JP2010539992A - トランスフェクション方法及びそのための組成物 - Google Patents
トランスフェクション方法及びそのための組成物 Download PDFInfo
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Abstract
【選択図】なし
Description
本発明は、一般的に、細胞生物学分野に関する。より具体的には、本発明は、細胞、特に、樹状細胞をトランスフェクションする方法、及び免疫応答を誘発する方法に向けられる。この目的を達成するために、本発明は、Toll様受容体(TLR)ファミリーの1つ又は複数のメンバーと相互作用する能力がある部分に連結した、核酸を結合する能力がある少なくとも1個の正荷電基を含む化合物を提供する。
本明細書に用いられる場合、用語「由来する」は、特定された完全体(integer)が、特定の供給源から得ることができるが、ただし、必ずしもその供給源から直接というわけではないことを示すものと解釈されるべきである。
1.Sambrook、Fritsch、及びManiatis、Molecular Cloning:A Laboratory Manual、Cold Spring Harbor Laboratories、New York、第2版(1989)、I巻、II巻、及びIII巻の全体、
2.DNA Cloning:A Practical Approach、I巻及びII巻(D.N.Glover編、1985)、IRL Press、Oxford、テキストの全体、
3.Oligonucletoide Synthesis:A Practical Approach(M.J.Gait編、1984)IRL Press、Oxford、テキストの全体、並びに特に、Gait、1〜22ページ;Atkinsonら、35〜81ページ;Sproatら、83〜115ページ;及びWuら、135〜151ページによるその中の論文
に記載されている。
本発明に繋がる研究において、本発明者らは、細胞に核酸をトランスフェクションする効率的な手段を生み出そうとした。それはまた、対象において、抗原提示細胞、特に樹状細胞をターゲットにし、体液性免疫応答及び細胞性免疫応答の両方を誘発する、DNAワクチン候補を作製しようとすることでもあった。
カチオン性ペプチドの合成
分岐ペプチド構築物については、リシン残基を用いて足場鋳型の分岐点を供給することによって合成した。足場内に存在する分岐点の数に依存して、ジカチオン性又はテトラカチオン性構造が生じた(図1)。これらの分岐構築物を、PEG−S RAM樹脂(Rapp Polymere、Tubingen、Germany;置換係数(substitution factor)0.27mmol/g)上で合成した。まず、等モル量のO−ベンゾトリアゾール−N,N,N,N’,N’−テトラメチル−ウロニウム−ヘキサフルオロホスフェート(HBTU;Novabiochem、Darmstadt、Germany)、1−ヒドロキシベンゾトリアゾール(HOBt)、及び1.5倍モル過剰のジイソプロピルエチルアミン(DIPEA;Sigma、Castle Hill、Australia)と共に、Fmoc−リシン(Mtt)−OH(Novabiochem、Laufelfingen、Switzerland)を4倍過剰で、樹脂に結合させた。アシル化を40分間行い、完全な反応をトリニトロベンジルスルホン酸(TNBSA)試験によって確認した(21)。α−アミノ基上のFmoc保護基の除去を2.5%ジアザビシクロ[5.4.0]ウンデカ−7−エン(Sigma、Steinheim、Germany)で達成し、Fmoc−リシン(Fmoc)−OH(Auspep、Melbourne、Australia)を、Fmoc基の除去後、2つの一級アミノ基を露出させて分岐点として働くように、結合させた。ジメチルホルムアミド(DMF;Auspep、Melbourne、Australia)を用いて、それぞれのアシル化段階と脱保護段階の間に樹脂を洗浄した。後続のアミノ酸を4倍過剰で結合させ、アシル化を60分間行った。四価の構築物の組み立てについて、アシル化のさらなるラウンドを、Fmoc−リシン(Fmoc)−OHを用いて60分間行い、4つの分岐点を生じさせた。アミノ酸を含む基の四価アルギニン構築物(R4)への化学選択的ライゲーションを可能にするために、システインを構築物のC末端に挿入し、R4−Cysを生じさせた。分岐ペプチド構築物上へのカルボキシフルオレセイン結合について、最後のアシル化反応後、N末端Fmoc基を除去しなかった。その代わり、C末端リシンのε−アミノ基上に存在するMtt保護基をジクロロメタン中1%TFA(Ajax Finechem、Seven Hills、Australia)での処理によって除去した。