前日、Ｖ７９−４(チャイニーズハムスター肺線維芽細胞)細胞をトリプシン処理し、４ｍｌのＤＭＥＭ(ダルベッコ修飾イーグル培地、ライフ・テクノロジーズ)および１０％ＦＢＳ(キャン・セラ、レキサデール、オンタリオ)中で６ウェルペトリ皿に２５００００個の割合で平板培養した。５０００００細胞を平板培養することにより、ＬＭ(ｔｋ−)セルラインで使用するためプロトコルを修正した。〜８００μｌの選別緩衝液中で選別された１×１０^６ＡＣｅｓに、脂質またはデンドリマー試薬を加えた。プロトコル変形の例を表１に示す。染色体およびトランスフェクション剤を静かに混合した。複合体を細胞に滴下し、プレートを渦状に混合した。プレートを定められたトランスフェクションの期間５％ＣＯ_２インキュベーター中で３７℃に保った。次いで、ウェル中の容量をＤＭＥＭおよび１０％ＦＢＳにより４〜５ｍｌとした。受容細胞を５％ＣＯ_２インキュベーター中３７℃で２４時間放置した。トランスフェクション細胞をトリプシン化した。ＩｄＵ標識染色体送達について分析すべき試料を、冷７０％エタノール中に固定し、−２０℃で貯蔵し、ＩｄＵ抗体染色に備えた。コロニー選択用に培養する試料を計数し、次にデュプリケイトで１００００および１０００００細胞の密度で１０ｃｍ皿に導入し、残りの細胞は１５ｃｍ皿に入れた。２４時間後、０.７％ｍｇ／ｍｌハイグロマイシンＢ、＃４０００５１(カルビオケム、サンディエゴ、カリフォルニア)と共にＤＭＥＭおよび１０％ＦＢＳを含む選択培地を加える。選択培地を２〜３日ごとに取り換える。このハイグロマイシンＢ濃度は、７日間選択後に野生型細胞を殺す。１０〜１４日目に、コロニーを拡大させ、次いで、ＦＩＳＨにより無傷染色体導入についてスクリーニングし、ベータガラクトシダーゼ発現について検定した。
【０２１５】
表１：送達トランスフェクションプロトコル
【表２】

【０２１６】
ＩｄＵ抗体標識
中和段階における何らかの修飾、変性中におけるデタージェントの存在および遮断緩衝液の組成以外は、標準ＢｒｄＵ染色フローサイトメトリープロトコル(Gratzer et al.Cytometry(１９８１)；６：３８５−３９３)を使用した。各段階間に試料を７〜１０分間３００ｇの遠心分離にかけ、上清を除去する。１００万〜２００万細胞の試料を冷７０％エタノールに固定する。次いで、細胞を室温で３０分間２ＮのＨＣＬ＋０.５％トリトンＸ１〜２ｍｌ中で変性させる。指示薬が中性になるまで試料を３〜４回冷ＤＭＥＭで洗浄する。冷ＤＭＥＭ＋５％ＦＢＳで最終洗浄する。ＰＢＳ、０.１％トリトンＸおよび４％ＦＢＳを含む遮断／透過化緩衝液を１０〜１５分間加えた後、遠心分離により試料を沈降させる。２０μｌのＩｄＵ／ＢｒｄＵ ＦＩＴＣコンジュゲートＢ４４クローン抗体(ベクトン・ディッキンソン・イムノサイトメトリー・システムズ、サンホセ、カリフォルニア)をペレットに加え、３０分ごとに攪拌しながら暗所中室温で２時間放置する。細胞を遮断／透過化緩衝液で洗浄し、フロー分析用にＰＢＳに再懸濁する。
【０２１７】
蛍光ＩｄＵｒｄ標識ＡＣｅｓのフローサイトメトリー検出
ＩｄＵ標識ＡＣｅｓを含むトランスフェクション細胞のパーセントを、アルゴンレーザーを４００ｍＷで４８８ｎｍに変えたフローサイトメトリーを用いて測定した。標準ＦＩＴＣ５３０／３０ｎｍバンドパスフィルターを通してＦＩＴＣ蛍光を集めた。細胞集団を側方散乱対前行散乱に基いてゲートすることにより、屑および二重線を排除した。データを蓄積することにより(１５０００事象)形成された２変量チャンネル分布は、前方散乱対緑色蛍光を示した(ＩｄＵ−ＦＩＴＣ)。細胞が陽性であると測定された蛍光レベルを、約１％が陽性領域に現れるように、陰性対照細胞のヒストグラムの視覚的検査により確立した。
【０２１８】
結果：
ＩｄＵ標識ＡＣｅｓのトランスフェクション送達結果を表２に示す。
表２
【表３】

【０２１９】
修飾は当業者には容易に理解できるものであるため、この発明は添付の請求の範囲によってのみ限定されるものとする。[0001]
(Related application)
U.S. Patent Application No. 09/815979, entitled "Methods of Delivering and Evaluating Nucleic Acid Molecules to Cells and Their Evaluation," by De Jong et al., March 22, 2001, by De Jong et al., March 22, 2001. US Patent Application No. 09/815881 entitled "Delivering Nucleic Acid Molecules to Cells and Evaluating the Same" and "Methods of Delivering Nucleic Acid Molecules to Cells and Evaluating the Same" by De Jong et al. Priority is claimed in US patent application Ser. Where permitted, the subject of each of these applications is incorporated by reference.
[0002]
(Field of the Invention)
The present invention relates to a method for delivering a nucleic acid molecule to a cell, a method for delivering a nucleic acid to a cell, and a method for measuring nucleic acid expression therein.
[0003]
(Background of the Invention)
Some methods have been developed for delivering nucleic acid molecules, particularly plasmid DNA and other small fragments of nucleic acid, to cells. These methods are not ideal for delivery of large nucleic acid molecules. That is, there is a need for a method of delivering nucleic acid molecules of increasing size and complexity, such as artificial chromosomes, to cells. There is a need for in vitro and in vivo procedures, such as methods for use in gene therapy and for the production of transgenic animals and plants. Further, there is a need for a quick and easy measurement and evaluation of the efficiency of DNA delivery to cells.
[0004]
Accordingly, it is an object of the present invention to provide a method for delivering nucleic acid molecules, especially large molecules, such as artificial chromosomes, to cells. Also provided are methods of evaluating delivery.
[0005]
(Summary of the Invention)
Methods for delivering large nucleic acid molecules to cells are provided. Any method that can be used to deliver nucleic acid molecules of any size is suitable for delivering large nucleic acid molecules to cells, such as natural and artificial chromosomes and fragments thereof. These methods include, but are not limited to, cell-based protein production, transgenic protein production, and in vitro, ex vivo and in vivo delivery of nucleic acids for applications including delivery of nucleic acid molecules to cells for gene therapy. Designed for delivery. Also provided are methods for producing proteins in cells and transgenic animals and plants, methods for producing transgenic animals and plants by introducing nucleic acids into cells, and methods for ex vivo and in vivo gene therapy.
[0006]
Although the methods provided herein are designed for delivery of large nucleic acid molecules to cells, they can also be used for delivery of small molecules. In some of these methods, the nucleic acid molecule is exposed to a first delivery agent, typically an agent that increases contact between the nucleic acid molecule and the cell, and the cell is exposed to a second delivery agent, typically a second agent. Different from one factor, some include agents that increase the permeability of cells, for example, exposure to energy. The selected delivery factors and combinations thereof deliver nucleic acids to cells to a greater extent than in the absence of the factors or in the presence of one factor alone. Generally, in all of these methods, where the permeability-enhancing agent is energy, such as electroporation or acoustic perforation, the cells are contacted with it in the absence of nucleic acid molecules.
[0007]
The cells may also be treated with lipid agents, especially dendrimers, such as SAINT-2® (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enenyl) pyridinium chloride, also 1-methyl-4- (Also referred to as 19-cis, cis-hepatritiaconta-9,28-dienyl) pyridinium chloride) and a method for contacting simultaneously or sequentially with energy application. The nucleic acid may be treated with a delivery agent if desired, and contacted with the cells treated as described above.
[0008]
The delivery method chosen will depend on the target cell (cell to which the nucleic acid is to be delivered), the nucleic acid molecule and the type (s) of delivery agent (s) selected. Examples of methods for delivering large nucleic acid molecules to cells provided by the present invention include, but are not limited to, methods including any of the following:
[0009]
Mixing the nucleic acid molecule with a delivery agent, eg, a cationic lipid that neutralizes the charge of the nucleic acid, and contacting the cell with the mixture of nucleic acid and delivery agent;
[0010]
Contacting the cell with the nucleic acid molecule and then contacting the cell with the delivery agent or contacting the cell with the delivery agent and then contacting the cell with the nucleic acid molecule;
[0011]
Contacting the cell with the delivery agent in the absence of the nucleic acid molecule, applying ultrasound or electrical energy to the cell contacted with the delivery agent, and contacting the cell with the nucleic acid molecule at the end of the energy application;
[0012]
Applying ultrasound or electrical energy to the cells and, at the end of the energy application, contacting the cells with a mixture of nucleic acid molecules and a delivery agent;
[0013]
Applying ultrasound or electrical energy to the cells and, at the end of the energy application, contacting the cells with the delivery agent and contacting the cells previously contacted with the delivery agent with the nucleic acid molecule;
[0014]
Applying ultrasound or electrical energy to the cells and bringing the cells into contact with the nucleic acid at the end of the energy application;
[0015]
The cells in the absence of the nucleic acid molecule are contacted with the delivery agent, ultrasound or electrical energy is applied to the cells contacted with the delivery agent, and at the end of the energy application, the cells are contacted with the mixture of nucleic acid and delivery agent.
[0016]
Also provided are methods that include a combination of the above methods. Generally, in the case of any of the methods or a combination of the methods, the application of energy to the cells occurs prior to the introduction of the nucleic acid molecule. However, in some cases, energy can be applied in the presence of a nucleic acid molecule, for example, if the integrity of the nucleic acid molecule is not compromised by the application of energy in the presence of the nucleic acid molecule.
[0017]
The methods provided herein are intended for the delivery of large nucleic acid molecules to cells in a variety of environments for a variety of purposes. For example, methods wherein nucleic acid molecules greater than about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and 100 megabase pairs are provided herein. Can be used to deliver cells. These methods can be used to deliver large nucleic acid molecules to cells in vitro or in vivo.
[0018]
In in vivo applications of the delivery methods, eg, in vivo gene therapy, large nucleic acid molecules can be delivered directly to cells in an animal subject. Such animals include, but are not limited to, mammals. For example, the animal subject can be a human or other primate, rodent, rabbit, dog, horse or monkey. The reagent may be administered locally or systemically (eg, in the bloodstream) in the subject. For example, topical administration of nucleic acids and / or delivery agents can be performed on areas such as, for example, joints, skin, tissues, tumors, and organs. For systemic administration, the nucleic acid molecule can be targeted to a cell or tissue of interest.
[0019]
Also, by using the delivery methods provided by the present invention, large nucleic acid molecules can be delivered to target cells in vitro, which are then introduced into an animal subject, especially a human subject, which is itself, for example, an ex vivo gene therapy method. Can be done. That is, the present invention provides in vivo and ex vivo gene therapy methods using the method for delivering large nucleic acid molecules to cells provided by the present invention.
[0020]
In certain embodiments of the method using a delivery agent, the delivery agent is a cationic compound. Cationic compounds include, but are not limited to, cationic lipids, cationic polymers, cationic lipid mixtures, cationic polymer mixtures, mixtures of cationic lipids and cationic polymers, mixtures of cationic lipids and neutral lipids, polycationic lipids, non-liposomes There are formed lipids, activated dendrimers, ethanolic cationic lipids, cationic amphiphiles and pyridinium chloride surfactants.
[0021]
Among the nucleic acid molecules that can be delivered to cells using the methods provided herein are artificial chromosomes, satellite DNA-based artificial chromosomes (SATAC, also referred to herein as ACes) and native chromosomes or any of these chromosomes Fragment.
[0022]
The ultrasound energy may be applied as a single continuous pulse or as two or more intermittent pulses. The intermittent pulse of ultrasonic energy may be applied at substantially the same length of time, at substantially the same energy level, or the energy level, the length of application time, or the energy level and the length of application time change I can do it. The ultrasonic energy range and the number of pulses can vary and can be empirically determined, depending on the instrument selected, and by the methods provided herein. Typically, the ultrasound is applied for about 30 seconds to about 5 minutes. The power used is a function of the sonoporator used.
[0023]
The effect of the ultrasonic energy can be enhanced by contacting the cavitation compound with cells (in vitro) or administering to the subject (in vivo) prior to applying the ultrasonic energy. That is, the provided methods can include the use of the cavitation compounds described above.
[0024]
When electric fields are used in the methods provided herein, they are preferably applied to cells suspended for about 20-50 msec (milliseconds), but the timing and voltage are a function of the instrument used and the particular parameters. (Function). Electrical energy may be applied as 1-5 intermittent pulses. As shown, the electric field range and number of pulses can vary with instrument specifications and can be determined empirically.
[0025]
Using the delivery methods provided herein to deliver a large nucleic acid molecule to an animal cell, particularly a non-human animal cell, and exposing the animal cell with the delivered large nucleic acid molecule to conditions that give rise to a transgenic animal. Provides a method for producing a transgenic animal, especially a non-human transgenic animal.
[0026]
The methods of delivering large nucleic acid molecules to cells provided by the present invention can also be used in methods of producing transplantable organs and tissues. Examples of transgenic animals, particularly non-human transgenic animals, or cells used in the method of producing transplantable organs include, but are not limited to, embryonic stem cells, nuclear transfer donor cells, stem cells and specific organs. Cells that can be developed are included. The method of delivering nucleic acid molecules to cells provided by the present invention can also be used in a method of generating a cell protein production cell line.
[0027]
Further, a method is provided for monitoring nucleic acid delivery to a cell. These methods allow for the rapid and accurate measurement of nucleic acid transfer to cells, thus screening the use of various delivery factors and protocols to deliver any nucleic acid to any cell type in vitro, ex vivo or in vivo. And can be optimized. Further provided are methods of monitoring nucleic acid delivery and expression in cells.
[0028]
In embodiments of the method of monitoring delivery of a nucleic acid to a cell, the labeled nucleic acid molecule, e.g., DNA, is delivered to the cell using the delivery agent (s) described herein or using delivery methods known to those of skill in the art. Deliver to The number of cells containing the label is then determined as an indicator of the ability of the delivery method to facilitate or accomplish delivery of the nucleic acid molecule by using a detection method, eg, flow cytometry. Other detection methods that can be used instead of, or in addition to, flow cytometry include, but are not limited to, fluorimetry, cell imaging, fluorescence microscopy and the above detection and, if necessary or desired, There are other methods known to those skilled in the art for quantification.
[0029]
In one example of an embodiment of a method of monitoring and quantifying the delivery of a nucleic acid molecule, such as DNA, to a cell, the nucleic acid molecule is a nucleoside or ribonucleoside analog, particularly a thymidine analog, such as iododeoxyuridine (IdU or IdUrd) and bromoside. Artificial chromosome labeled with deoxyuridine (BrdU), the delivery factor is a cationic compound, used alone or in combination with energy.
[0030]
Because of the ease of collecting the number of events, the monitoring methods provided in the present invention, particularly those based on flow cytometry techniques, provide statistically superior methods of delivering nucleic acid molecule delivery data than prior methods of assessing nucleic acid molecule migration. Provide a collection method. Since a clear value is instrumentally guided, it is less likely to make a judgment error.
[0031]
With the monitoring methods provided in the present invention, the delivery of small numbers of nucleic acid molecules to cells can be detected quickly, easily and accurately. Such a small number may be sufficient for gene transfer, gene therapy, cellular protein production purposes and other goals of gene transfer. In addition, according to the above monitoring method, the difference in the delivery efficiency between different delivery methods can be quickly quantified, so that the method for delivering a nucleic acid molecule, for example, DNA to cells can be easily developed and optimized.
[0032]
Also, by using these methods, the efficiency of transfection into cells for which a delivery protocol has not been established or for which transfection is not easy can be optimized. These methods also allow for the rapid screening of delivery protocols and agents for the ability to facilitate or enable the delivery of nucleic acid molecules of any size, eg, DNA, to cells.
[0033]
Also provided is a method of combining a method of monitoring nucleic acid molecule delivery with a method of monitoring nucleic acid molecule expression. In addition to assessing the efficiency of nucleic acid molecule delivery to cells, it is possible to monitor subsequent expression of the delivered nucleic acid molecule in the same cell population. That is, these methods also use a marker gene, such as, but not limited to, a fluorescent protein, such as a red, green or blue fluorescent protein, to map a biological event between nucleic acid molecule delivery and early gene expression. Provide a method.
[0034]
In certain embodiments of these combination methods, delivery and expression of nucleic acid molecules, such as delivery of chromosomes and expression of genes encoded therein, is accomplished using IdU labeling of nucleic acid molecules comprising a sequence encoding a green fluorescent protein. Monitor.
[0035]
In certain embodiments, a method of monitoring delivery and expression of a nucleic acid molecule comprises introducing a labeled nucleic acid molecule encoding a reporter gene into a cell, detecting the labeled cell as an indicator of nucleic acid delivery to the cell, and detecting DNA in the cell. Detecting or measuring delivery and expression of the nucleic acid molecule in the cell by measuring the reporter gene product as an indicator of expression. Labeled cells can be detected, for example, by flow cytometry, fluorimetry, cell imaging or fluorescence microscopy. The label can be, for example, iododeoxyuridine (IdU or IdUrd) or bromodeoxyuridine (BrdU), and the reporter gene can, for example, encode a fluorescent protein, an enzyme, such as luciferase, or an antibody. Nucleic acid molecules delivered include, but are not limited to, RNA, eg, ribozymes, DNA, eg, naked DNA and chromosomes, plasmids, chromosomal fragments, typically containing at least one gene or at least 1 Kb, naked. DNA or natural chromosome. Methods by measuring the delivery and expression of the artificial chromosome expression system (ACes) are mentioned herein as examples. Any type of eukaryotes and prokaryotes, including cell lines, primary cells, primary cell lines, plant cells and animal cells, and including stem cells, embryonic cells, and other cells to which nucleic acid molecules can be delivered. Cells are also contemplated and can be used in the methods provided herein.
[0036]
(Detailed description of the invention)
A. Definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications and publications cited herein are hereby incorporated by reference.
[0037]
The term "nucleic acid" as used herein refers to a polynucleotide comprising at least two covalently linked nucleotides or nucleotide analog subunits. The nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA. Nucleotide analogs are commercially available and methods for producing polynucleotides containing such nucleotide analogs are known (Lin et al. (1994) Nucl. Acids Res. 22: 5220-5234; Jellinek et al. (1995). Biochemistry 34: 11363-11372; Pagratis et al. (1997) Nature Biotechnol. 15: 68-73). The nucleic acid can be single-stranded, double-stranded, or a mixture thereof. For the purposes of the present invention, unless otherwise specified, the nucleic acid is double-stranded or apparent from the context.
[0038]
The term "nucleic acid" refers to single-stranded and / or double-stranded polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and analogs or derivatives of RNA or DNA. The term "nucleic acid" also includes analogs of nucleic acids, such as peptide nucleic acids (PNA), thiophosphate DNA, and other such analogs and derivatives.
[0039]
As used herein, DNA is intended to include DNA molecules of all types and sizes, including cDNA, plasmids and DNA, including modified nucleotides and nucleotide analogs.
[0040]
Nucleotides as used herein include nucleoside mono-, di- and triphosphates. Nucleotides also include modified nucleotides, such as, but not limited to, thiophosphate nucleotides and deazapurine nucleotides and other nucleotide analogs.
[0041]
As used herein, the term “large nucleic acid molecule” or “large nucleic acid” refers to a nucleic acid molecule having a size of at least about 0.5 megabase pairs (M bases), greater than 0.5 M bases, Include nucleic acid molecules of at least about 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and 100, 200, 300, 500 M bases. Large nucleic acid molecules can typically range from about 10 to about 450 or more M bases, and come in a variety of sizes, such as about 250 to about 400 M bases, about 150 to about 200 M bases, about 90 to about M bases. It may have 120M base, about 60 to about 100M base and about 15 to 50M base.
[0042]
Examples of large nucleic acid molecules include, but are not limited to, native chromosomes and fragments thereof, particularly mammalian chromosomes and fragments thereof that retain centromeres and telomeres, artificial chromosome expression systems (ACes, satellite DNA-based artificial Also referred to as chromosomes (SATAC); see US Pat. Nos. 6,025,155 and 6077697), mammalian artificial chromosomes (MAC), plant artificial chromosomes, insect artificial chromosomes, avian artificial chromosomes and mini-chromosomes (eg, US Pat. See). The large nucleic acid molecule may comprise a desired nucleic acid fragment encoding a particular nucleotide sequence, such as a single copy of a gene of interest, or may carry multiple copies or multiple genes or different heterologous nucleotide sequences. For example, ACes may carry 40 or more copies of the gene of interest. Large nucleic acid molecules can be associated with proteins, eg, chromosomal proteins, which typically function to regulate gene expression and / or participate in determining overall structure.
[0043]
Artificial chromosomes, as used herein, are nucleic acid molecules that can be stably replicated and separated together with endogenous chromosomes in a cell. It has the capacity to act as a gene delivery vehicle by adapting and expressing the foreign genes contained therein. Mammalian artificial chromosome (MAC) refers to a chromosome that has an active mammalian centromere (s). Plant artificial chromosome, insect artificial chromosome and avian artificial chromosome refer to chromosomes including plants, insects and avian centromeres, respectively. Human artificial chromosome (HAC) refers to a chromosome containing the human centromere. For examples of artificial chromosomes, see, for example, U.S. Patent Nos. 6,025,155, 6077697, 5,288,625, 5,712,134, 5,695,967, 5869294, 5891691, and 5721118 and published International PCT applications WO97 / 40183 and WO98 / 08964.
[0044]
As used herein, the term "satellite DNA-based artificial chromosome (SATAC)" is interchangeable with the term "artificial chromosome expression system (ACes)." These artificial chromosomes are virtually all neutral non-coding sequences (heterochromatin), with the exception of foreign heterologous, typically gene-encoding nucleic acids, which are interspersed within heterochromatin, where Expressed (see U.S. Patent Nos. 6,025,155 and 6077697 and International PCT Application No. WO 97/40183). Foreign genes included in these artificial chromosome expression systems include, but are not limited to, traceable marker proteins (reporter genes), such as fluorescent proteins such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively). ) Or other reporter genes, such as β-galactosidase and a protein that confers drug resistance, such as a nucleic acid encoding a gene encoding hygromycin resistance. Other examples of heterologous DNA include, but are not limited to, DNA encoding therapeutically active agents, such as anticancer agents, enzymes and hormones, and DNA encoding other types of proteins, such as antibodies.
[0045]
As used herein, the terms "heterologous" and "foreign" with respect to nucleic acids, such as DNA and RNA, are used interchangeably and refer to the position (s) at which they exist or where they originally exist. And / or a nucleic acid that is not naturally present as part of the genome or cell found at a location (s) and / or quantity in the genome or cell that is different from the quantity. It can be a nucleic acid that is not endogenous to the cell but has been introduced into the cell from the outside. Examples of heterologous DNA include, but are not limited to, a gene product or genes of interest introduced into cells for the purpose of, for example, gene therapy, production of transgenic animals or production of an encoded protein. DNA encoding the product is included. Other examples of heterologous DNA include, but are not limited to, a DNA encoding a traceable marker protein, eg, a protein that confers drug resistance, a DNA encoding a therapeutically active substance, eg, an anti-cancer agent, an enzyme, and a hormone. , And other types of proteins, such as DNA encoding antibodies.
[0046]
As used herein, "delivery" is used interchangeably with "transfection" and refers to the process by which exogenous nucleic acid molecules are introduced into a cell such that they are located inside the cell. . Delivery of nucleic acids is a process that is distinct from the expression of nucleic acids.
[0047]
"Expression" as used herein refers to the process by which a nucleic acid is translated into a peptide or transcribed into RNA, which can be translated, for example, into a peptide, polypeptide or protein. If the nucleic acid is derived from genomic DNA, expression can include splicing of the mRNA if a suitable eukaryotic host cell or organism is selected. In the case of a heterologous nucleic acid that is expressed in a host cell, it must first be delivered to the cell and then, once inside the cell, ultimately be present in the nucleus.
[0048]
As used herein, cell yields are calculated after the specified time frame, after 24 hours for purposes of the present invention, and when used for calculation of clonal fractions (fractions, fractions). Refers to “total cell yield”.
[0049]
Cell recovery time as used herein refers to the time frame for cells to reach equilibrium for new conditions.
[0050]
Cell viability as used herein refers to a cytotoxic event, such as cell viability following a delivery procedure.
[0051]
Control plate efficiency (CPE), as used herein, refers to the fraction of untreated cells under standard optimal growth conditions for a particular cell that survives the plating procedure. Plate efficiency refers to the fraction of treated cells that survive despite the plating procedure.
[0052]
As used herein, the clonal fraction is a measure of cell yield and cell plating efficiency after delivery of exogenous nucleic acid to cells.
[0053]
Transduction efficiency, as used herein, is the percentage of the total number of cells to which the nucleic acid has been delivered and which contains the delivered nucleic acid.
[0054]
Transfection efficiency, as used herein, is the percentage of the total number of cells that have delivered a nucleic acid containing a selectable marker and survive the selection.
[0055]
As used herein, a potential transfection efficiency index refers to the theoretical maximum transfection efficiency for a particular cell type under particular conditions, for example, a particular concentration or amount of a particular delivery agent.
[0056]
The term "cell" as used herein is intended to include all types of eukaryotic and prokaryotic cells, including animals and plants.
[0057]
As used herein, a "delivery agent" refers to a composition, condition or physical treatment that can facilitate delivery of a nucleic acid to a cell by exposing the cell and / or nucleic acid to the process of introducing the nucleic acid into the cell. Say. Delivery agents include compositions, conditions and physical treatments that facilitate contact of cells with nucleic acids and / or increase permeability of cells to nucleic acids. Generally, nucleic acids are not directly processed by energy, for example, acoustic perforation.
[0058]
As used herein, a cationic compound is a compound having a polar group that is positively charged at or near physiological pH. These compounds facilitate delivery of the nucleic acid molecule to the cell. It is believed that this is achieved by exerting their ability to neutralize the charge on the nucleic acids. Examples of cationic compounds include, but are not limited to, cationic lipids or cationic polymers or mixtures thereof, with or without neutral lipids, polycationic lipids, non-liposome-forming lipids, ethanolic cationic lipids And cationic amphiphiles. Also anticipated cationic compounds are activated dendrimers, i.e. spherical cationic polyamidoamines having a well-defined globular structure of charged amino groups that are branched from the central core and can interact with the negatively charged phosphate groups of nucleic acids. There are polymers (eg, starburst dendrimers).
[0059]
Cationic compounds used as delivery agents also include mixtures of cationic compounds, including peptide and protein fragments. The additional component may be non-covalently or covalently bound to the cationic compound or otherwise associated with the cationic compound.
[0060]
Ultrasonic energy as used herein shall include sound waves (for external applications) and lithotripter-generated shock waves (for internal applications).
[0061]
Electrical energy as used herein shall include piercing a membrane to deliver a molecule to a cell by applying an electric field to the cell, such as electroporation techniques.
[0062]
Cavitation compounds as used herein shall include contrast agents commonly used in ultrasound imaging devices, and include gas-filled and non-gaseous agents. These cavitation compounds enhance the efficiency of acoustic or shock wave energy delivery.
[0063]
As used herein, "pharmaceutically acceptable" refers to compounds, compositions suitable for administration to a subject without inducing excessive toxicity, irritation, allergic response or other undesirable complications. And dosage forms.
[0064]
Embryonic stem cells, as used herein, are primitive immature cells that are precursors to stem cells.
[0065]
Stem cells, as used herein, are primitive immature cells that are precursors to mature tissue-specific cells.
[0066]
As used herein, a nuclear transfer donor cell is a cell that is a source of nuclei that is introduced into an enucleated oocyte during the nuclear transfer process.
[0067]
The term "subject" as used herein refers to animals, plants, insects and birds into which large DNA molecules can be introduced. Higher organisms include, for example, mammals and birds, such as humans, primates, rodents, cows, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chickens, and the like.
[0068]
As used herein, “administering to a subject” is a procedure in which one or more delivery factors and / or large nucleic acid molecules are introduced or applied to a subject, either together or separately, and present in the subject. The purpose is to finally contact the target cells to be contacted with the above factors and / or large nucleic acid molecules.
[0069]
As used herein, "applying to a subject" is a procedure in which target cells present in the subject are ultimately brought into contact with energy, such as ultrasound or electrical energy. The application is made in such a way that the energy can be applied.
[0070]
Gene therapy, as used herein, is necessary to correct or regulate a disease or disorder by introducing or inserting nucleic acid molecules, and particularly large nucleic acid molecules, into certain cells, also referred to as target cells. To produce a specific gene product. The nucleic acid is introduced into the selected target cells such that the nucleic acid is expressed, thereby producing the encoded product. Alternatively, the nucleic acid may somehow convey the expression of the DNA encoding the therapeutic product. The product can be a therapeutic compound, produced in a therapeutically effective amount or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some way directly or indirectly transmits the expression of the therapeutic product. Expression of nucleic acids by target cells in an organism affected by a disease or disorder provides a means of modulating the disease or disorder. The nucleic acid encoding the therapeutic product can be modified prior to introduction into the cells of the diseased host to enhance or otherwise alter the product or its expression.
[0071]
When used in gene therapy, the transfected cells can be introduced into a subject's body after transfection of the cells in vitro. This is often referred to as ex vivo gene therapy. Alternatively, the cells can be directly transfected in vivo in a subject.
[0072]
Flow cytometry, as used herein, refers to a method using laser-based instruments that can analyze and sort cells and / or chromosomes based on size and fluorescence.
[0073]
The term reporter gene as used herein encompasses all genes that express a detectable gene product, which can be RNA or protein. Preferred reporter genes are those that can be easily detected. Examples of reporter genes include, but are not limited to, fluorescent proteins, CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems. For example, beta-galactosidase, firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7: 725-737), bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. al. (1984), Biochemistry 23: 3663-3667), and alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101).
[0074]
A reporter gene construct, as used herein, is a DNA molecule that includes a reporter gene operably linked to a transcription control sequence. Transcription control sequences may be promoters and other optional regulatory regions, such as enhancer sequences that regulate the activity of the promoter, or control sequences that regulate the activity or efficacy of RNA polymerase that recognizes the promoter, or controls that are recognized by effector molecules. Sequences include those specifically induced by the interaction of cell surface proteins with extracellular signals. For example, modulation of the activity of a promoter can be accomplished by modifying the RNA polymerase attached to the promoter region or, alternatively, by interfering with the initiation or elongation of mRNA. The sequences are collectively referred to herein as transcription control elements or sequences. In addition, the construct can include a sequence of nucleotides that alters the amount of the reporter gene product by altering the translation of the produced mRNA.
[0075]
As used herein, a promoter refers to a region of DNA that is upstream of the transcription start site in the direction of transcription. It is an RNA polymerase binding and transcription initiation site and other regions, such as, but not limited to, a repressor or activator protein binding site, a calcium or cAMP responsive site, and directly or indirectly from a promoter. Includes the above nucleotide sequences known to those of skill in the art to alter the amount of transcription.
[0076]
As used herein, a promoter that is regulated or transmitted by the activity of a cell surface protein is activated when a cell is exposed to a particular extracellular signal due to the presence of a cell surface protein whose activity is affected by the extracellular protein. Is a variable promoter.
[0077]
B. Method for delivering DNA to cells
Various methods of delivery of nucleic acids, particularly large nucleic acid molecules, such as artificial chromosomes, such as ACes (previously referred to as SATAC), have been provided. These methods generally involve exposing the nucleic acid molecule to a factor that increases contact between the nucleic acid molecule and the cell, and exposing the cell to a permeability enhancer. Each of the methods provided herein requires the use of one or both of these factors, which are applied in a different order. Generally, factors that increase the permeability of the cell, such as energy, are applied prior to contacting the cell with the nucleic acid.
[0078]
In the methods provided herein, large nucleic acid molecules may include factors including, but not limited to, delivery agents that increase contact between the nucleic acid molecule and cells and / or agents and treatments that increase cell permeability. Delivered using. Generally, nucleic acid molecules are delivered by using factors that increase contact between the nucleic acid and the cell by neutralizing the charge on the nucleic acid molecule, and by using energy to increase the permeability of the cell. These factors can be used individually and in various combinations and order of application. In general, energy, such as acoustic and electroporation, is not applied to the cells before applying the nucleic acid molecule to it.
[0079]
The method chosen to deliver a particular nucleic acid molecule, eg, DNA, to a target cell may vary depending on the particular nucleic acid molecule being introduced and the particular recipient cell. Preferred methods for particular nucleic acid molecules, eg, DNA, and recipient cells are those that result in the greatest amount of nucleic acid molecule, eg, DNA, introduced into the cell nucleus to an extent acceptable for cell survival. The appropriate method of delivery of a nucleic acid molecule, eg, DNA, for a specific pairing and recipient cell can be determined using the methods of monitoring nucleic acid molecules, eg, DNA delivery and screening methods and factors and conditions provided herein, or It can be determined empirically using methods known to those skilled in the art.
[0080]
The method chosen requires consideration of some parameters discussed in detail below. A method for detecting a delivered nucleic acid is provided. This method can be used to evaluate the delivery of any nucleic acid molecule and can be used as a rapid screening tool to optimize nucleic acid, eg, chromosome transfer conditions.
[0081]
In particular, delivery methods are evaluated for the ability to first introduce a nucleic acid molecule, eg, DNA, into a cell and to identify a method that provides a sufficient number of viable cells to express the delivered nucleic acid molecule, eg, DNA. obtain. Once the above methods have been identified, they can be optimized using the delivery monitoring methods provided herein and then evaluated for their ability to effect expression of the delivered nucleic acid molecule.
[0082]
Delivery factor
Delivery agents include compositions, conditions and physical treatments that enhance the contact of cells with nucleic acid molecules, eg, DNA, and / or increase the permeability of cells to nucleic acid molecules, eg, DNA. Such factors include, but are not limited to, cationic compounds, peptides, proteins, energies such as ultrasonic energy and electric fields, cavitation compounds.
[0083]
Delivery agents used in the methods provided herein include cells and / or nucleic acid molecules, such as DNA, to facilitate delivery of nucleic acid molecules, such as DNA, to cells. There are compositions, conditions, or physical treatments that can be exposed in the process of introducing the compound. For example, compounds and chemical compositions such as, but not limited to, calcium phosphate, DMSO, glycerin, chloroquine, sodium butyrate, polybrene and DEAE dextran, peptides, proteins, temperature, light, pH, radiation and pressure are all Is a possible delivery factor.
[0084]
Cationic compound
The cationic compounds used in the methods provided in the present invention are commercially available or can be synthesized by those skilled in the art. Cationic compounds can be used to deliver nucleic acid molecules, eg, DNA, to particular cell types using the provided methods. One of skill in the art would be able to readily determine which cationic compounds are best suited for delivery of a specific nucleic acid molecule, eg, DNA, to a specific target cell type using the provided screening procedures.
[0085]
(a) Cationic lipid
Cationic lipid reagents can be classified into two general classes based on the number of positive charges on the lipid head group: single positive charges or multipositive charges, usually 5 or less. Cationic lipids are often mixed with neutral lipids before being used as delivery agents. Neutral lipids include lecithins, phosphatidylethanolamine, phosphatidylethanolamines such as DOPE (dioleoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine) and distearoyl. Phosphatidylethanolamine, phosphatidylcholine, phosphatidylcholines, such as DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine, fatty acid esters, glycerin esters, sphingolipids, cardiolipin , Cerebrosides, and ceramides, and There mixtures thereof, but is not limited thereto. Neutral lipids also include cholesterol and other 3βOH-sterols.
[0086]
Other lipids contemplated by the present invention include phosphatidyl glycerin, phosphatidyl glycerins, such as DOPG (dioleoyl phosphatidyl glycerin), DPPG (dipalmitoyl phosphatidyl glycerin), and distearoyl phosphatidyl glycerin, phosphatidyl serine, phosphatidyl serines, such as There are dioleoyl- or dipalmitoyl phosphatidylserine and diphosphatidylglycerins.
[0087]
Examples of cationic lipid compounds include Lipofectin (Life Technologies, Inc., Burlington, Ontario) (cationic lipid N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) 1: 1 (w / w) formulation, lipofectamine (See Life Technologies, Inc., Burlington, Ontario, US Pat. No. 5,334,761) (polycationic lipids) 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and dioleoylphosphatidylethanolamine (DOPE): 1 (w / w) formulation), Lipofe Tamin Plus (see Life Technologies, Burlington, Ontario, US Pat. Nos. 5,334,761 and 5,736,392; see also US Pat. No. 6,051,429) (Lipofectamine and Plus Reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario, International PCT application) No. WO00 / 27795) (cationic lipids), effecten (Qiagen, Inc., Mississauga, Ontario) (non-liposomal lipid formulation), metafectene (Biotex, Moonich, Germany) (polycationic lipids), Eufectins (Promega Biosciences, Inc., San Luis Obispo, CA) (Ethanolic cationic lipids 1 to 12: C ₅₂ H ₁₀₆ N ₆ O ₄ ・ 4CF ₃ CO ₂ H, C ₈₈ H ₁₇₈ N ₈ O ₄ S ₂ ・ 4CF ₃ CO ₂ H, C ₄₀ H ₈₄ NO ₃ P ・ CF ₃ CO ₂ H, C ₅₀ H ₁₀₃ N ₇ O ₃ ・ 4CF ₃ CO ₂ H, C ₅₅ H ₁₁₆ N ₈ O ₂ ・ 6CF ₃ CO ₂ H, C ₄₉ H ₁₀₂ N ₆ O _Three ・ 4CF ₃ CO ₂ H, C ₄₄ H ₈₉ N ₅ O ₃ ・ 2CF ₃ CO ₂ H, C ₁₀₀ H ₂₀₆ N ₁₂ O ₄ S ₂ ・ 8CF ₃ CO ₂ H, C ₁₆₂ H ₃₃₀ N _{twenty two} O ₉ ・ 13CF ₃ CO ₂ H, C ₄₃ H ₈₈ N ₄ O ₂ ・ 2CF ₃ CO ₂ H, C ₄₃ H ₈₈ N ₄ O ₃ ・ 2CF ₃ CO ₂ H, C ₄₁ H ₇₈ NO ₈ P), cytofectene (Bio-Rad, Hercules, CA) (mixture of cationic and neutral lipids), Geneporter (Gene Therapy Systems, Inc., San Diego, CA) (Dope and cationic lipids) ) And Fugene 6 (Roche Molecular Biochemicals, Indianapolis, Indiana) (a multi-component lipid based on non-liposomal reagents).