その後、等モル量のHOBt、HBTU、及び1.5倍モル過剰のDIPEAの存在下で、5(6)−カルボキシフルオレセイン(Fluka BioChemika、Switzerland)を4倍過剰用いて、暗闇中18時間、露出したε−アミノ基に結合させた。アシル化後、樹脂をDMFで洗浄し、N末端Fmoc基を除去した。全てのペプチドを同時に、固相支持体及び側鎖保護基から切断した。ウォーターズ(Waters)3 HPLCクロマトグラフィーシステムに設置されたバイダック(Vydac)C4カラム(4.6mm×250mm)又はC8カラム(4.6mm×250mm)を用いて逆相分析クロマトグラフィーによってペプチドの純度を評価した。限界溶媒としてH2O中0.1%TFA及びアセトニトリル中0.1%TFAを用いる1ml/分の流速を用いて、クロマトグラムを展開した。必要な場合、セミ分取用ウォーターズ又はGBC HPLCシステム及びセミ分取用バイダックC4カラム(10mm×300mm)を用いて2.5ml/分の流速でペプチドを精製した。ペプチド含有量の推定を、蛍光ペプチドの吸光度を496nmで測定するUV分光光度法によって測定し、83,000M−1cm−1のモル吸光係数を用いて濃度を計算した。合成された蛍光分岐ペプチド構築物の模式図は、図1に示されている。
R4によるDNA移動の遅延
分岐ペプチドR4とDNAの間の会合を調べるために、緑色蛍光タンパク質及びインフルエンザHAタンパク質をそれぞれコードするDNAプラスミドpEGFP−N1又はpCI−HAの一定量を、様々な量のR4とインキュベートし、アガロースゲル電気泳動によって分析した(図2)。
R4−DNA複合体の形成
R4のDNAへの結合を確認するために、漸増量のR4及びDNAを含む遠心分離した混合物の上清におけるDNAの量を、260nmにおいて吸光度を測定することによって決定した(図3A)。DNAをその電荷の中和によって溶液から沈殿させるR4の能力は、溶液中のDNAの減少を生じさせ、それにしたがって、それら2つの間の会合を示すことになるだろう。遠心分離された上清におけるDNAの量は、0.1μg〜5μgのR4とインキュベートした場合、劇的には変化しなかった。しかしながら、7.5μgのR4を用いた場合、わずかな減少が生じ、これより多い量を含む溶液中において、DNAは検出されなかった。この結果は、存在するR4の量がDNAの溶液からの沈殿と比例することを示す。吸光度の測定値をまた、いかなる遠心分離された不溶性物質も再懸濁するために用いられた洗浄上清についても取った。これらの試料中にDNAがほとんど存在しないことは、R4−DNA沈殿物が安定であること、及びDNAがこの複合体から解離する可能性は低いことを示唆する。
R4(S2Pam2Cys)−DNAでのDCのトランスフェクション
R4構築物又はR4(S2Pam2Cys)構築物がトランスフェクションされたDNAの発現を誘導する能力があるかどうかを決定するために、マウスDC系、D1にpEGFPプラスミドをR4又はR4(S2Pam2Cys)を用いてトランスフェクションした(図4A)。これらの細胞における緑色蛍光タンパク質(GFP)の発現レベルを、市販されているトランスフェクション試薬FUGENEを用いてトランスフェクションしたD1細胞における緑色蛍光タンパク質の発現レベルと比較した。
ヒト単球由来樹状細胞(MoDC)のトランスフェクション
ヒト単球由来樹状細胞(MoDC)のトランスフェクションを増強するR4(S2Pam2Cys)の能力を調べた。MoDCにpEGFPプラスミドをトランスフェクションした実験において(図6A)、R4(S2Pam2Cys)のみが、GFPの発現を誘導することにおいて効率的であった。FUGENEか又はR4のいずれかを加えたpEGFPでの細胞のトランスフェクションは、相対的に非効率であり、これらの細胞におけるトランスフェクションレベルは、プラスミド単独で処理されたものと類似していた。この結果は、MoDCに、各トランスフェクション試薬と共にpCI−HAプラスミドをトランスフェクションした実験において、繰り返された(図6B)。
R4(S2Pam2Cys)−DNAでの非DC細胞系のトランスフェクション