[0088]
(b) Non-lipid cationic compound
Non-lipid cationic reagents include SUPERFECT (registered trademark) (QIAGEN, Inc., Mississauga, Ontario) (activated dendrimer (cationic polymer: charged amino group) and CLONfectin (registered trademark) (CLONfectin®) The medium is Nt-butyl-N′-tetradecyl-3-tetradecyl-aminopropionamidine (Clontech, Palo Alto, Calif.), But is not limited thereto.
[0089]
Pyridinium amphiphiles are double-stranded pyridinium compounds that are essentially non-toxic to cells and show little preference for their ability to transfect cells. Examples of pyridinium amphiphiles include pyridinium chloride surfactants, such as SAINT-2 (1-methyl-4- (1-octadec-9-enyl-nonadeca-10-enylenyl) pyridinium chloride) (e.g., Van der Woude et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1160). The pyridinium chloride surfactant is typically mixed with a neutral helper lipid compound such as dioleoylphosphatidylethanolamine (DOPE) in a 1: 1 molar ratio. Other Saint derivatives with different chain lengths, saturations and head groups can be made by those skilled in the art and included within the scope of the present method.
[0090]
energy
Delivery agents also include the treatment or exposure of cells and / or nucleic acid molecules, but generally cells, to a source of energy, such as acoustic and electrical energy.
[0091]
Ultrasound
For in vitro and in vivo transfections, the ultrasound source should be able to provide the appropriate frequency and energy output to facilitate transfection. For example, the output device may generate ultrasonic energy in a frequency range from 20 kHz to about 1 MHz. The power of the ultrasonic energy is, for example, about 0.05 w / cm ² ~ 2w / cm ² Or about 0.1 w / cm ² ~ About 1w / cm ² Range. Ultrasound may be administered in one continuous pulse or as two or more intermittent pulses, which may be the same or may vary in time and intensity.
[0092]
Ultrasound energy can be applied locally to the body, or ultrasound-based extracorporeal shockwave lithotripsy can be used for "widespread" applications. Ultrasound energy can be applied to the subject's body using various ultrasound devices. In general, ultrasound is performed by direct contact using a standard or custom made ultrasound imaging probe or needle with or without the use of other medical devices, such as scopes, catheters and surgical instruments, or It may be administered through an ultrasonic bath that partially or completely surrounds the tissue or organ with the flowing medium. The source of the ultrasound may be external to the subject's body, for example, applying ultrasound to the subject's body, an ultrasound probe applied to the subject's skin, or internal, such as ultrasound placed within the subject's body It can be a catheter with a transducer. Suitable ultrasound systems are known (see, eg, International PCT Application No. WO 99/21584 and US Pat. No. 5,676,151).
[0093]
When a cationic compound and a nucleic acid molecule, for example, DNA, are administered systemically, simultaneous application of ultrasound to one or several organs or tissues can facilitate delivery of the nucleic acid molecule to multiple regions of the subject's body. Alternatively, selective application of ultrasound to specific regions or tissues may facilitate selective uptake of nucleic acid molecules, eg, DNA.
[0094]
Ultrasound transfection efficiency also depends on the contrast agent acting as an artificial cavitation nucleus, such as Albnex (Molecular Biosystems, San Diego, CA), Imagent (Alliance Pharmaceutical, San Diego, CA), Levovist -Use of SHU (Schering AG, Berlin, Germany), Definity (EI Dupont de Nemours, Wilmington, Delaware), STUC (Washington University, St. Louis, MO) and introduction of gaseous microbubbles. Can be enhanced by The contrast agent can be introduced locally, e.g., into a joint, systemically, can enhance cavitation efficiency by concentrating the lithotripter shock wave in a particular area, or can target the contrast agent to a particular site and then lithotripter Cavitation efficiency can be increased by concentrating the shock waves.
[0095]
Electroporation
Electroporation temporarily pierces the outer membrane of cells by using a pulsed rotating electric field. Methods and devices used for in vitro and in vivo electroporation are known (see, e.g., U.S. Pat. Standard protocols can be used.
[0096]
C. Cell targeting and delivery
The methods provided herein can be used for delivery of nucleic acids to cells, such as, but not limited to, eukaryotic and prokaryotic cells. Examples of cells that can be used in these methods include cell lines, primary cells, primary cell lines, plant cells and animal cells, including but not limited to stem cells and embryonic cells. For example, fibroblasts, such as lung and dermal fibroblasts, fibroblast-like cells, synovial cells, fibroblast-like synoviocytes, stem cells, such as embryonic and adult stem cells, such as mesenchymal stem cells, myoblasts, Lymphoblasts, carcinomas and hepatoma cells are among the many cells to which nucleic acids and especially large nucleic acids and artificial chromosomes can be delivered and monitored using the methods provided herein. Specific cells include mammalian cells, for example, A9 cells (mouse fibroblasts, HPRT, ATCC accession number CCL-1.4), CHO-S cells and DG44 cells (Chinese hamster ovary cells), V79 cells (Chinese hamster lung). Fibroblasts, ATCC accession number CCL-39), LMTK cells (mouse fibroblasts, ATCC accession number CCL-1.3), skin fibroblasts, L8 cells (rat myoblasts, ATCC accession number CRL-1769) , CCD1043 SK cells (human fibroblasts, ATCC accession number CRL-2056), adult-derived mesenchymal stem cells (eg, human bone marrow-derived, Cambrex Biosciences, East Rutherford, NJ), synovial cells (rat And human), Detroit 551 cells (human embryonic dermal fibroblasts, ATCC accession number) CL-110), NS0 (murine myeloma, ECACC accession number 85110503), 293 cells (human embryonic kidney cells transformed by type 5 (Ad5) DNA (ATCC accession number CRL-1573)), P46-Fl ( There are bovine lymphoid cell lines), DT40 (chicken lymphoblasts), EJ30 cells (human bladder cancer), HepG2 cells (human hepatome) and murine and bovine embryos.
[0097]
In certain embodiments, the methods of delivering nucleic acids to cells provided herein can be used to deliver nucleic acids to cells to treat a disease or disorder, for example, in gene therapy applications. For gene therapy applications, the nucleic acid delivered to the cell may encode a therapeutic molecule, eg, a protein. In many cases, effective gene therapy applications are complicated by the need to deliver large nucleic acids to cells. It may also be desirable to provide multiple copies of the nucleic acid encoding one or more therapeutic molecules. Compounding the difficulty in gene therapy methods is the difficulty that cells that are preferred for use in gene therapy applications often cannot be easily transfected. The methods provided herein are particularly suited for the delivery of large nucleic acids, which may be in the form of artificial chromosomes or fragments thereof, to cells for use in therapeutic applications.
[0098]
In vitro delivery
The cationic compound and the nucleic acid molecule, eg, DNA, can be added to cells in vitro, separately or mixed together, with or without the application of ultrasound or electrical energy. Generally, when applying energy, it is applied before contacting the nucleic acid molecule with the cell.
[0099]
Generally, nucleic acid molecules, eg, DNA, mixed with the cationic lipid / compound can be added to the cells as described in the Examples. Parameters important in optimizing the delivery of a nucleic acid molecule, eg, DNA, to a target cell are readily apparent to those skilled in the art. These parameters include, for example, cation compound, cation compound concentration, nucleic acid molecule, eg, DNA, nucleic acid molecule concentration, cell growth medium, cell culture conditions, length of time cells are exposed to the cation compound, and target cell type. The toxicities of the cationic compounds, and the amount and time of use of ultrasound or electroporation are particularly included. It may be necessary to optimize these parameters for various nucleic acid molecules, such as DNA and target cell types. The above optimization is performed by a routine procedure using the guidance provided in the present invention. In addition, rapid screening methods may provide direction as to what parameters need to be adjusted to optimize delivery (see “Examples”). Modifications of culture conditions, times, reagent concentrations and other parameters for use with different combinations of cationic compounds and target cell types, and to optimize delivery can be determined empirically. If it is necessary to use ultrasonic energy to increase transfection efficiency, it can be applied as described below and in the Examples. Electroporation can be performed according to the following procedure or by suitable protocols known to those skilled in the art.
[0100]
Contact of the cell with the cationic compound and the nucleic acid molecule, eg, DNA, at separate and distinct stages can be performed generally as described in the Examples. One of skill in the art could readily alter the order of application of components to target cells based on the disclosure herein.
[0101]
Ex vivo gene therapy
Delivery of the nucleic acid molecule, eg, DNA, is performed according to the above-described procedures for in vitro delivery. After selection is complete, the cells containing the nucleic acid molecule, eg, DNA, are introduced into the target target by various means, including injection, eg, subcutaneous, intramuscular, intraperitoneal, intravascular, and intralymphatic and intraarticular injection. The cells can be administered with or without the aid of a medical device, such as an arthroscope, other scopes, or various types of catheters.
[0102]
In vivo gene therapy
In one method of delivering a nucleic acid molecule, eg, DNA, to target cells in a subject's body in vivo, a cationic compound is first delivered to a target area (eg, a tissue, organ, tumor or joint). After an appropriate amount of time, the target area is subjected to an ultrasonic frequency at an appropriate energy level for an appropriate time, depending on the target area in the device, tissue type and body. Alternatively, electrical energy is delivered to the target area. The nucleic acid molecule, eg, DNA, is then delivered to the same area. If desired, by repeating this procedure, the nucleic acid molecule, eg, DNA, can be delivered to different regions simultaneously by multiple injections or multiple administrations over time.
[0103]
The cationic compound can be mixed with the nucleic acid molecule, eg, DNA, and delivered to the target area. The target area may then be subjected to ultrasound frequencies at the appropriate energy level for the appropriate time. Depending on the nucleic acid molecule, e.g., DNA, location in vivo, the cationic compound used and other variables, it may be necessary to use ultrasound or electroporation to achieve adequate transduction efficiency into cells at the target area. It may not be. Delivery of the contrast agent to the target area prior to ultrasound application can facilitate the introduction of nucleic acid molecules, eg, DNA.
[0104]
Nucleic acid molecules, such as DNA, can be found in body organs or tissues such as skin, muscle, stomach, intestine, lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen, prostate and joints (e.g., knees, elbows , Shoulders, wrists, hips, fingers, ankles, etc.). The molecule is delivered to, for example, primary cells and cell lines, such as fibroblasts, muscle, stomach, intestine, lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen, prostate, and mimics in vivo systems I can do it.
[0105]
The cationic compound and the nucleic acid molecule, e.g., DNA, may be separately or together injected (e.g., subcutaneous, intramuscular, intraperitoneal, intravascular, intraarticular and intralymphatic), instillation, cannulation, slow infusion, topical application. And can be delivered to target areas of the body by various means, including and other methods of administration. They can be administered in a suitable manner systemically (eg, by intravenous injection), eg, locally by delivery to a specific target area (tissue or area), eg, using a catheter, or by direct injection. They can be administered with or without the aid of medical instruments, such as arthroscopes, other scopes or various types of catheters.
[0106]
Cationic compounds can also be administered by coating a medical device, such as a catheter, for example, an angioplasty balloon catheter, with the cationic compound formulation. Coating can be accomplished, for example, by immersing the medical device in a mixture of a cationic lipid or cationic compound formulation and a suitable solvent, such as an aqueous buffer, aqueous solvent, ethanol, methylene chloride, chloroform, and other suitable solvents. . An aliquot of the formulation attaches spontaneously to the surface of the device and is subsequently administered to the subject as appropriate. Alternatively, a lyophilized mixture of the cationic lipid formulation can be specifically bound to the device surface. Such conjugation techniques are known (see, eg, Ishihara et al. (1993) Journal of Biomedical Materials Research 27: 1309-1314).
[0107]
Cationic compounds and nucleic acid molecules, eg, DNA, can be formulated in pharmaceutically acceptable carriers, eg, saline or other pharmaceutically acceptable solutions, for in vivo delivery. Nucleic acid molecules, such as DNA and cationic compounds, regardless of route of administration, are formulated into pharmaceutically acceptable dosage forms by standard methods known to those skilled in the art.
[0108]
In the case of gene therapy, the dosage level of the nucleic acid molecule, eg, DNA, can be varied to achieve an optimal therapeutic response for a particular subject. This depends on various factors, such as the mode of administration, the activity of the nucleic acid molecule, eg, DNA, the properties of the protein produced, the transfection efficiency of the target cell (ability to take up the nucleic acid molecule, eg, DNA), the route of administration, the Depends on location and other factors.
[0109]
As will be readily appreciated by those skilled in the art, the dose administered and the particular mode of administration will depend on the age, weight and particular animal and area thereof to be treated, the particular nucleic acid molecule and the cationic compound used, the anticipated therapy or It will vary depending on the diagnostic application and on the form of the formulation, for example, suspensions, emulsions or liposomes. Typically, doses are administered at low levels and are increased until the desired therapeutic effect is achieved. The amount of cationic compound administered will vary and will generally depend on the amount of nucleic acid molecule, eg, DNA, being administered. For example, the weight ratio of cationic compound to nucleic acid molecule can be from about 1: 1 to about 15: 1, for example, from about 5: 1 to about 1: 1. Generally, the amount of cationic compound administered will vary from about 0.1 milligram (mg) to about 1 gram (g). According to general guidance, for compositions typically with a body weight of 70 kg and about 1-10 million chromosomes per ml, a single dose ranging from 1-20 ml is administered as a single or multiple dose.
[0110]
In the case of topical treatment of disease, administration to diseased joints in rheumatoid arthritis, psoriasis and diabetes is of course possible as well as injection into muscle in the treatment of diseases such as hemophilia or other genetic diseases. Except for local treatment, a targeted delivery step is required.
[0111]
Also provided are cells for treating a disease. For example, provided is a composition comprising cells selected for therapeutic treatment of joint or rheumatoid arthritis, wherein the cells contain large heterologous nucleic acid molecules. For example, the cells of the composition can include artificial chromosomes. In certain embodiments of the above composition, the cells may include ACes.
[0112]
Gene therapy in connective tissue and rheumatic diseases
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint inflammation and progressive cartilage and (hard) bone destruction. Treatment of RA is problematic with current strategies because relatively high systemic doses are required to achieve therapeutic levels of anti-rheumatic drugs in joints. In addition, the available treatments are associated with significant troublesome side effects. That is, gene therapy is more about delivery of therapeutic molecules to sites of inflammation in the treatment of connective tissue diseases, rheumatic diseases and chronic erosive joint diseases such as RA, osteoarthritis, ankylosing spondylitis and juvenile chronic arthritis. It is an effective system.
[0113]
In synovial connections, smooth connections are ensured by the macromolecular structure of the articular cartilage that covers the epiphysis. The cavity or connection space found at the location of the adjacent bone is lined with tissue called the synovium. The synovium consists of macrophage-like type A cells (which appear to be derived from macrophages / mononuclear progenitors and exhibit phagocytosis) and fibroblast-like type B cells (appearance is more fibroblastic, hyaluronic acid And the production of other components of synovial fluid). Underneath the synovium are scattered subcellular subsynovium, which may be fibrous in nature and fat or ring-shaped. Fibroblast-like synovial cells (FLS) can be distinguished from normal fibroblasts in the subintimal synovium by different gene expression patterns. FLS has high levels of uridine diphosphoglucose dehydrogenase (UDPGD), high levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and CD44 (hyaluronic acid receptor), It has been shown to express fibronectin receptors and β3 integrins. Subline (subline) fibroblasts or fibroblasts from other sources do not or only express low levels of these markers (e.g., Edwards (1995) Ann.Rheum.Dis. 54: 395-397, Firestein (1996) Arthritis Rheum. 39: 1781-1790, Edwards (2000) Arthritis Res. 2: 344-347).
[0114]
Progression of the pathology in RA involves thickening of the synovial lining due to proliferation of fibroblast-like synovial cells (FLS) and infiltration of inflammatory cells (eg, lymphocytes, macrophages and mast cells). The normal biology of synovial cells is also altered in the pathological processes of RA, including the invasion and destruction of joint cartilage and bone. In addition to producing elastase and collagenase, synovial cells mediate the pathophysiological processes of RA by expressing cell surface proteins involved in the recruitment and activation of lymphocytes and macrophages within the synovium. Proliferation of synovial cells induces pannus tissue that infiltrates cartilage and overgrows, leading to bone destruction and destruction of joint structures and functions. Inflammatory (inducible) cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) play important roles in inflammation and joint damage associated with RA. Pathological effects elicited by these cytokines include leukocyte infiltration leading to synovial hyperplasia, cell activation, cartilage breakdown and inhibition of cartilage matrix synthesis.
[0115]
By introducing nucleic acids into rheumatoid synovial tissue, mediators can be produced that provide a damaging substance that blocks inflammation or hyperplasia or specifically destroys the diseased synovium. Ex vivo retroviral delivery of nucleic acids encoding an interleukin-1 receptor antagonist (IL1-RA) and transduction of synovial cells have been used to inhibit inflammation in gene therapy of human RA (eg, Evans (1996) Human Gen. Ther. 7: 1261-1280 and Del Vecchio et al. (2001) Arthritis Res. 3: 259-263). Adenovirus vectors have been proposed for delivery of nucleic acids encoding IL-1 receptor antagonists to synovial cells in in vivo transduction methods [eg, US Pat. No. 5,747,072 and PCT Application Publication No. WO 00/52186. reference].
[0116]
Artificial chromosomes offer advantages over viral-based systems for gene therapy. For example, the artificial chromosome expression systems (ACes), and other artificial chromosomes reported in US Pat. Nos. 6,025,155 and 6077697 and PCT application WO 97/40183, have large nucleic acids and / or have been identified both in vitro and in vivo. Serves as a non-integrative non-viral vector with great capacity for delivery of multiple copies of the nucleotide sequence to cells, eg, synovial cells. The artificial chromosome systems provide an additional advantage in that they allow for stable and predictable expression of genes that produce single or multiple proteins over an extended period of time.
[0117]
The methods provided in the present invention provide for the introduction of large nucleic acids, such as artificial chromosomes, into primary cells, such as synovial cells (eg, fibroblast-like synovial cells) and skin fibroblasts, and skeletal myofibroblast cell lines. Can be used to That is, a method for introducing a heterologous nucleic acid into a synovial cell by introducing a chromosome, for example, an artificial chromosome, into the synovial cell is also included in the method provided by the present invention. In one embodiment above, the artificial chromosome is ACes. Synovial cells can be, for example, fibroblast-like synovial cells.
[0118]
A particular method provided herein for introducing large nucleic acid molecules into synovial cells involves exposing the nucleic acid molecules to a delivery agent and contacting the synovial cells with the nucleic acid molecules. In certain embodiments of the method, the delivery factor is not energy. In one embodiment, the large nucleic acid molecule is a chromosome. For example, the nucleic acid can be an artificial chromosome, for example, ACes. In certain embodiments, the synovial cells are fibroblast-like synovial cells. Any delivery agent, such as those described herein, can be used in the above methods. For example, the delivery agent can include a cationic compound.
[0119]
Exposing the nucleic acid molecule to the delivery agent, exposing the synovial cells to the delivery agent, and contacting the synovial cell with the nucleic acid molecule, thereby delivering the nucleic acid molecule to the synovial cell. There is also provided a method of introducing nucleic acid molecules into a nucleic acid, wherein the steps are performed sequentially or simultaneously in some order. In some embodiments of the method, when the delivery agent is energy, it is not applied to the nucleic acid molecule, and is not applied to the synovial cells after contacting the nucleic acid molecule with the synovial cells. The nucleic acid may be any nucleic acid. In certain embodiments, the nucleic acids are large nucleic acids, chromosomes, artificial chromosomes, or ACes. In yet another embodiment, the synovial cells are fibroblast-like synovial cells. Delivery agents, such as those described herein, can be used in the above methods. For example, the delivery agent can include a cationic compound.
[0120]
Another method of delivering a nucleic acid molecule to a synovial cell provided herein involves contacting the synovial cell with a delivery agent in the presence or absence of the nucleic acid molecule and transferring ultrasonic or electrical energy to the synovial cell. Wherein the contacting and the application are performed sequentially or simultaneously, and include the step of contacting the synovial cells with the nucleic acid molecules to deliver the nucleic acid molecules to the synovial cells. The nucleic acid may be any nucleic acid. In certain embodiments, the nucleic acids are large nucleic acids, chromosomes, artificial chromosomes, or ACes. In yet another embodiment, the synovial cells are fibroblast-like synovial cells. Numerous delivery factors, such as those described herein, can be used in the above methods. For example, the delivery agent can include a cationic compound. In one embodiment, the energy is ultrasound.
[0121]
That is, provided herein is a method for delivering nucleic acids, particularly large nucleic acids, such as chromosomes, including artificial chromosomes, such as ACes, to primary cells, including synovial cells and fibroblasts. These methods can be used in vitro and in vivo.
[0122]
Also provided herein is a synovial cell comprising a large heterologous nucleic acid, a heterologous chromosome or a portion thereof, or an artificial chromosome. In one embodiment, the artificial chromosome is ACes. The synovial cells include fibroblast-like synovial cells. Synovial cells can be from any species, including but not limited to mammals. For example, synovial cells containing large nucleic acids, such as artificial chromosomes (eg, ACes), include primate synovial cells, as well as rodents, rabbits, monkeys, dogs, horses, and human synovial cells.
[0123]
The ability to achieve large nucleic acid delivery to the cells demonstrates that the method is useful in gene therapy applications and testing in diseased animal models for therapeutic molecules that can be used in gene therapy. That is, provided herein is a method of treating a disease or modulating a condition, comprising introducing a large nucleic acid molecule, a chromosome or a portion thereof, or an artificial chromosome into an affected subject.
[0124]
Provided herein are methods of treating or modulating a rheumatic disease condition in a subject. In one embodiment, the method comprises introducing a large nucleic acid into the subject, wherein the large nucleic acid is a factor that modulates or encodes a pathology of rheumatic disease. For example, the nucleic acid can be or encode a molecule having an anti-rheumatic effect. Conditions associated with rheumatic diseases are known in the art and are described herein. For example, one such condition is an inflammatory process that involves the processes of cell activation, invasion, proliferation and recruitment. In a specific embodiment of this method, the disease is rheumatoid arthritis. The nucleic acid can be, for example, a chromosome or a portion thereof or an artificial chromosome, for example, ACes. In certain embodiments, large nucleic acids are introduced into a subject at a site of inflammation. One potential site of inflammation is a joint.
[0125]
Also provided is a method of treating rheumatic disease in a subject into which a large nucleic acid is introduced, wherein the large nucleic acid is a therapeutic agent or includes a nucleic acid that encodes it. For example, the nucleic acid can be or encode a molecule having an anti-rheumatic effect. In certain embodiments of this method, the disease is rheumatoid arthritis. The nucleic acid can be, for example, a chromosome or a portion thereof or an artificial chromosome, for example, ACes. In an embodiment of this method, the large nucleic acid is introduced into the subject at the site of inflammation. One potential site of inflammation is a joint.
[0126]
In methods of modulating the condition of or treating rheumatic diseases, the methods can be practiced in any format, including ex vivo and in vivo formats. That is, for example, a nucleic acid can be introduced into a cell in vitro and then introduced into a subject. Alternatively, nucleic acids can be introduced into cells in vivo. In certain embodiments, the nucleic acid is introduced into synovial cells, which can be, for example, fibroblast-like synovial cells. The introduced nucleic acid may be a molecule having or having an anti-rheumatic effect in a subject or may comprise a nucleic acid encoding the same. For example, the molecule may alter, counteract or reduce the condition. The molecules may improve the symptoms of the disease. Molecules with anti-rheumatic effects in subjects with RA are known in the art [see, eg, Vervoordeldonk and Tak (2001) Best Prac. Res. Clin. Rheumatol. 15: 771-788 and WO 00/52186]. Such molecules include anti-inflammatory or immunomodulatory molecules. For example, interleukin-1 receptor antagonist, soluble interleukin-1 receptor, soluble tumor necrosis factor receptor, interferon-β, interleukin-4, interleukin-10, interleukin-13, transforming growth factor β, Dominant negative I kappa B-kinase, FasL, Fas associated death domain protein or CTLA-4 are among the molecules that may have anti-rheumatic effects.
[0127]
Also provided are methods for identifying, evaluating or testing nucleic acids as potential therapeutics in the treatment of connective tissue or rheumatic diseases by introducing large nucleic acid molecules into connective tissue or rheumatic disease animal models. The nucleic acid molecule can be a candidate therapeutic or include a nucleic acid that encodes it. The method may include determining whether the nucleic acid molecule has any effect on the animal, in particular, an antirheumatic effect. For example, when determining whether a nucleic acid molecule has any effect on an animal, one or more conditions can be assessed, eg, improvement or reduction in adverse conditions. In certain embodiments, the disease is a rheumatic disease, eg, rheumatoid arthritis. The animal can be any animal for which the disease can be modeled. For example, the animal can be a mammal. In certain embodiments, the animal is a monkey, rodent, rabbit, dog, cat, horse, cow, pig or primate. The large nucleic acid can be, for example, a chromosome or a portion thereof or an artificial chromosome, for example, ACes. In certain embodiments, the nucleic acid molecule is in a synovial cell, eg, a fibroblast-like synovial cell. In yet another embodiment, the nucleic acid is introduced into a joint of an animal. Nucleic acid molecules can be introduced into animals using in vitro or in vivo formats. For example, nucleic acids can be introduced into cells in vitro and then into animals. In another embodiment, the nucleic acid is introduced into the cell in vivo.
[0128]
Animal models include, for example, animal models of RA. Several animal models of RA, and methods of making such models, are known in the art. The models described above include adjuvant-induced arthritis (AA) [eg, see Kong et al. (1999) Nature 4023: 304-309] and collagen type II-induced arthritis [eg, Tak et al. (1999) Rheumatology 38: 362-369. , Han et al. (1998) Autoimmunity 28: 197-208, Gerlag et al. (2000) J. Immunology 165: 1652-1658]. For example, experimental induction of adjuvant-induced arthritis in Lewis rats results in severe inflammation of bone marrow and peri-articular soft tissue with extensive local bone and cartilage destruction, loss of bone density and limb disability [eg, Bendele et al. (1999) Arthritis Rheum. 42: 498-506].
[0129]
D. Evaluation of nucleic acid delivery to cells
Microscopy and colony formation assays that can be used to assess stable nucleic acid molecule delivery rely on manual visualization or measurement of nucleic acid molecule (e.g., selectable marker gene) expression, a process separate from delivery. It is. The above methods involve a delay in obtaining an evaluation of the delivery method. Microscopy techniques for visualizing chromosome or plasmid transfer using bromodeoxyuridine (BrdU) (see, eg, Pittman et al. J Immunol Methods 103: 87-92 (1987)) are time consuming and detect low levels of migration. However, there are limitations on the required large sample size, which is limited by the need to score manually. Colony forming transfection analysis can take 4-6 weeks to generate and evaluate marker-expressing transfection colonies.
[0130]
Conversely, the method provided by the present invention is based on fast, automated, sensitive and accurate analytical techniques, such as flow cytometry, which is time consuming, labor intensive and error prone. No manual detection of individual transfected cells, for example by microscopic techniques, is required. The above method allows analysis of nucleic acid molecule delivery data within 48 hours after transfection. In addition, data collected by flow cytometry analysis is statistically superior because many events such as nucleic acid molecule introduction can be easily collected. Since clear data obtained by these methods is instrument guidance, it is less susceptible to determination errors. That is, these methods increase the accuracy in evaluating nucleic acid molecule delivery. Conversely, microscopy is limited by the time it takes to score a distinct event and the sample size is also limited.
[0131]
Methods for monitoring nucleic acid molecule delivery are hampered by the problem of cell autofluorescence and wild-type cell differentiation from cells that express low levels of the reporter gene product to detect nucleic acid molecules, eg, DNA, but not reporter gene expression products. It is possible to measure the absolute value of a nucleic acid molecule introduced within 24 hours without being performed (see, for example, Ropp et al. (1995) Cytometry 21: 309-317).
[0132]
1. Factors to consider when working on nucleic acid delivery
Delivery of a nucleic acid, eg, DNA, to a cell is the process by which the nucleic acid is introduced inside the cell. Methods of delivering nucleic acids can be evaluated in a variety of ways, including:
[0133]
a. Introduction efficiency
Delivery methods can be evaluated by measuring the percentage of recipient cells in which the nucleic acid, including DNA, is present (ie, transduction efficiency). However, when evaluating delivery methods for the ultimate goal of producing cells that express the introduced nucleic acid, there are additional factors that are more important than the mere presence of the nucleic acid in the recipient cell to be considered. These additional factors include cell viability. Chronogenicity is an excellent measure of viability when assessing a growing cell population. Metabolic integrity can be monitored when the target cell population is not dividing or is growing slowly.
[0134]
b. Chronogenicity (clonogenicity)
Chronogenicity represents a measure of cell viability with respect to delivery procedures, growth conditions and cell manipulation (eg, plating). It is important to assess chronogenicity to determine whether the delivery procedure results in a sufficient number of viable cells to achieve the desired number of cells containing the introduced nucleic acid.
[0135]
Chronogenicity can be expressed as a clonal fraction. The clonal fraction is an index calculated by multiplying two separate fractions and normalizing against a control plate efficiency correction factor (CPE). The two separate fractions multiplied by this calculation are the fraction of cells surviving the delivery procedure (population cell yield) and the fraction of cells surviving plating. That is, the calculation is performed as follows:
Clone fraction = (number of viable colonies after plating / number of plating cells) × {number of cells after transfection / (number of transfected cells × CPE)}
[0136]
The values used in this calculation for the number of cells after transfection (ie, after delivery) and the number of colonies after plating are based on the number of cells or colonies at some point in the process. For example, a value for cell number after transfection represents a cell number at a time after nucleic acid delivery that is sufficient to complete the delivery process. This time can be determined empirically. Typically, this time ranges from 4-48 hours, and is generally about one day after transfection. Similarly, the value of the number of viable colonies after plating is determined at some point after nucleic acid delivery long enough to exclude non-viable cells and establish viable cells as colonies. Represents the number of colonies. This time can be determined empirically. Typically, this is the point in time when at least the average colony is made up of about 50 cells, or generally after 5 cell cycles.
[0137]
A correction factor is included to take into account the plating efficiency of control wells, a ratio determined by dividing the number of colonies counted by the initial plating cell number (typically 600-1000 cells). . For LM (tk-) and V79-4 cells, the value of the correction factor typically ranges from about 0.7 to about 1.2, and may be, for example, 0.9.
[0138]
The number of plated cells should be constant at 1000 (Simplified Plate Efficiency Assay) by performing duplicates, except that if the CPE is less than 0.3, the number of seeded cells should be 5,000-50,000. Should be increased to the range. If the CPE is less than 0.1 to 0.2, viable fraction analysis should be considered.
[0139]
c. Viable fraction
If the target cell population is not dividing or is dividing slowly, the replication or chronogenicity assays are not directly relevant. To measure cell killing, a less direct cell viability assay that monitors metabolic death rather than loss of replication competence must be used. These procedures include, for example, (1) membrane integrity as measured by dye exclusion, (2) inhibition of nucleic acid synthesis as measured by incorporation of nucleic acid precursors, (3) radioactive chromium release, and (4) MTT assays. (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide). Because these methods reflect only immediate changes in metabolism, they are different from measuring replication competence and can be reversed or delayed, which can lead to errors in assessing cell viability. To minimize these errors, a correlation of the duplicate procedure has been suggested.
[0140]
d. Measurement of potential transfection efficiency (PTE) and chromos index (CI)
Cells that are viable and contain the nucleic acid from the total number of cells to which the nucleic acid has been delivered when assessing the delivery method used to introduce the nucleic acid into the cell, targeting expression of the nucleic acid, eg, DNA, in the cell It is desirable to obtain an indication of the theoretical maximum percentage of This is referred to as potential transfection efficiency and can be calculated from existing or historical experimental data sets and is determined as follows:
Potential transfection efficiency (PTE) = transduction efficiency x (clone fraction or viable fraction) x correction factor (CF)
[0141]
The Chromos Index (C.I.) is a measure of the proliferating population by using experimental values of labeled nucleic acids, such as ACes delivery%, and clonal fractions measured using a simplified chronogenetic assay to measure transduction efficiency. Is an efficient and rapid method of determining the potential transfection efficiency of DNA.
Chromos index (CI) =% labeled ACes delivered x Clone fraction evaluated x CF
[0142]
The transfer efficiency and the values of the clonal and viable fractions are calculated as described above. The correction factor (CF) takes into account sample size, sampling time and control plate efficiency. If all of these factors are constant for each variable, ie, sampling time and magnitude, the correction factor approaches the reciprocal of the value for CPE, that is, the clonal fraction or transfer efficiency is low even at low CF. If it is still close to 100%, or in other words, 100% delivery and viability, the maximum potential transfection efficiency will be equal to the plating efficiency of the control cells. The calculation of the CI determines the optimization of each variable, with the goal that the parameters such as transfer efficiency, clonal fraction and CF approach 1 (or 100%). If the sample size or collection time varies for either clonal fraction or transfer efficiency, CF represents an extrapolation based on slope or rate of change. The application of this evaluation method is provided in the "Examples".
[0143]
A stable transfection efficiency of about 1% is in the range (1-100%) that is considered useful for introducing large nucleic acid molecules into target cells. Using the methods provided herein, achieve the desired transfection efficiency without the need for long-term culturing of transfectants under selection conditions and measuring the number of surviving cells for selectable marker expression It is possible to predict which delivery method must be selected in order to do so. This analysis involves the calculation of the Chromos Index (CI) which integrates the "biological" value (clonal fraction) with the measurement of chromosomal "uptake" or transduction efficiency (percent of cells containing ACes delivered).
[0144]
2. Labeling of nucleic acid molecules for introduction
In the method of monitoring nucleic acid molecule delivery provided herein, labeling of the nucleic acid molecule to be delivered, for example, DNA, enables detection of the nucleic acid molecule from the recipient cell after introduction into the cell. Nucleic acid molecules can be labeled by incorporation of nucleotide analogs. Any nucleic acid molecule analog that can be detected in cells can be used in these methods. The analog may be directly detectable by, for example, radioactivity, or may be specifically recognized by the analog and detected in the recipient cell to an endogenous nucleic acid molecule, for example, an analog that distinguishes it from nucleotides constituting DNA. It can be detected upon binding of a possible molecule. Analogs that are directly detectable have unique properties that allow them to be detected using standard analytical methods. An analog may also be detectable upon binding to a detectable molecule, eg, a labeled antibody that specifically binds to the analog. The label on the antibody is one that can be detected using standard analytical methods. For example, the antibodies are fluorescent and can be detected by flow cytometry or microscopy.
[0145]
In certain embodiments of these methods, the nucleic acid molecule to be delivered, eg, DNA, is labeled with a thymidine analog, eg, iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdU). In a preferred embodiment, IdUrd is used to label a nucleic acid molecule to be delivered, eg, DNA. The introduced IdUrd-labeled nucleic acid molecule, eg, DNA, can be immunologically labeled with a FITC-conjugated anti-BrdU / IdUrd antibody and quantified by flow cytometry. That is, introduction of a labeled nucleic acid molecule, eg, DNA, into a recipient cell can be detected within hours after transfection.
[0146]
E. FIG. Stability of the delivered nucleic acid molecule
It is also interesting to assess the stability of the nucleic acid molecule, eg, DNA, under selected delivery conditions. Some delivery conditions and factors can have deleterious effects on nucleic acid molecular structure. Furthermore, labeling techniques used in certain monitoring methods of nucleic acid molecules, eg, DNA delivery, can also impact the structure and function of nucleic acid molecules, eg, DNA.
[0147]
The effect of delivery conditions on nucleic acid molecules can be assessed in a variety of ways, including microscopic analysis. In one example of a specific assay for the stability of artificial chromosomes, for example ACes, fluorescence microscopy for the ability to expose the chromosome to conditions of interest, for example, IdU labeling, and to remain intact and condensed after incorporation of the nucleotide analog. Analyze below.
[0148]
Methods for monitoring nucleic acid molecule delivery and expression
Also, the method of monitoring delivery of the nucleic acid molecule delivery methods provided herein can be combined with the evaluation of nucleic acid molecule, eg, DNA expression, in recipient cells to provide an overall process of nucleic acid molecule introduction for expression purposes. Further information regarding may be provided.
[0149]
For example, to facilitate the analysis of nucleic acid molecules, for example, DNA expression, it may be desirable to include in the introduced nucleic acid molecule, for example, DNA, a reporter gene that encodes a product that is easily detected. The reporter gene products for direct detection include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), luciferases, and CAT. Reporter gene products for indirect detection include, but are not limited to, β-galactosidase and cell surface markers.
[0150]
For example, the use of a GFP reporter gene, such as, but not limited to, an artificial chromosome containing a GFP coding sequence, such as ACes, in combination with labeling of ACes with a DNA analog, such as IdU, results in rapid delivery and expression. Can be monitored accurately. For example, following delivery of Ids-labeled GFP gene-containing ACes to target cells by any of the reported methods, the ACes-containing cells are divided into two populations. One population is fixed, stained for IdU, and analyzed by flow cytometry to determine percent delivery. The other population undergoes 4-5 cell divisions (about 72 hours) and GFP fluorescence is measured as an indicator of expression.
[0151]
Since the above studies indicate that the incorporation of the analog label does not affect GFP protein expression, these methods can be combined to monitor the delivery and initial expression of ACes, thereby reducing the effectiveness of the delivery method. It indicates that further information can be provided for a quick assessment. Also, by using these methods in combination, the biological events between the initial delivery stage and the initial gene expression can be mapped.
[0152]
The examples described below are for illustrative purposes only and are not intended to limit the scope of the invention.
【Example】
[0153]
Example 1
Manufacture of artificial chromosomes
A. GFP chromosome contained in A9 cell line
Plasmid
Plasmid pIRES-EGFP (see SEQ ID NO: 13, a known plasmid obtained from Clontech, California, e.g., see U.S. Pat. No.). This plasmid contains an internal ribosome entry site of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region (IRES; Jackson (1990) Trends Biochem. 15: 477-483; Jang et al. 1988) J. Virol. 62: 2636-2643). This allows the gene of interest (cloned into MCS) and the EGFP gene to be translated from a single bicistronic mRNA transcript. Plasmid pIRES2-EGFP is designed for the selection of transiently transfected mammalian cells expressing EGFP and the protein of interest by flow cytometry and other methods. This vector can also be used to express EGFP alone or to obtain a stable transfection cell line without drug and clonal selection.
[0154]
Enhanced GFP (EGFP) is a mutant of GFP that has a 35-fold increase in fluorescence. This variant has a Ser to Thr mutation at amino acid position 65 and a Phe to Leu mutation at position 64 and is encoded by a gene with optimized human codons (see, eg, US Patent No. 6,053,312). . EGFP is a red shift mutant of wild-type GFP optimized for brighter fluorescence and higher expression in mammalian cells (Yang et al. (1996) Nucl. Acids Res. 24: 4592-4593; Haas et al. (1996) Curr. Biol. 6: 315-324; Jackson et al. (1990) Trends Biochem. 15: 477-483) (maximum excitation = 488 nm; maximum emission = 507 nm). EGFP encodes a GFPmut1 mutant (Jackson (1990) Trends Biochem. 15: 477-483) containing a double amino acid substitution from Phe-64 to Leu and from Ser-65 to Thr. The coding sequence of the EGFP gene contains over 190 silent base changes corresponding to human codon usage preference (Jang et al. (1988) J. Virol. 62: 2636-2643). Conversion of sequences flanking both sides of EGFP into the Kozak consensus translation initiation site (Huang et al. (1990) Nucleic Acids Res. 18: 937-947) further increased translation efficiency in eukaryotic cells. .
[0155]
Plasmid pIRES-EGFP was derived from PIRESneo (which was originally called pCIN4) by replacing the neo gene downstream of the IRES sequence with the EGFP coding region. The IRES sequence allows for the translation of two reading frames from one mRNA transcript. The expression cassette for pIRES-EGFP consists of the human cytomegalovirus (CMV) major immediate early promoter / enhancer, followed by a multiple cloning site (MCS), a synthetic intron (IVS; Huang et al. (1990) Nucleic Acids Res. 18: 937-). 947), after the EMCV IRES, contains the EGFP coding region and the bovine growth hormone polyadenylation signal.
[0156]
Position of the major component (for SEQ ID NO: 13):
Human cytomegalovirus (CMV) immediate early promoter: 232-820;
MCS 909-974;
IVS 974-1269;
ECMV IRES 1299-1884;
Enhanced green fluorescent protein (EGFP) gene 1905-2621;
Fragment containing a bovine poly A signal 2636-2913;
Col E1 origin of replication 3343-4016, and
Ampicillin resistance gene 5026-4168
[0157]
Propagation in Escherichia coli (E. coli)
Suitable host strains: DH5a, HB101, and other multipurpose strains. Single-stranded DNA production requires a host containing an F plasmid, such as JM101 or XL1-Blue.
Selectable marker: The plasmid confers resistance to kanamycin (30 μg / ml) to the E. coli host.
Escherichia coli origin of replication: pUC
Number of copies: ~ 500
Plasmid incompatibility group: pMB1 / ColE1
[0158]
pCHEGFP2
Plasmid pCHEGFP2 was constructed by deletion of the Nsi1 / SmaI fragment from pIRES-EGFP. Plasmid pIRES-EGFP contains the coding sequence for the 2.1 kB Nru1 / Xho fragment of pCHEGFP2 containing the CMV promoter, synthetic intron, EGFP coding sequence and bovine growth hormone polyadenylation signal. Digestion of pIRES-EGFP with Nru1 and Sma1 resulted in a 2.1 kb fragment. The digested DNA was fractionated by agarose gel electrophoresis, the separated bands were excised and then eluted from the gel using a Qiaex11 gel purification system (Qiagen, Mississauga, Ontario).
[0159]
pFK161
Cosmid pFK161 was obtained from Dr. Gyula Hadlaczky and contains a 9 kb NotI insert derived from a murine rDNA repeat (for a report on this cosmid, see PCT Application Publication No. WO 97/40183 by Hadlaczky et al. Clone 161). This cosmid is called clone 161 and contains the sequence corresponding to nucleotides 10232-15000 in SEQ ID NO: 16. It is a fragment of a giant chromosome (see U.S. Patent No. 6,077,697 and International PCT Application No. WO 97/40183, e.g., H1D3 as described in the European Collection of Animal Cell Culture (ECACC) under accession number 96040929). , A mouse-hamster hybrid cell line with this giant chromosome) was inserted into plasmid pWE15 (Stratagene, La Jolla, CA). Half of a 100 μl low melting point agarose block (large plug) containing the isolated SATAC was digested with NotI at 37 ° C. overnight. Plasmid pWE15 was similarly digested with NotI overnight. The large plug was then thawed and mixed with the digested plasmid, ligation buffer and T4 ligase. Ligation was performed at 16 ° C. overnight. Bacterial DH5α cells were transformed with the ligation product and the transformed cells were plated on LB / Amp plates. For a total of 189 colonies, 15-20 colonies were cultured on each plate. Plasmid DNA was isolated from surviving colonies for culture in LB / Amp medium and analyzed by Southern blot hybridization for the presence of DNA that hybridized with the pUC19 probe. This screening method ensured that all clones, even those lacking the insert but containing the pWE15 plasmid, were detected.
[0160]
Cosmid minipreps were prepared for restriction site analysis in the insert DNA by using liquid cultures of all 189 transformants. Six of the original 189 cosmid clones contained the insert. These clones were named as follows: 28 (〜9 kb insert), 30 (〜9 kb insert), 60 (〜4 kb insert), 113 (〜9 kb insert), 157 (〜9 kb insert) and 161 (~ 9 kb insert). Restriction enzyme analysis showed that three of the clones (113, 157 and 161) contained the same insert.
[0161]
For sequence analysis, the insert of cosmid clone # 161 was subcloned as follows. To obtain the terminal fragment of the insert of clone 161 the clone was digested with NotI and BamHI and ligated with NotI / BamHI digested pBluescript KS (Stratagene, La Jolla, CA). Two fragments of the insert of clone # 161 were obtained: the 0.2 kb and 0.7 kb insert fragments. The same digest was ligated with BamHI digested pUC19 to subclone the internal fragment of the insert of clone # 161. Three fragments of the insert of clone 161: 0.6 kb, 1.8 kb and 4.8 kb insert fragments were obtained.
[0162]
The insert corresponds to the inner portion of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 set forth in Genbank Accession No. X82564, provided as SEQ ID NO: 5. Sequence data obtained for the insert of clone 161 is shown in SEQ ID NOs: 6-12. Specifically, the individual subclones corresponded to the following positions in GenBank Accession No. X82564 (ie, SEQ ID NO: 5) and SEQ ID NOs: 6-12.
[0163]
[Table 1]

[0164]
The sequences shown in SEQ ID NOs: 6-12 differ in some positions from the sequence presented at positions 7551-1670 of Genbank Accession No. X82564. The divergence can be attributed to random mutations between the rDNA repeat units.
[0165]
For use herein, an rDNA insert from the clone was prepared by digesting a cosmid with NotI and BglII and purified as described above. Culture and maintenance of bacterial stocks and purification of plasmids are performed using standard known methods (eg, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press). Performed and plasmids were purified from bacterial cultures using Midi- and Maxi-Preps kits (Qiagen, Mississauga, Ontario).
[0166]
B. Preparation of GFP, murine A9 cell line
Cell culture and transfection
A murine A9 cell line was obtained from the ATCC and cells were thawed and maintained as described below. Briefly, cells were cultured in 15 cm tissue culture dishes (Falcon, Becton, Ontario) containing 90% DMEM (Canadian Life Technologies Burlington, Ontario) and 10% FBS (Cancella, Lexdale, Ontario). Dickinson Lovewear, Franklin Lakes, New Jersey) 2 × 10 per piece ⁶ Plate at cell density, 37 ° C, 5% CO ₂ Maintained. When the cells reached 70-80% of the culture saturation density, the culture was passaged using routine procedures. The subculture was performed as follows. The medium was aspirated off. 10 ml of 1 × trypsin-EDTA (Canadian Life Technologies Burlington, Ontario) was dispensed into the cell monolayer and the dish was gently rotated to distribute the trypsin-EDTA. Finally, the trypsin-EDTA mass was aspirated off and the dishes were placed at 37 ° C. for 5 minutes. To quench trypsin-EDTA, 10 ml of growth medium was added to the dish and the single cell suspension was transferred to a 50 ml conical tube. Cell counting was performed using a cell counter (Beckman-Coulter, Hialeah, FL). The cells were diluted and re-cultured as described above. Cultures were harvested by trypsin-EDTA treatment for cold storage, counted, and the cell suspension was centrifuged at 500 xg for 5 minutes in a swinging bucket centrifuge. Cell pellet is 1 × 10 ⁷ Resuspended in a freezing medium containing 90% DMEM, 20% FBS and 10% DMSO (Sigma-Aldrich, Oakville, Ontario) at a density of cells / ml. A 1 ml aliquot of the cell suspension was then dispensed into cryovials (Nunc, Rochester, NY), frozen overnight in isopropanol-filled containers (Nunc, Rochester, NY), stored at -70 ° C, and then stored for long periods. Therefore, it was transferred to the gas phase of a liquid nitrogen freezer.
[0167]
Ca ₂ PO ₄ Coprecipitation methods (eg, Graham et al. (1978) Virology 52: 456-457; Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1373-1376; and (1990) Current Protocols in Molecular Biology A9 cells were transfected (see, Vol. 1, Willie Inter-Science, Addendum 14, units 9.1.1-9.1.9). One day before transfection, A9 cells were plated at 2 x 10 cells per 10 cm dish. ⁶ Cells were plated at density and the medium was replaced with fresh growth medium 3 hours before transfection. 140 μg of 9 kb rDNA, NotI and 5 μg of 2.1 kB CMV-EGFP XhoI / NruI fragment were mixed, co-precipitated and used with Ca ₂ PO ₄ A coprecipitate (calcium phosphate transfection system, Canadian Life Technologies Burlington, Ontario) was prepared and distributed to two 10 cm dishes of subconfluent A9 cells. DNA-Ca ₂ PO ₄ After allowing the complexes to stand on the cells for 18 hours, the precipitate was aspirated off and the cells were subjected to a glycerin shock for 1.5 minutes. After glycerin shock, the cell monolayer was gently washed with 2 × 10 ml dPBS (Canadian Life Technologies Burlington, Ontario) and 10 ml of pre-warmed growth medium was added. Finally, return the dish to the incubator, 37 ° C, 5% CO ₂ Maintained. After recovery for 3 hours, each dish was passaged in a 3 × 15 cm tissue culture dish.
[0168]
Culture GFP fluorescence in culture using an inverted microscope with epifluorescence illumination (Axiovert 25, Zeiss, (North York, Ontario) and # 41017 Endow GFP filter set (Chroma Technologies, Brattleboro, Vermont)). Monitored visually. Enrichment of the GFP-expressing population was performed as follows.
[0169]
Enrichment of GFP-expressing cell population by fluorescence-activated cell sorting
Cell sorting was performed using a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a turbo-sort option and two Innova 306 lasers (Coherent, Palo Alto, CA). For cell sorting, a 70 μm nozzle was used. The sheath buffer was changed to PBS (maintained at 20 psi). GFP was excited with a 488 nm laser beam and the excitation was detected with FL1 using a 500 EFLP filter. Forward and side scatter were adjusted to sort for viable cells. Only viable cells were then analyzed for GFP fluorescence. Gating parameters were adjusted using wild type A9 cells as a negative control and GFP CHO cells as a positive control.
[0170]
For the first round of sorting, A9 cells were harvested 4 days after transfection, resuspended in 10 ml of growth medium, and sorted for a GFP-expressing population using the above parameters. GFP positive cells were distributed in 5 × 10 ml volumes of growth medium (Canadian Life Technologies Burlington, Ontario) supplemented with 1 × penicillin / streptomycin, and non-expressing cells were instructed to be discarded. Expressing cells were further diluted to 50 ml using the same medium, plated on 2 × 15 cm dishes, and cultured as described in the previous section. When the sorted populations reached confluent growth, they were re-sorted to enrich for GFP-expressing cells. By performing a total of four consecutive selections, a high enrichment of GFP-expressing cells as high as 89% was achieved after final selection. The final GFP expressing population was expanded for cryopreservation and fluorescent in situ hybridization screening (see below). Single cell clones were established from the population of interest by directing GFP-expressing single cells to individual wells of a 96-well plate using a flow cytometer. These were cultured as described above.
[0171]
Fluorescent In-Situ hybridization
Fluorescence In-Situ Hybridization (FISH) screening was performed on GFP enriched populations and single cell clones to detect amplification and / or artificial chromosome formation. Preparation and hybridization of metaphase spreads was performed (see Telenius et al. (1999) Chromosome Res 7: 3-7). Probes used include pSAT1 recognizing mouse major repeats (eg, Wong et al. (1988) Nucl. Acids Res. 16: 11645-11661), pFK161 hybridizing with mouse rDNA containing regions and mouse small repeats PCR-generated probes are included.
[0172]
That is, one method provided herein for the generation of artificial chromosomes, for example, ACes, employs a method based on a selectable marker, such as a fluorescent protein or flow cytometry, or other methods, such as fluorometry, cell imaging or fluorescence microscopy. Heterologous nucleic acids encoding other proteins that can be readily detected using are introduced into cells. For example, rDNA and DNA encoding enhanced green fluorescent protein (EGFP) can be introduced into cells, eg, A9 cells. Transfected cells can be sorted based on flow cytometry-based or other methods, such as fluorimetry, cell imaging, or properties that can be detected by fluorescence microscopy, such as fluorescence properties. For example, cells containing a fluorescent protein can be isolated from non-transfected cells using a fluorescence activated cell sorter (FACS). If sorting is performed prior to chromosome analysis of cells for the presence of artificial chromosomes, subsequent cell chromosome analysis and cells containing artificial chromosomes, such as ACes, will provide a population of transfected cells that can be enriched for artificial chromosomes. Identification and selection. For example, cells may be analyzed for an indication of chromosome segment amplification, the presence of structures that may occur in connection with amplification and new artificial chromosome formation, and / or the presence of artificial chromosomes, such as ACes. Cell analysis is typically a method of visualizing chromosomal structure as described herein and / or using techniques known to those of skill in the art, including, but not limited to, G- and C-band methods and FISH analysis. including. The analysis may use specific labeling of specific nucleic acids, for example, satellite DNA sequences, heterochromatin, rDNA sequences and heterologous nucleic acid sequences, at which amplification may be performed. Changes in the number of chromosomes during analysis of transfected cells and / or the appearance of unique chromosomal structures due to increased splitting resulting, for example, from amplification of repetitive units, aid in the identification of artificial chromosome-containing cells.
[0173]
C. Purification of artificial chromosomes by flow cytometry and preparation of DNA from flow-selected chromosomes
Artificial chromosomes were purified from host cells by flow cytometry (see de Jong (1999) Cytometry 35: 129-133). Briefly, purification was performed on a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a turbo-sort option and two Innova 306 lasers (Coherent, Palo Alto, CA). Was. Turbo-sort option adjustment is 20 lb / in ² From 60 lb / in ² Increasing the maximum system pressure, the drop drive frequency from 50,000 drops / sec to a maximum of 99000 drops / sec, and increasing the deflection surface voltage up to 6000V-8000V. Other adjustments are made to the device to accommodate higher pressures. Hoechst 35258 was excited with a first UV laser beam and the excitation was detected with FLI by using a 420 nm handpass filter. Chromomycin A3 was excited by a second laser set at 458 nm and the fluorescence was detected by FL using a 475 nm long pass filter. The power was 200 mW for both lasers. A bivariate distribution (1024 x 1024 channels) was accumulated during each sort. For whole chromosome selection, a sheath pressure of 30 lb / in ² And a 50 μm diameter nozzle was attached. The fall delay profile was performed every morning and repeated after the main plug. Instrument alignment was performed daily using 3.0 μm diameter sphero rainbow beads (SpheroTech, Libertyville, Ill.). Alignment was considered optimized when a CV of 2.0% or less was achieved for FL1 and FL4.
[0174]
Condensing agents (hexylene glycol, spermine and spermidine) were added to the sheath buffer to maintain the condensed chromosomes after sorting. Sheath buffer contains 15 nm Tris HCl, 0.1 mM EDTA, 20 mM NaCl, 1% hexylene glycol, 100 mM glycine, 20 μM spermine and 50 μM spermidine. The selected chromosomes were placed at 4 ° C, about 1 × 10 ⁶ At a concentration of chromosomes / ml, they were collected in 1.5 ml screw-cap Eppendorf tubes, which were then stored at 4 ° C.
[0175]
For the preparation of purified genomic DNA, selected chromosomal samples were transferred to 0.5% SDS, 50 mM EDTA and 100 μg / ml proteinase K, and then incubated at 50 ° C. for 18 hours. After adding 1 μg of a 20 mg / ml glycogen solution (Boehringer Mannheim) to each sample, it was extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). After centrifugation at 21000 × g for 10 minutes, the aqueous phase was transferred to a new microcentrifuge tube and re-extracted as above. 0.2 volume of 10M NH ₄ OAC, 1 μl of 20 mg / ml glycogen and 1 volume of isopropanol were added to the extracted aqueous phase twice, then vortexed and centrifuged at 30,000 × g (room temperature) for 15 minutes. The pellet was washed with 200 μl of 70% ethanol and re-centrifuged as above. The washed pellets are air dried and then 0.5-2 × 10 ⁶ Resuspended in 5 mM Tris-Cl, pH 8.0 at chromosome equivalents / μl.
[0176]
PCR on DNA prepared from chromosomal samples selected using primer sets specific for EGFP and RAPSYN, essentially as described above (see Co et al. (2000) Chromosome Research 8: 183-191). Was carried out. Briefly, 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 200 μM dNTPs, 500 nM forward and reverse primer, 1.5 mM MgCl ₂ A 50 μl PCR reaction was performed on genomic DNA equal to 10,000 or 1000 chromosomes in a solution containing 1.25 units of Taq polymerase (Ampli-Taq, Perkin-Elmer Cetus, CA). Separate reactions were performed for each primer set. The reaction conditions were as follows: one cycle of 95 ° C. for 10 minutes, then 35 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and finally 72 ° C. for 10 minutes. One cycle. Upon completion, the samples were kept at 4 ° C. until analysis by agarose gel electrophoresis using the following primers (SEQ ID NOs: 1-4, respectively):
EGFP forward primer 5'-cgtccaggagcgcaccatcttctt-3 '
EGFP reverse primer 3'-atcgcgcttctcgttggggtcttt-3 '
RAPSYN forward primer 5'-aggactgggtggcttccaactcccagacac-3 ', and
RAPSYN reverse primer 5'-agcttctcattgctgcgcgccaggttcagg-3 '.
All primers were obtained from Canadian Life Technologies (Burlington, Ontario).
[0177]
Example 2
Preparation of cationic vesicles
Vesicles were prepared at a lipid concentration of 700 nmol / ml lipid (cationic lipid / DOPE 1: 1) according to the following procedure. In a glass tube (10 ml), 350 nmol of cationic lipid (SAINT-2) is mixed with 350 nmol dioleoylphosphatidylethanolamine (DOPE) and both are mixed with an organic solvent (chloroform, methanol or chloroform / methanol 1: 1, v / V). Dioleoylphosphatidylethanolamine (DOPE; Avanti Polar Lipid, Alabaster, Alabama) forms an inverted hexagonal phase in the membrane, weakening the membrane. Other effectors that can be used are cis-unsaturated phosphatidylethanolamine, cis-unsaturated fatty acids and cholesterol. Cis unsaturated phosphatidylcholine is less effective.
[0178]
The solvent was evaporated under a stream of nitrogen (15 min at room temperature / 250 μl solvent). The remaining solvent was completely removed by drying the lipid in a desiccator under high vacuum from a vacuum pump for 15 minutes. 1 ml of ultra-pure water was added to the dry mixture. This was vigorously vortexed for about 5 minutes. The resulting solution was sonicated in an ultrasonic bath (Laboratory Surprise, Inc., New York) until a clear solution was obtained. The resulting suspension contained a population of unilamellar vesicles with a size distribution between 50-100 nm.
[0179]
Example 3
Preparation of cationic vesicles by alcohol injection
In a glass tube (10 ml), 350 nm cationic lipid (Saint-2) was mixed with 350 nmol of DOPE and both were solubilized in an organic solvent (chloroform, methanol or chloroform / methanol 1/1). The solvent was evaporated under a stream of nitrogen (15 min at room temperature / 250 μl solvent). The remaining solvent was completely removed by drying the lipid under high vacuum for 15 minutes. This was then reconstituted in 100 μl pure ethanol.
[0180]
Example 4
Transfection of beta ACes into V79-4 cell line
Transfection procedures for various transfection agents
All compounds were tested in a Chinese hamster lung fibroblast cell line (V79-4, ATCC No. CCL-39). About 17 hours prior to transfection (double cell doubling), exponentially growing cells are trypsinized and contain Dulbecco's modified Eagle's medium (Life Technologies, Burlington, Ontario) and 10% FBS (Cancella, Lexadel, Plated 250,000 cells per well in 6-well petri dishes supplemented with Ontario). At the time of transfection, the number of cells per well was estimated to be about 1 million. For transfection, the individual manufacturer's protocols for complexation to naked DNA were followed, with the exception that by varying the amount of transfection agent used, different amounts and types of DNA present, and Different ionic strengths of complex formation were reflected. Typically one million ACes (in a volume of 800 μl) were combined with the transfection agent at a wide range of concentrations (between 5 and 100 times the lowest suggested by the manufacturer). The ACes / transfection mixture was conjugated in a volume ranging from 0.8 ml to 1.9 ml for the time recommended by the manufacturer. Some manufacturers recommend adding media to the complex formation reaction. The complex mixture was then applied to recipient cells and transfection continued according to the manufacturer's protocol. Details regarding the various conditions used with the different drugs are given in Table 1.
[0181]
Transfection procedure for Superfect
Superfect was tested on a Chinese hamster lung fibroblast cell line (V79-4, ATCC No. CCL-39). About 17 hours prior to transfection (double cell doubling), exponentially growing cells are trypsinized and contain Dulbecco's modified Eagle's medium (Life Technologies, Burlington, Ontario) and 10% FBS (Cancella, Lexadel, Plated 250,000 cells per well in 6-well petri dishes supplemented with Ontario). One million ACes in 800 μl of sorted buffer complexed with 10 μl of Superfect reagent. The complex was incubated at room temperature for 10 minutes. At the time of transfection, the number of cells per well was estimated to be about 1 million. The medium was removed from the wells and 600 μl of DMEM and 10% FBS were added. Superfect: ACes complex was added dropwise to the wells and incubated at 37 ° C. for 3 hours. After incubation, the transfected cells were trypsinized, transferred to a 15 cm dish containing 25 ml of DMEM and 10% FBS, and allowed to bind for 24 hours. Twenty-four hours later, selective medium containing 0.7 mg / ml hygromycin B was added to each well. The selection medium was changed every 2-3 days. After 10-12 days, colonies were screened for beta-galactosidase expression and / or FISH for intact chromosome detection.
[0182]
Application example of Chromos index measurement
About 1 × 10 ⁶ Of V79-4 cells complexed with delivery factors (i.e., Lipofectamine plus and Lipofectamine or Superfect) ⁶ Was transfected with IdUrd-labeled ACes. The transfected cells were then fixed in ethanol. The fixed cells were denatured and exposed to a FITC-conjugated antibody that specifically binds to BrdU / IdUrd-labeled nucleic acids.
[0183]
The percentage of transfected cells containing IdUrd-labeled ACes was measured using flow cytometry and collecting FITC fluorescence. Upon accumulation of the data, a bivariate channel distribution was formed showing forward scatter versus green fluorescence (IdUrd-FITC). Visual inspection of the histograms of the negative control cells established the fluorescence level at which the cells were determined to be positive, with the gate for the negative cells set such that 1% appeared in the positive area.
[0184]
The number of cells recovered 24 hours after transfection was determined by counting aliquots using a Coulter counter. To determine the control plate efficiency of the recipient cell line, untreated cells are plated at 600-1000 cells per 10 cm Petri dish in growth medium to form an average colony for 5 cell cycles or 50 cells 5% CO at 37 ° C up to ₂ Placed in the incubator. At this point, the number of viable colonies was determined. If the CPE exceeded 0.1-0.2, the treated cells were seeded at 1000 cells. When CPE was low, seeding density was increased to 5000-50,000 cells per dish.
[0185]
Example 5
Ultrasonic transfer transfection of LMTK (-) cells with lipofectamine
LM (tk-) cells were incubated at 37 ° C., 5% CO 2 in DMEM containing 4500 mg / L D-glucose, L-glutamine, pyridoxine hydrochloride and 10% fetal bovine serum. ₂ And cultured. Twenty-four hours before use, corner wells of a 12-well dish were seeded with 200,000 cells per dish (to ensure that there was no interference from ultrasound from other wells).
[0186]
By counting GFP chromosomes, about 1 × 10 ⁶ ACes have been demonstrated. The chromosomes were resuspended in tubes by rapid movement. 10 μl of the chromosome suspension was removed and mixed with an equal volume of 30 mg / ml PI (propidium iodide) dye. 8 μl of the stained chromosomes were loaded into a Petrov-Hauser counting chamber and the chromosomes were counted.
[0187]
The medium was removed from the cells and the cells were washed twice with HBSS (phenol-free, Gibco BRL) warmed to 37 ° C. 500 μl of warmed HBSS was added to each well of cells (1 μl) and Lipofectamine (Gibco BRL) was added to each well. The plate was then sealed with parafilm tape and gently shaken at 20 rpm for 30 minutes at room temperature (stagger plate-10 minutes for ease of operation).
[0188]
After incubation, Ultrasound Gel (Ather-Sonic Generic Ultrasound Transmission Gel, Pharmaceutical Innovations, Inc., Newark, NJ) was applied to a 2.5 cm sonoporator head. Ultrasound was passed through the bottom of the plate for 60 seconds with an ImaRX sonoporator 100 at 2.0 watts / cm. ² Applied to the cells at the output energy. After ultrasonication of the wells, seeded cells (2 × 10 ⁵ 200 μl of GFP ACes per chromosome or sheath buffer (15 nM Tris HCl, 0.1 mM EDTA, 20 mM NaCl, 1% hexylene glycol, 100 mM glycine, 20 μM spermine and 50 μM spermidine) per chromosome immediately Added to wells. (Repeat until all samples on the plate that require ultrasound have been processed). The plate was then resealed with parafilm tape and gently shaken (20 rpm) at room temperature for 1 hour.
[0189]
After incubation, 1 ml (1 × of penicillin and streptomycin from DMEM, 4500 mg / L D-glucose, L-glutamine and pyridoxine hydrochloride, 10% fetal bovine serum, and 10,000 units / ml penicillin and 10,000 mg / ml streptomycin) Solution, containing 100 × stock solution) was added to each well and the cells were incubated at 37 ° C. for 18-24 hours.
[0190]
The cells in the plate were then washed with antibiotic-containing medium and 2 ml of medium was placed in each well. Cells are transfected / 5% CO 2 up to 48 hours after acoustic perforation ₂ , 37 ° C. The cells were then trypsinized and 1x10 in DMEM. ⁶ And reanalyzed by flow cytometry.
[0191]
Results: Flow analysis was performed on a FACS Vantage (BDIS, San Jose, CA) equipped with a turbo-sort option and two Innova 305 lasers (Coherent, Palo Alto, CA). GFP signal excitation was 488 nm and emission was detected on FL1 using a 500 nm long pass filter. Analysis of transfected cells generated a population of GFP positive cells ranging from 13-27%. The non-acoustic perforation control value was 5%.
[0192]
Example 6
Ultrasonic transmission transfection with Saint-2
A. Ultrasonic transfer transfection of CHO-KI cells with Saint-2
CHO-KI cells were cultured in CHO-S-SFM2 medium (Gibco BRL, Paisley, UK) at 37 ° C., 5% CO 2. ₂ And cultured. 2 × 10 ⁵ And 5 × 10 ⁵ Cells in between were plated on sterile glass slides in 12-well plates 24 hours before use.
[0193]
Transfection of cells was performed as follows. The medium was removed from the cells, and the cells were washed twice with HBSS (Hank's balanced salt solution without phenol red (Gibco BRL, UK)) at 37 ° C. Then, 500 μl of HBSS at 37 ° C. was added per well, and then 10 μl of a freshly prepared vesicle solution (prepared in Example 2) was added to a final concentration of 23.3 nmol / ml.
[0194]
Alternatively, the medium was removed from the cells and the cells were washed twice with HBSS. 500 μl of 37 ° C. HBSS / lipid solution was added to each well. An HBSS / lipid solution was prepared by adding 1 μl of the ethanolic lipid solution (prepared as described above) to 500 μl of HBSS with vigorous vortexing. The plate was then sealed with parafilm tape and gently shaken at room temperature for 30 minutes. After the incubation, the ultrasonic wave was applied at 0.5 watt / cm. ² Cells were applied through the bottom of the plate for 60 seconds with an output energy of. Ultrasound was transmitted by an ultrasonic gel (Aquasonic 100, Parker, NJ) between the transducer and the plate. Ultrasound was applied with an ImaRX sonoporator 100. Immediately after ultrasonic application, seeded cells (2 × 10 ⁵ ~ 5 × 10 ⁵ ) (Prepared in Example 1) and one GFP chromosome was added. The plate was then resealed and gently shaken at room temperature for 1 hour. After incubation, 1 ml of medium (CHO-S-SFM2, containing 10% fetal bovine serum, 10000 μg / ml penicillin and 10000 μg / ml streptomycin (Gibco BRL, Paisley, UK)) was added to each well and the cells were incubated at 37 ° C. Incubated for 24 hours. The cells were then washed with medium, 1 ml of medium was added and the cells were incubated for an additional 24 hours at 37 ° C. The detection of the expressed gene was then evaluated microscopically or the detection of the introduced chromosome was evaluated by FISH analysis. Negative control tests were performed in the same manner, except that no chromosomes were added to the cells.
[0195]
result
After transfection, using visual inspection, 30% of the cells remained on the glass slides, of which 10% were positive for green fluorescent protein expression after 48 hours (3% of the original population). After two weeks in culture, FISH was performed on the cells, and 1.4% of the cells contained intact artificial chromosomes.
[0196]
B. Ultrasonic transmission transfection of Hep-G2 cells with Saint-2
Hep-G2 cells were incubated at 37 ° C., 5% CO 2 in DMEM supplemented with 4500 mg / l glucose, pyridoxine / HCl, 10% fetal bovine serum, 10,000 μg / ml streptomycin and 1000 μg / ml penicillin. ₂ And cultured. 2 × 10 ⁵ And 5 × 10 ⁵ Cells in between were plated on sterile glass slides in 12-well plates 24 hours before use.
[0197]
Cells were transfected with the GFP chromosome using the procedure of Example 6A, except replacing the CHO-KI medium with Hep-G2 medium.
[0198]
result
After transfection, 30% of the cells remained on the glass slide. 80% of these cells were positive for green fluorescent protein expression.
[0199]
C. Ultrasonic transfection of A9 cells with Saint-2
A9 cells were incubated at 37 ° C. in DMEM (Gibco BRL, Paisley, UK) supplemented with 4500 mg / l glucose, pyridoxine / HCl, 10% fetal bovine serum, 10,000 μg / ml streptomycin and 10,000 μg / ml penicillin. 5% CO ₂ And cultured. 2 × 10 ⁵ And 5 × 10 ⁵ Cells in between were plated on sterile glass slides in 12-well plates 24 hours before use.
[0200]
Cells were transfected with the GFP chromosome using the procedure of Example 6A, except replacing the CHO-KI medium with A9 medium.
[0201]
result
After transfection, 30% of the cells remained on glass slides, of which 50% were positive for green fluorescent protein expression.
[0202]
Example 7
Delivery of ACes to synovial cells, skeletal myofibroblasts and dermal fibroblasts
Mammals comprising a primary murine pericentric heterochromatin, comprising a reporter gene (lacZ) and a hygromycin B selectable marker gene, prepared as described in US Patent Nos. 6,025,155 and 6077697 and PCT Application Publication No. WO / 9740143 ( Murine) ACes artificial chromosomes (染色体 60 Mb) were delivered to primary rat fibroblast-like synoviocytes, rat dermal fibroblasts and rat skeletal myofibroblast line (L8 cells; ATCC accession number CRL-1769). Prior to delivery, ACes were labeled with iododeoxyuridine (IdUrd) as described herein.
[0203]
Cell preparation
Primary fibroblast-like synovial cells and rat skin fibroblasts were obtained from rats using standard methods [eg, Aupperle et al. (1999) J. Immunol. 163: 427-433 and Alvaro-Garcia et al. al. (1990) J. Clin. Invest. 86: 1790]. Such methods include the isolation of synovial cells by removing skin and muscle from the rodent of a rodent animal, generally, and then mincing the knee joint tissue. The minced tissue is then incubated with collagenase, filtered through a nylon mesh and washed thoroughly. Cells can be cultured overnight, at which time non-adherent cells are removed. Adherent cells can be cultured and passaged by replating at a constant dilution when the culture reaches confluent growth. Cells are plated at a rate of 50,000-75,000 cells per 6-well dish in medium containing low glucose DMEM, 1-glutamine, penicillin / streptomycin and 20% FBS. Cells are grown for 3-5 days at 37 ° C. in 5% CO 2 to approximately 80% confluence or 500,000 cells per well. ₂ Cultured in an incubator.
[0204]
Transfection of cells with ACes
One million IdUrd-labeled ACes were complexed with 2, 5 or 10 μl of Superfect (Qiagen) or Lipofectamine Plus (Life Technologies; Gibco) as follows. Complex formation with Superfect was performed at room temperature for 10 minutes. For complex formation with Lipofectamine plus, the indicated amount of plus reagent was added to 1 million ACes and allowed to complex for 15 minutes at room temperature. Next, the indicated amount of lipofectamine was added to 200 μl of low glucose DMEM (without FBS) and combined with the ACes / plus reagent complex for 15 minutes at room temperature. The complexed ACes was then added dropwise to the cells in 600 μl of medium (approximately 1.4 ml final volume). 5% CO ₂ After 3 hours at 37 ° C. in an incubator, a total of 3 ml of culture medium (low glucose DMEM, 1-glutamine, penicillin / streptomycin and 20% FBS) was added. After 24-48 hours, the cells were trypsinized to form a single cell suspension, the supernatant was removed by centrifugation and then fixed in cold 70% ethanol for a minimum of 1 hour. Aliquots of fixed cells were set aside for microscopic analysis.
[0205]
ACes FITC-conjugated antibody labeling
Following transfection, ACes were labeled with a FITC-conjugated antibody that specifically binds BrdU- or IdUrd-labeled nucleic acids, and cells were analyzed by FACs for FITC fluorescence and microscopic staining. Fixed cells were denatured in 2N HCl and 0.5% Triton-X for 30 minutes at room temperature. After denaturation induction, cells were neutralized by a series of washing steps at 4 ° C. Samples were resuspended in PBS and 4% FBS or BSA and 0.1% Triton-X (blocking buffer) for a minimum of 15 minutes to minimize background staining. The cells were then sedimented and exposed to FITC-conjugated antibody for 2 hours at room temperature. After washing the cells with blocking buffer, samples were prepared for flow cytometric analysis. Samples for microscopic analysis were dried on slides and the staining protocol was followed, except for BrdU / IdUrd antibody, which was diluted 1/5 and exposed to cells for 24 hours.
[0206]
result
Delivery of intact ACes was detected within 24-48 hours after transfection. The number of cells recovered 24 hours after transfection was determined by counting aliquots using a Coulter counter. To determine the control plating efficiency of the recipient cell line or the plating efficiency of transfected cells, cells were plated in growth medium at a rate of 1000-10000 cells per 10 cm Petri dish and 5 days CO2 at 37 ° C for 10 days. ₂ Placed in the incubator. At that point, the number of viable colonies was determined. Normalized plate efficiency was calculated as described herein.
[0207]
When Superfect was used as the delivery agent, the percent delivery to fibroblast-like synovial cells as measured by flow cytometry ranged from ２４24% to .66.3%. The% normalized plate efficiency when using 2 μl of Superfect was ３６36% and 〜16% when using 5 μl Superfect. Higher doses of Superfect are associated with toxicity and higher numbers of ACes per cell than lower doses. When lipofectamine plus was used as the delivery agent, the percent delivery to fibroblast-like synovial cells as measured by flow cytometry ranged from １１11% to ２７27%, with increasing doses of the agent delivering The percentage has increased.
[0208]
L8 and rat dermal fibroblasts (RSF) transfected with ACes were cultured under hygromycin B selection and analyzed for lacZ expression. In this example, the hygromycin selection gene was included in the ACes, but there are also a variety of other selectable marker genes that may be involved in introducing heterologous nucleic acids into cells where it is desirable to include the gene. Can be used. Such selection systems are known to those skilled in the art. Selection of a selectable marker gene can take into account, for example, the level of toxicity of the selection agent to the host cell for transfection. Identification of a suitable selectable marker gene is performed using routine procedures using the guidance applied herein.
[0209]
Clones of L8 and RSF cells expressing lacZ were identified. These results demonstrate that IdUrd-labeled ACes can be efficiently delivered to primary cells and cell lines, and that the transgene contained in ACes is expressed in transfected cells.
[0210]
Example 8
Ex vivo transfer of reporter gene to rat joint
To examine the introduction of the heterologous gene into the in vivo environment and the expression of the gene in vivo, L8 cells transfected with the ACes described above were injected into the ankle joint of a rat suffering from adjuvant-induced arthritis. On day 0, adjuvant induction of arthritis was performed in Lewis rats. Methods for inducing adjuvants in animal models are known in the art [eg, Kong et al. (1999) Nature 4023: 304-309]. In one example of a protocol for adjuvant induction of arthritis, Lewis rats were immunized at the base of the tail on day 0 with 1 mg of Mycobacterium tuberculosis H37RA (Difco, Detroit, Michigan) in 0.1 ml mineral oil. Become Forefoot swelling typically begins around day 10.
[0211]
On day 12, transfected L8 cells into the right ankle joint (~ 0.7 x 10 ⁶ Cells) or untransfected control cells were injected intra-articularly. On day 14, rats were sacrificed for joint analysis for the presence of implanted transfected L8 cells.
[0212]
Different tissues of sacrificed rats were examined for the presence of lacZ mRNA by RT-PCR analysis. Total mRNA was extracted from the tissue and RT-PCR was performed using primers specific for the lacZ gene. Amplification products were detected only in the synovium but not in other tissues (liver, kidney, heart, spleen and lung). In addition, synovium from sacrificed rats was analyzed by in situ enzymatic staining and X-gal staining for β-gal activity. After snap freezing of the synovium, 8 μm and 20 μm sections were cut, counterstained with Mayer's hematoxylin, and analyzed for blue staining of the cells. Staining was detected from the synovium injected with ACes-transfected L8 cells, but not the synovium injected with non-transfected cells. These results demonstrate the potential of effective ex vivo gene transfer in a rat adjuvant arthritis model using ACes containing the marker gene, a potential treatment of arthritis and other connective tissue diseases using ACes as a non-viral vector for gene therapy. ing.
[0213]
Example 9
Flow cytometry technique for measurement of artificial chromosome delivery
The production cell line (see Example 1) was incubated with 0.168 μg / ml hygromycin B (Calbiochem, San Diego, Calif.) And 10% fetal bovine serum (Canberra, Lexdale, Ontario) in MEM medium (Gibco BRL). Cultured in Iododeoxyuridine or bromodeoxyuridine was added directly to the culture medium of the production cell line (CHO E42019) in the exponential growth phase. Stock iododeoxyuridine was prepared in Tris-based pH 10 and bromodeoxyuridine stock was prepared in PBS. A final concentration of 0.05-1 μM was used for a 20-24 hour continuous labeling of 5-50 μM with a 15 minute pulse. Twenty-four hours later, exponentially growing cells were mitotically blocked with corticin (1.0 μg / ml) for 7 hours before harvesting. Chromosomes were then isolated and stained with Hoechst 33258 (2.5 μg / ml) and chromomycin A3 (50 μg / ml). Purification of artificial chromosomes was performed using a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA). Chromomycin A3 was excited with a first laser set at 457 nm and emission was detected using a 475 nm longpass filter. Hoechst was excited by a second UV laser and the emission was detected using a 420/44 nm bandpass filter. The output of both lasers was 150 mW. A bivariate distribution indicating cell karyotype was accumulated from each sort. ACes were gated from other chromosomes and selected. Condensing agents (hexylene glycol, spermine and spermidine) were added to the sheath buffer to maintain the condensed intact chromosomes after sorting. The IdU labeling index of the selected chromosomes was measured with a microscope. Aliquots (2-10 μl) of the selected chromosomes were fixed in 0.2% formaldehyde solution for 5 minutes and then dried on uncontaminated microscope slides. Microscope samples were fixed with 70% ethanol. Air-dried slides were denatured in coplin jar with 2N HCl for 30 minutes at room temperature and washed 2-3 times with PBS. Non-specific binding was blocked with PBS and 4% BSA or serum for a minimum of 10 minutes. A 1/5 dilution of the FITC-conjugated IdU / BrdU antibody (Becton Dickinson) in a final volume of 60-100 μl was applied to the slides. Plastic strips, Durra seals (Diversified Biotech, Boston, Mass.) Were placed over the slides and the slides were kept in a humidified shielded box at 4% C in the dark for 8-24 hours. DAPI (Sigma) 1 μg / ml in VectorShield was used as counterstain. Fluorescence was detected using a Zeiss Axioplan 2 microscope equipped for epifluorescence. A minimum of 100 chromosomes were scored to determine% labeling. Unlabeled chromosomes were used as negative controls.
[0214]
The day before transfection, V79-4 (Chinese hamster lung fibroblasts) cells were trypsinized and incubated in 4 ml of DMEM (Dulbecco's Modified Eagle's Medium, Life Technologies) and 10% FBS (Cancella, Lexdale, Ontario). Plated at a rate of 250,000 cells in a well Petri dish. The protocol was modified for use in the LM (tk-) cell line by plating 500,000 cells. 1x10 sorted in ~ 800 [mu] l sorting buffer ⁶ ACes were supplemented with lipid or dendrimer reagents. Table 1 shows an example of the protocol modification. The chromosome and the transfection agent were mixed gently. The complex was dropped onto the cells and the plate was vortex mixed. Plates are given 5% CO for a defined transfection period ₂ It was kept at 37 ° C. in the incubator. The volume in the wells was then made up to 4-5 ml with DMEM and 10% FBS. 5% CO ₂ It was left at 37 ° C. for 24 hours in an incubator. Transfected cells were trypsinized. Samples to be analyzed for IdU-labeled chromosome delivery were fixed in cold 70% ethanol, stored at -20 ° C, and prepared for IdU antibody staining. Samples to be cultured for colony selection were counted and then introduced in duplicate into 10 cm dishes at a density of 10,000 and 100,000 cells, and the remaining cells were placed in 15 cm dishes. Twenty-four hours later, selective medium containing DMEM and 10% FBS with 0.7% mg / ml hygromycin B, # 40051 (Calbiochem, San Diego, CA) is added. The selection medium is changed every 2-3 days. This hygromycin B concentration kills wild type cells after 7 days of selection. On days 10-14, colonies were expanded and then screened for intact chromosome transfer by FISH and assayed for beta-galactosidase expression.
[0215]
Table 1: Delivery transfection protocol
[Table 2]

[0216]
IdU antibody labeling
A standard BrdU staining flow cytometry protocol (Gratzer et al. Cytometry (1981); 6: 385-393) was used, except for any modifications during the neutralization step, the presence of detergent during denaturation, and the composition of the blocking buffer. The sample is centrifuged at 300 g for 7-10 minutes between each step and the supernatant is removed. A sample of 1 to 2 million cells is fixed in cold 70% ethanol. The cells are then denatured for 30 minutes at room temperature in 1-2 ml of 2N HCL + 0.5% Triton X. Wash the sample 3-4 times with cold DMEM until the indicator is neutral. Final wash with cold DMEM + 5% FBS. After addition of blocking / permeabilization buffer containing PBS, 0.1% Triton X and 4% FBS for 10-15 minutes, the samples are sedimented by centrifugation. Add 20 μl of IdU / BrdU FITC conjugate B44 clone antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) to the pellet and leave for 2 hours at room temperature in the dark with stirring every 30 minutes. Wash cells with blocking / permeabilization buffer and resuspend in PBS for flow analysis.
[0219]
Flow cytometric detection of fluorescent IdUrd-labeled ACes
The percentage of transfected cells containing IdU-labeled ACes was measured using flow cytometry with the argon laser changed to 488 nm at 400 mW. FITC fluorescence was collected through a standard FITC 530/30 nm bandpass filter. Debris and doublets were eliminated by gating the cell population based on side scatter versus forward scatter. The bivariate channel distribution formed by accumulating the data (15000 events) showed forward scatter versus green fluorescence (IdU-FITC). The fluorescence level at which the cells were determined to be positive was established by visual inspection of the histogram of the negative control cells so that about 1% appeared in the positive area.
[0218]
result:
Table 2 shows the results of transfection delivery of IdU-labeled ACes.
Table 2
[Table 3]

[0219]
The invention is to be limited only by the appended claims, as modifications are readily apparent to those skilled in the art.

Claims (231)

A method for introducing a large nucleic acid molecule into a cell,
(a) exposing the nucleic acid molecule to a delivery agent;
(b) exposing the cells to a delivery agent; and
(c) contacting the cell with the nucleic acid molecule to deliver the nucleic acid molecule to the cell, wherein steps (a) to (c) are performed sequentially and sequentially or simultaneously, provided that the delivery factor is A method wherein, if energy, it does not apply to the nucleic acid molecule and after contact of the nucleic acid molecule with the cell it does not apply to the cell. 2. The method of claim 1, wherein the nucleic acid molecule is exposed to an agent that increases contact between the nucleic acid molecule and the cell, and the cell is exposed to an agent that increases cell permeability. 2. The method of claim 1, wherein the nucleic acid molecule is larger than about 0.6 megabases. 2. The method of claim 1, wherein the nucleic acid molecule is larger than about 1 megabase. 2. The method of claim 1, wherein the nucleic acid molecule is larger than about 5 megabases. 2. The method of claim 1, wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, or a fragment of a chromosome larger than about 0.6 megabases or naked DNA larger than about 0.6 megabases. 2. The method according to claim 1, wherein the nucleic acid molecule is an artificial chromosome. The method according to claim 1, wherein the nucleic acid molecule is an artificial chromosome expression system (ACes). 2. The method of claim 1, wherein the nucleic acid molecule is exposed to the delivery agent in vitro, ex vivo or in vivo. 2. The method of claim 1, wherein the contacting of the cell with the nucleic acid molecule exposed to the delivery agent is performed in vitro, ex vivo or in vivo. The exposure of the nucleic acid to the delivery agent is performed by mixing the nucleic acid with the delivery agent, and the exposure of the cell to the agent that enhances permeability comprises the application of ultrasound or electrical energy to the cell. 3. The method according to 1 or 2. 2. The method of claim 1, wherein the delivery agent comprises a cationic compound. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 13. The method of claim 12, wherein the method is selected from the group consisting of pyridinium surfactants. A composition wherein the delivery factor comprises one or more cationic compounds, wherein the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) , dioleyl phosphatidyl ethanolamine (DOPE), 2,3-dioleyloxy-N-[2 (spermine carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate (DOSPA), C ₅₂ H _{106 N 6 O 4 · 4CF 3} CO 2 H, C 88 H 178 N 8 O 4 S 2 · 4CF 3 CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O _{3 · 4CF 3 CO 2 H,} C 55 H 116 N 8 O 2 · 6CF 3 CO 2 H, C 49 H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO ₂ H, C ₁₀₀ H _{206 N 12 O 4 S 2 ·} 8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 · 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C ₄₃ H ₈₈ N ₄ O ₃ .2CF ₃ CO ₂ H, and (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride, The method according to claim 12. 2. The method of claim 1, wherein the delivery factor is energy. 17. The method of claim 15, wherein the cells are treated with energy. The method of claim 15, wherein the energy is ultrasonic energy. 18. The method of claim 17, wherein the ultrasonic energy is applied to the cells for about 30 seconds to about 5 minutes. 18. The method of claim 17, wherein the ultrasonic energy is applied as a single pulse. 18. The method of claim 17, wherein the ultrasonic energy is applied as two or more intermittent pulses. 21. The method of claim 20, wherein the intermittent pulses of ultrasonic energy are applied for substantially the same amount of time and at substantially the same energy level. 21. The method of claim 20, wherein the energy level, duration of application, or energy level and duration of application vary for intermittent pulses. 12. The method of claim 11, wherein the cells are contacted with a cavitation compound prior to applying ultrasonic energy to the cells. 18. The method of claim 17, wherein the cells are contacted with a cavitation compound prior to applying ultrasonic energy to the cells. 12. The method of claim 11, wherein the permeability enhancing agent comprises the application of electrical energy. (a) applying ultrasound or electrical energy to the cells, and
2. The method of claim 1, comprising (b) delivering the nucleic acid molecule to the cell by contacting the cell with a mixture of the nucleic acid molecule and a delivery agent after termination of the application of ultrasound or electrical energy. 27. The method of claim 26, wherein the agent is a cationic compound. 26. The method of claim 25, wherein the energy is ultrasound. 29. The method of claim 28, wherein the cells are contacted with a cavitation compound prior to applying ultrasonic energy. The method according to claim 1, wherein the cell is a plant cell or an animal cell. 2. The method of claim 1, wherein the cells are selected from the group consisting of nuclear transfer donor cells, stem cells, primary cells, cells from immortalized cell lines, and cells capable of developing a particular organ. 2. The method of claim 1, wherein the cells are selected from the group consisting of primary cells, immortalized cells, embryonic cells, stem cells, transformed cells and tumor cells. 2. The method of claim 1, wherein the cells are selected from the group consisting of nuclear transfer donor cells, stem cells, and cells capable of developing a particular organ. (a) contacting the cell with the delivery agent in the absence of the nucleic acid molecule, and applying ultrasonic or electrical energy to the cell, provided that the contacting and applying occur sequentially or simultaneously;
(b) A method for delivering a nucleic acid molecule to a cell, comprising delivering the nucleic acid molecule to the cell by contacting the cell with the nucleic acid molecule. 35. The method of claim 34, wherein the delivery agent comprises a cationic compound. A composition wherein the delivery factor comprises one or more cationic compounds, wherein the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) , dioleyl phosphatidyl ethanolamine (DOPE), 2,3-dioleyloxy-N-[2 (spermine carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate (DOSPA), C ₅₂ H _{106 N 6 O 4 · 4CF 3} CO 2 H, C 88 H 178 N 8 O 4 S 2 · 4CF 3 CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O _{3 · 4CF 3 CO 2 H,} C 55 H 116 N 8 O 2 · 6CF 3 CO 2 H, C 49 H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO ₂ H, C ₁₀₀ H _{206 N 12 O 4 S 2 ·} 8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 · 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C ₄₃ H ₈₈ N ₄ O ₃ .2CF ₃ CO ₂ H, and (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride; 35. The method of claim 34. 35. The method of claim 34, wherein the delivery agent is (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 35. The method of claim 34, wherein the nucleic acid molecule is greater than about 1 megabase. 35. The method of claim 34, wherein the nucleic acid molecule is selected from the group consisting of artificial chromosomes, artificial chromosome expression systems (ACes) and native chromosomes or fragments thereof that are at least greater than about 0.6 megabases. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 36. The method of claim 35, wherein the method is selected from the group consisting of a pyridinium surfactant. 35. The method of claim 34, wherein the energy is ultrasound. Ultrasonic energy, between about 0.1 and 1 watt / cm ^2, is applied for about 30 seconds to about 5 minutes cells, The method of claim 41, wherein. 42. The method of claim 41, wherein the ultrasonic energy is applied as one continuous pulse or as two or more intermittent pulses. 44. The method of claim 43, wherein the pulse is an intermittent pulse, and wherein the intermittent pulse of ultrasonic energy is applied for substantially the same amount of time at substantially the same energy level. 44. The method of claim 43, wherein the pulse is an intermittent pulse, and for the intermittent pulse, the energy level, duration of application, or energy level and duration of application vary. 35. The method of claim 34, wherein the cells are contacted with a cavitation compound prior to applying ultrasonic energy. 35. The method of claim 34, wherein the cells are selected from the group consisting of embryonic stem cells, nuclear transfer donor cells, stem cells and cells capable of developing a particular organ. A method of delivering a nucleic acid molecule to a cell in a subject, comprising:
(a) administering a delivery agent to the subject in the absence of the nucleic acid molecule;
(b) applying the ultrasound or electrical energy to the subject after administering the agent, and
and (c) delivering the nucleic acid molecule to the cells by administering the nucleic acid molecule to the subject after the application of the ultrasound or electric energy is terminated. 49. The method of claim 48, wherein the agent is a cationic compound. 49. The method of claim 48, wherein the administration of the delivery factor and the nucleic acid molecule and the application of energy are directed directly to a local area of the subject where the cells are present. A composition wherein the delivery factor comprises one or more cationic compounds, wherein the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) , dioleyl phosphatidyl ethanolamine (DOPE), 2,3-dioleyloxy-N-[2 (spermine carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate (DOSPA), C ₅₂ H _{106 N 6 O 4 · 4CF 3} CO 2 H, C 88 H 178 N 8 O 4 S 2 · 4CF 3 CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O _{3 · 4CF 3 CO 2 H,} C 55 H 116 N 8 O 2 · 6CF 3 CO 2 H, C 49 H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO ₂ H, C ₁₀₀ H _{206 N 12 O 4 S 2 ·} 8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 · 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C ₄₃ H ₈₈ N ₄ O ₃ .2CF ₃ CO ₂ H, and (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride; 49. The method of claim 48. 51. The method of claim 50, wherein the region of interest is selected from the group consisting of a joint, a tumor, an organ, and a tissue. 49. The method of claim 48, wherein the nucleic acid molecule is greater than about 1 megabase. 49. The method of claim 48, wherein the nucleic acid molecule is larger than about 5 megabases. 49. The method of claim 48, wherein the nucleic acid molecule is selected from the group consisting of artificial chromosomes, satellite artificial chromosomes, and native chromosomes or fragments thereof. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 50. The method of claim 49, wherein the method is selected from the group consisting of pyridinium surfactants. 49. The method of claim 48, wherein the energy is ultrasound, and wherein the cavitation compound is administered to the subject before administering the ultrasound energy to the subject. A method for delivering a large nucleic acid molecule to a cell,
(a) contacting a nucleic acid molecule with a composition comprising a cationic lipid,
(b) A method wherein the nucleic acid molecule is contacted with a cell, wherein steps (a) and (b) are performed simultaneously or sequentially. The cationic lipid composition comprises 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and dioleyl phosphatidylethanolamine (DOPE) 59. The method of claim 58, comprising: 59. The method of claim 58, wherein the nucleic acid molecule is greater than 0.6 megabase pairs in size. 59. The method of claim 58, wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, a fragment of a chromosome, or naked DNA. 59. The method of claim 58, wherein said cells are selected from the group consisting of plant cells and animal cells. 59. The method of claim 58, wherein the cells are selected from the group consisting of primary cells, immortalized cells, embryonic cells, stem cells, transformed cells and tumor cells. 59. The method of claim 58, wherein the nucleic acid molecule is contacted with the cell in vitro, ex vivo or in vivo. A method of delivering a nucleic acid molecule to a cell in a subject, comprising:
(a) mixing the nucleic acid molecule with a delivery agent; and
(b) A method comprising administering a mixture of a nucleic acid molecule and an agent to a subject, thereby delivering the nucleic acid molecule to cells to a greater extent than using the agent or energy alone. 66. The method of claim 65, wherein the agent is a cationic compound. 67. The method of claim 66, wherein the mixture of the cationic compound and the nucleic acid molecule is applied topically. 68. The method of claim 67, wherein the mixture is applied to a joint, tumor, organ, or tissue. 66. The method of claim 65, wherein the nucleic acid molecule is greater than about 1 megabase. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 67. The method of claim 66, wherein the method is selected from the group consisting of a pyridinium surfactant. 66. The method of claim 65, wherein the nucleic acid molecule is selected from the group consisting of artificial chromosomes, artificial chromosome expression systems (ACes), natural chromosomes, or fragments thereof that are at least greater than about 0.6 megabases. 66. The method of claim 65, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least about 0.6 megabases. A method of delivering a nucleic acid molecule to a cell in a subject, comprising:
(a) applying ultrasound or electrical energy to the subject, and
(b) delivering the nucleic acid molecule to the cells by administering the nucleic acid molecule and a delivery agent to the subject at the end of the application of the ultrasound or electrical energy,
In that case, the delivery agent and the nucleic acid are administered continuously or as a single composition. 74. The method of claim 73, wherein the nucleic acid molecule is administered to the cell by administering the nucleic acid molecule after termination of the application of ultrasound or electrical energy and administering a delivery agent. 74. The method of claim 73, wherein the cavitation compound is administered to the subject prior to applying the ultrasonic energy. 78. The method of claim 75, wherein the energy is ultrasound, and wherein the cavitation compound is administered to the subject prior to applying the ultrasound energy. 74. The method of claim 73, wherein the agent is a cationic compound. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 78. The method of claim 77, wherein the method is selected from the group consisting of pyridinium surfactants. A composition wherein the delivery factor comprises one or more cationic compounds, wherein the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) , dioleyl phosphatidyl ethanolamine (DOPE), 2,3-dioleyloxy-N-[2 (spermine carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate (DOSPA), C ₅₂ H _{106 N 6 O 4 · 4CF 3} CO 2 H, C 88 H 178 N 8 O 4 S 2 · 4CF 3 CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O _{3 · 4CF 3 CO 2 H,} C 55 H 116 N 8 O 2 · 6CF 3 CO 2 H, C 49 H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO ₂ H, C ₁₀₀ H _{206 N 12 O 4 S 2 ·} 8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 · 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C ₄₃ H ₈₈ N ₄ O ₃ .2CF ₃ CO ₂ H, and (1-methyl-4- (1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride; 74. The method according to claim 73. A method of delivering a nucleic acid molecule to a cell in a subject, comprising:
(a) applying ultrasound or electrical energy to the subject, and
(b) delivering the nucleic acid molecule to the cells by administering the nucleic acid molecule to the subject at the end of the application of ultrasound or electrical energy. 81. The method of claim 80, wherein the energy is ultrasound, and wherein the cavitation compound is administered to the subject prior to applying the ultrasound energy. 81. The method of claim 80, wherein the nucleic acid molecule is an artificial chromosome expression system (ACes). 81. The method of claim 80, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least about 0.6 megabases. An ex vivo gene therapy method,
(a) delivering a nucleic acid molecule to a cell in vitro by contacting the cell in the absence of the nucleic acid molecule with a composition comprising a compound that delivers the nucleic acid molecule to the cell;
(b) applying ultrasound or electrical energy to the cells that have been first contacted with the compound;
(c) delivering the nucleic acid molecule to the cell by contacting the cell with the nucleic acid molecule at the end of the application of ultrasound or electrical energy; and
(d) a method comprising the step of introducing cells into a subject. 85. The method according to claim 84, wherein the compound is a cationic compound. The cationic compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 86. The method of claim 85, wherein the method is selected from the group consisting of pyridinium surfactants. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N 8 _{O 4 S 2 · 4CF 3 CO} 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · 6CF _{3 CO 2 H, C 49 H} 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 O _{4 S 2 · 8CF 3 CO 2} H, C 16 _{2 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 86. The method according to claim 85, wherein the method is selected from the group consisting of-(1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 85. The method of claim 84, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least greater than about 0.6 megabases. 85. The method according to claim 84, wherein the cells are plant cells or animal cells. 85. The method of claim 84, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell line, and a cell capable of developing a particular organ. 85. The method of claim 84, wherein the cells are selected from the group consisting of immortalized cells, embryonic cells, stem cells, transformed cells, and tumor cells. An ex vivo gene therapy method,
(a) delivering a nucleic acid molecule to a cell in vitro by contacting the cell with the nucleic acid molecule mixed with a composition comprising a compound that delivers the nucleic acid molecule to the cell;
(b) delivering the nucleic acid molecule to the cell to a greater extent than in the presence of the compound alone by applying ultrasound or electrical energy to the cell that has been contacted with the nucleic acid molecule and the compound;
(c) A method comprising the step of introducing cells into a subject. The compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and pyridinium chloride. 93. The method of claim 92, wherein the method is selected from the group consisting of a surfactant. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 93. The method of claim 92, wherein the method is selected from the group consisting of-(1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 93. The method of claim 92, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least about 0.6 megabases. 93. The method according to claim 92, wherein the cells are plant cells or animal cells. 93. The method of claim 92, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 93. The method of claim 92, wherein said cells are selected from the group consisting of embryonic cells, stem cells, transformed cells and tumor cells. 93. The method of claim 92, wherein the energy is ultrasound and the cell is contacted with a cavitation compound prior to applying the ultrasound to the cell. An ex vivo gene therapy method,
(a) contacting a cell in the absence of a nucleic acid molecule in vitro with a composition comprising a compound that delivers the nucleic acid molecule to the cell;
(b) contacting the cell previously contacted with the compound with a nucleic acid molecule,
(c) delivering the nucleic acid molecule to the cell to a greater extent than in the presence of the compound alone by applying ultrasound or electrical energy to the cell contacted with the compound and the nucleic acid molecule; and
(d) a method comprising the step of introducing cells into a subject. The compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and pyridinium chloride. The method of claim 100, wherein the method is selected from the group consisting of surfactants. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 The method of claim 100, wherein the method is selected from the group consisting of-(1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 110. The method of claim 100, wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, a fragment of a chromosome or naked DNA having a size of at least about 0.6 megabases. The method of claim 100, wherein the cells are plant cells or animal cells. 110. The method of claim 100, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 110. The method of claim 100, wherein said cells are selected from the group consisting of embryonic cells, transformed cells and tumor cells. 110. The method of claim 100, wherein the energy is ultrasound and the cell is contacted with a cavitation compound prior to applying the ultrasound to the cell. An ex vivo gene therapy method,
(a) applying ultrasound or electrical energy to the cells in vitro,
(b) delivering the nucleic acid molecule to the cell at the end of the application of ultrasound or energy by contacting the cell with a mixture of the nucleic acid molecule and a composition comprising the compound that delivers the nucleic acid molecule to the cell;
(c) A method comprising the step of introducing cells into a subject. The compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and pyridinium chloride. 111. The method of claim 108, wherein the method is selected from the group consisting of a surfactant. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 109. The method according to claim 108, wherein the method is selected from the group consisting of-(1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 109. The method of claim 108, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least greater than about 0.6 megabases. 109. The method according to claim 108, wherein the cells are plant cells or animal cells. 109. The method of claim 108, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 111. The method of claim 108, wherein said cells are selected from the group consisting of embryonic cells, transformed cells and tumor cells. 111. The method of claim 108, wherein the energy is ultrasound and the cell is contacted with a cavitation compound prior to applying the ultrasound to the cell. An ex vivo gene therapy method,
(a) applying ultrasound or electrical energy to the cells in vitro,
(b) contacting the cell with a composition comprising at least one compound that delivers a nucleic acid molecule to the cell upon termination of the application of ultrasound or electrical energy;
(c) delivering the nucleic acid molecule to the cell by contacting the cell previously contacted with the compound with the nucleic acid molecule; and
(d) a method comprising the step of introducing cells into a subject. The compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and pyridinium chloride. 117. The method of claim 116, wherein the method is selected from the group consisting of a surfactant. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 117. The method of claim 116, wherein the method is selected from the group consisting of-(l-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 117. The method of claim 116, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least greater than about 0.6 megabases. 117. The method according to claim 116, wherein the cells are plant cells or animal cells. 117. The method of claim 116, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 117. The method of claim 116, wherein said cells are selected from the group consisting of embryonic cells, transformed cells and tumor cells. 117. The method of claim 116, wherein the energy is ultrasound and the cell is contacted with a cavitation compound prior to applying the ultrasound to the cell. An ex vivo gene therapy method,
(a) contacting a cell with a nucleic acid molecule in vitro;
(b) delivering the nucleic acid molecule to the cell by applying ultrasound or electrical energy to the cell contacted with the nucleic acid molecule; and
(c) a method comprising the step of introducing cells into a subject. 125. The method of claim 124, wherein the energy is ultrasound, and wherein the cells are contacted with a cavitation compound prior to applying the ultrasound energy. 125. The method of claim 124, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least greater than about 0.6 megabases. 125. The method of claim 124, wherein said cells are plant cells or animal cells. 125. The method of claim 124, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 125. The method of claim 124, wherein said cells are selected from the group consisting of embryonic cells, transformed cells and tumor cells. 125. The method of claim 124, wherein the energy is ultrasound and the cell is contacted with a cavitation compound prior to applying the ultrasound to the cell. An ex vivo gene therapy method,
(a) applying ultrasound or electrical energy to the cells in vitro,
(b) delivering the nucleic acid molecule to the cell by contacting the cell with the nucleic acid molecule at the end of the application of ultrasound or electrical energy;
(c) a method comprising the step of introducing cells into a subject. 142. The method of claim 131, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least about 0.6 megabases. 142. The method of claim 131, wherein the cells are plant cells or animal cells. 134. The method of claim 131, wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, an immortalized cell, and a cell capable of developing a particular organ. 134. The method of claim 131, wherein the cells are selected from the group consisting of embryonic cells, transformed cells, and tumor cells. 134. The method of claim 131, wherein the energy is ultrasound, and wherein the cells are contacted with a cavitation compound prior to applying the ultrasound energy. The compound is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and pyridinium chloride. 134. The method of claim 131, wherein the method is selected from the group consisting of a surfactant. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 134. The method of claim 131, wherein the method is selected from the group consisting of-(l-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. A kit for delivering a nucleic acid to a cell,
A delivery agent comprising a composition comprising a delivery agent,
A kit comprising reagents for performing acoustic or electroporation and optional instructions for delivering nucleic acids to cells. 140. The kit of claim 139, further comprising a composition comprising an artificial chromosome. The delivery factor is a cationic lipid, cationic polymer, cationic lipid mixture, cationic polymer mixture, cationic lipid and cationic polymer mixture, cationic lipid and neutral lipid mixture, polycationic lipid, non-liposome-forming lipid, activated dendrimer, and chloride. 141. The kit of claim 140, comprising a compound selected from the group consisting of a pyridinium surfactant. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 142. The kit of claim 141, wherein the kit is selected from the group consisting of-(l-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. A method for detecting or measuring the delivery and expression of a nucleic acid introduced into a cell,
Introducing a labeled nucleic acid molecule encoding a reporter gene into a cell,
A method comprising detecting or measuring delivery and expression of a nucleic acid molecule in a cell by detecting a labeled cell as an indicator of nucleic acid delivery to the cell and measuring a reporter gene product as an indicator of DNA expression in the cell. 144. The method of claim 143, wherein the labeled cells are detected by flow cytometry, fluorimetry, cell imaging, or fluorescence microscopy. 144. The method of claim 143, wherein the labeled cells are detected by flow cytometry. 144. The method of claim 143, wherein the nucleic acid molecule is DNA. 144. The method of claim 143, wherein the label is iododeoxyuridine (IdU or IdUrd) or bromodeoxyuridine (BrdU). 144. The method of claim 143, wherein the reporter gene encodes a fluorescent protein, enzyme or antibody. 149. The method of claim 148, wherein the enzyme is luciferase, [beta] -galactosidase or alkaline phosphatase. 149. The method of claim 148, wherein the fluorescent protein is a red, green or blue fluorescent protein. 144. The method of claim 143, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least greater than about 0.6 megabases. 144. The method of claim 143, wherein the nucleic acid molecule is an artificial chromosome, a plasmid, a chromosome fragment, naked DNA or a natural chromosome. 144. The method of claim 143, wherein the nucleic acid molecule is an artificial chromosome expression system (ACes). 144. The method of claim 143, wherein the cell is a eukaryotic cell. 156. The method of claim 154, wherein said cells are primary cells, cell lines, plant cells or animal cells. 160. The method of claim 155, wherein the cells are stem cells, nuclear transfer donor cells, tumor cells or transformed cells. A method for monitoring delivery of a nucleic acid molecule to a cell, comprising:
(a) labeling a nucleic acid molecule,
(b) delivering the labeled nucleic acid molecule to a cell; and
(c) A method comprising detecting a labeled nucleic acid molecule in a cell by flow cytometry, fluorimetry, cell imaging or fluorescence microscopy as an indicator of nucleic acid molecule delivery to the cell. 157. The method of claim 157, wherein the nucleic acid molecule is labeled with a thymidine analog. 160. The method of claim 158, wherein the thymidine analog is iododeoxyuridine or bromodeoxyuridine. 160. The method of claim 157, wherein the nucleic acid molecule is treated with a delivery agent comprising a cationic compound. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 163. The method of claim 160, wherein the method is selected from the group consisting of-(1-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 158. The method of claim 158, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA having a size of at least about 0.6 megabases. A method of screening for the ability of an agent to deliver a nucleic acid molecule to a cell, comprising:
(a) delivering a labeled nucleic acid molecule to a cell in the presence of an agent; and
(b) A method comprising measuring the number of cells containing a label as an indicator of the ability of the agent to deliver the nucleic acid molecule to the cells. 163. The method of claim 163, wherein the cell number is determined by flow cytometry, fluorimetry, cell imaging or fluorescence microscopy. 163. The method of claim 163, wherein the label is iododeoxyuridine or bromodeoxyuridine. 163. The method of claim 163, wherein the agent comprises a cationic compound. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 169. The method of claim 166, wherein the method is selected from the group consisting of-(l-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 163. The method of claim 163, wherein step (a) comprises contacting the nucleic acid molecule with a delivery agent comprising a cationic compound. When the compound is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy- N-[2 (spermine - carboxamido) ethyl] -N, N-dimethyl-1-propane aminium trifluoroacetate _{(DOSPA), C 52 H 106} N 6 O 4 · 4CF 3 CO 2 H, C 88 H 178 N _{8 O 4 S 2 · 4CF 3} CO 2 H, C 40 H 84 NO 3 P · CF 3 CO 2 H, C 50 H 103 N 7 O 3 · 4CF 3 CO 2 H, C 55 H 116 N 8 O 2 · _{6CF 3 CO 2 H, C 49} H 102 N 6 O 3 · 4CF 3 CO 2 H, C 44 H 89 N 5 O 3 · 2CF 3 CO 2 H, C 41 H 78 NO 8 P, C 100 H 206 N 12 _{O 4 S 2 · 8CF 3 CO} 2 H, C _{162 H 330 N 22 O 9 ·} 13CF 3 CO 2 H, C 43 H 88 N 4 O 2 · 2CF 3 CO 2 H, C 43 H 88 N 4 O 3 · 2CF 3 CO 2 H and (1-methyl-4 168. The method of claim 168, wherein the method is selected from the group consisting of-(l-octadeca-9-enyl-nonadeca-10-enylenyl) pyridinium chloride. 163. The method of claim 163, wherein the nucleic acid molecule is a natural chromosome, artificial chromosome, chromosome fragment or naked DNA, or plasmid. 163. The method of claim 163, wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, a fragment of a chromosome or naked DNA having a size of at least about 0.6 megabases. 144. The method of claim 143, wherein the cells are selected from the group consisting of primary cells, immortalized cells, embryonic cells, stem cells, transformed cells, and tumor cells. 157. The method of claim 157, wherein said cells are selected from the group consisting of primary cells, immortalized cells, embryonic cells, stem cells, transformed cells and tumor cells. 163. The method of claim 163, wherein the cells are selected from the group consisting of primary cells, immortalized cells, embryonic cells, stem cells, transformed cells and tumor cells. Synovial cells containing large heterologous nucleic acids. 178. The synovial cell of claim 175, wherein the heterologous nucleic acid is at least greater than about 0.6 megabases in size. Synovial cells containing artificial chromosomes. 180. The synovial cell of claim 177, wherein the artificial chromosome is ACes. 178. The synovial cell of claim 175 or 177, which is a fibroblast-like synovial cell. 180. The synovial cell of claim 175 or 177, which is a mammalian synovial cell. 180. The synovial cell of claim 180, which is a primate synovial cell. 180. The synovial cell according to claim 180, which is a rodent, rabbit, monkey or human synovial cell. A method for introducing a heterologous nucleic acid into synovial cells, comprising introducing an artificial chromosome into synovial cells. 183. The method of claim 183, wherein the artificial chromosome is ACes. 183. The method of claim 183, wherein the synovial cells are fibroblast-like synovial cells. A method for introducing a large nucleic acid molecule into synovial cells,
Exposing the nucleic acid molecule to a delivery agent and contacting the synovial cells with the nucleic acid molecule. 189. The method of claim 186, wherein the large nucleic acid molecule is an artificial chromosome. 187. The method of claim 187, wherein the artificial chromosome is ACes. 187. The method of claim 186, wherein the synovial cells are fibroblast-like synovial cells. 189. The method of claim 186, wherein the delivery agent comprises a cationic compound. A method for introducing a large nucleic acid molecule into synovial cells,
(a) exposing the nucleic acid molecule to a delivery agent;
(b) exposing the synovial cells to a delivery agent; and
(c) A method comprising the steps of: delivering a nucleic acid molecule to a synovial cell by contacting the synovial cell with the nucleic acid molecule, wherein the steps (a) to (c) are performed sequentially or sequentially. 192. The method of claim 191, wherein the synovial cells are fibroblast-like synovial cells. 192. The method of claim 191, wherein the delivery agent comprises a cationic compound. 192. The method of claim 191, wherein the nucleic acid molecule is at least greater than about 0.6 megabases in size. A method for delivering a nucleic acid molecule to synovial cells, comprising:
(a) contacting the synovial cells with the delivery agent in the absence of the nucleic acid molecule, and applying ultrasonic or electrical energy to the synovial cells, provided that the contacting and application occur sequentially or simultaneously;
(b) delivering the nucleic acid molecule to the synovial cells by contacting the synovial cells with the nucleic acid molecules. A method for delivering a nucleic acid molecule to synovial cells, comprising:
(a) contacting the nucleic acid molecule and the delivery agent with the synovial cells, and applying ultrasonic or electrical energy to the synovial cells, wherein the contacting and applying are performed sequentially or simultaneously, thereby synovializing the nucleic acid molecules. How to deliver to cells. 196. The method of claim 195 or 196, wherein the delivery agent comprises a cationic compound. 196. The method of claim 195 or 196, wherein the synovial cells are fibroblast-like synovial cells. 195. The method of claim 195 or 196, wherein the energy is ultrasound. A method of modulating a rheumatic disease course in a subject, comprising introducing a large nucleic acid into the subject, wherein the large nucleic acid is an agent that modulates or encodes a rheumatic disease course. 200. The method of claim 200, wherein the nucleic acid is introduced to the site of inflammation in the subject. 200. The method of claim 200, wherein the rheumatic disease course is inflammation. 200. The method of claim 200, wherein the rheumatic disease is rheumatoid arthritis. 200. The method of claim 200, wherein the large nucleic acid is an artificial chromosome. 205. The method of claim 204, wherein the artificial chromosome is ACes. 220. The method of claim 201, wherein the site of inflammation is in a joint. 200. The method of claim 200, wherein the nucleic acid is introduced into the cell in vitro and then transferred to the subject. 200. The method of claim 200, wherein the nucleic acid is introduced into the cell in vivo. 210. The method of claim 207 or claim 208, wherein the nucleic acid is introduced into synovial cells. 210. The method of claim 209, wherein the synovial cells are fibroblast-like synovial cells. 200. The method of claim 200, wherein said nucleic acid comprises a nucleic acid encoding an anti-inflammatory or immunomodulatory molecule. The nucleic acid is an interleukin-1 receptor antagonist, a soluble interleukin-1 receptor, a soluble tumor necrosis factor receptor, interferon-β, interleukin-4, interleukin-10, interleukin-13, transforming growth factor β 200. The method of claim 200, comprising a nucleic acid encoding a dominant negative I kappa B-kinase, FasL, Fas-associated death domain protein or CTLA-4 or a combination thereof. A method for identifying a candidate agent for treating a connective tissue disease,
Introducing a large nucleic acid molecule comprising a nucleic acid that is or encodes a candidate agent into an animal model of disease and determining whether the candidate agent ameliorates one or more conditions in the animal. Including methods. 220. The method of claim 213, wherein the disease is a rheumatic disease. 220. The method of claim 213, wherein the disease is rheumatoid arthritis. 220. The method of claim 213, wherein the animal is a mammal. 220. The method of claim 213, wherein the animal is a rodent, rabbit, dog, horse, or primate. 220. The method of claim 213, wherein the large nucleic acid molecule is an artificial chromosome. 218. The method of claim 218, wherein the artificial chromosome is ACes. 220. The method of claim 213, wherein the nucleic acid molecule is in a synovial cell. 220. The method of claim 220, wherein the synovial cells are fibroblast-like synovial cells. 220. The method of claim 213, wherein the nucleic acid is introduced into a joint of an animal. 213. The method of claim 213, wherein the nucleic acid is introduced into the cell in vitro and then transferred to the animal. 220. The method of claim 213, wherein the nucleic acid is introduced into a cell in vivo. A composition comprising cells selected for therapeutic treatment of a joint, wherein the cells comprise a large heterologous nucleic acid. A composition comprising cells selected for therapeutic treatment of a joint, wherein the cells comprise artificial chromosomes. 229. The composition of claim 226, wherein the artificial chromosome is ACes. A composition comprising cells selected for therapeutic treatment of rheumatoid arthritis, wherein the cells comprise a large heterologous nucleic acid. A composition comprising cells selected for therapeutic treatment of rheumatoid arthritis, wherein the cells comprise artificial chromosomes. 230. The composition of claim 229, wherein the artificial chromosome is ACes. 74. The method of claim 48 or claim 73, wherein the nucleic acid molecule is delivered to the cell to a greater extent than when using a delivery agent or energy alone.