DNAと複合体を形成した、R4構築物又はR4(S2Pam2Cys)構築物が非DC細胞系をトランスフェクションする能力があるかどうかを調べるために、ヒト肺上皮細胞系A549及びマウス肺胞マクロファージ細胞系MH−Sに、R4又はR4(S2Pam2Cys)を用いてpEGFPプラスミドをトランスフェクションした(図7A及び7B)。ヒト肺上皮細胞系A549(A)又はマウス肺胞マクロファージ細胞系MH−S(B)を、異なる希釈度のR4、R4Pam2Cys、又はFUGENEと複合体を形成した、緑色蛍光タンパク質をコードするプラスミドの非存在下又は存在下において2×105個の細胞/mlの濃度で、37℃、5%CO2で48時間、培養した。トランスフェクション効率については、フローサイトメーターで細胞の蛍光を測定することによって決定した。これらの細胞系の両方とも他の研究においてTLR−2を発現することが報告されている(Oshikawa 2003、Slevogt 2007)。未処理のA549又はMH−S細胞、及びDNAと共にR4で処理された細胞において、皆無かそれに近い蛍光が観察された。しかしながら、R4(S2Pam2Cys)及びDNAをトランスフェクションされたA549又はMH−S細胞は、pEGFPプラスミド及びFUGENE試薬をトランスフェクションされた細胞において観察された発現レベルと、より高くはないにしても、匹敵するレベルでGFPを発現した。
R4(S2Pam2Cys)−DNAで免疫処置されたマウスにおける特異的抗体産生
DNAと複合体を形成したR4(S2Pam2Cys)の抗体を誘導する能力を評価した。BALB/cマウス(週齢6〜8週間)に、単独か、又は1:3若しくは1:5のモル比でR4(S2Pam2Cys)と複合体を形成した、インフルエンザ血球凝集素をコードするDNAを含む50μgのDNAプラスミドを0日目及び28日目に尾の付け根に皮下接種した。陽性対照として、マウスをまた、スプリットPR8ウイルス(3μg/マウス)で鼻腔内に免疫処置した。非免疫処置マウスは陰性対照としての役割を果たした。最後の接種後14日目にマウスから血清を採取し、ELISAを行って、インフルエンザPR8ウイルスに特異的な抗体の存在を検出した。血清抗HA抗体をELISAによって測定した。非免疫処置マウス又は「ナイーブ」マウスにおいてHA特異的抗体は検出されなかったが、1:3及び1:5のモル比で混合された2つの用量のR4(S2Pam2Cys)及びDNAで免疫処置されたマウスにおいて、インフルエンザウイルスに結合することができる実質的レベルの抗体が検出された(図8)。
R4(S2Pam2Cys)−DNAで免疫処置されたマウスにおけるインフルエンザ核タンパク質特異的IFN−γ陽性CD8+T細胞の誘導
DNAと複合体を形成したR4(S2Pam2Cys)の細胞性免疫応答を誘導する能力を評価した。BALB/cマウス(週齢6〜8週間)に、単独か、又は1:1、1:2、若しくは1:5のNH3 +:PO4 −モル比でR4(S2Pam2Cys)と複合体を形成した、インフルエンザ核タンパク質をコードする20μgのDNAプラスミドを尾の付け根に皮下接種した。R4(S2Pam2Cys)を含む溶液の10μlのアリコートをDNAの溶液に2分間ごとに合計1時間、ゆっくり加えることによって、複合体形成を達成した。全ての接種材料を0.7MのNaClに溶解した。免疫処置後7日目又は10日目にマウスから脾臓及び鼠径リンパ節を摘出し、細胞内サイトカイン染色を行って核タンパク質147〜155に特異的なIFN−γ分泌CD8+T細胞の存在を検出した。それぞれの棒は2匹のマウスからの平均及び標準誤差を表す。
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Claims (20)
- 少なくとも1個のTLRリガンドに連結した正荷電基を含む化合物。
- 前記正荷電基がTLRリガンドに共有結合している、請求項1に記載の化合物。
- 前記正荷電基が1個又は複数のアミノ酸を含む、請求項1又は請求項2に記載の化合物。
- 前記正荷電基が分岐ペプチド又は直鎖ペプチドである、請求項1〜3のいずれか一項に記載の化合物。
- 前記ペプチドが少なくとも1個のアルギニン残基又はリシン残基を含む、請求項4に記載の化合物。
- 前記ペプチドが少なくとも4個のアルギニン残基又は少なくとも4個のリシン残基を含む、請求項4又は請求項5に記載の化合物。
- 前記TLRリガンドが脂質、又はペプチドグリカン、又はリポタンパク質、又はリポ多糖を含む、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがパルミトイル、ミリストイル、ステアロイル、ラウロイル、オクタノイル、又はデカノイルを含む、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがPam2Cys、Pam3Cys、Ste2Cys、Lau2Cys、及びOct2Cysからなる群より選択される、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがTLR−2か又はTLR−6のいずれかに結合する、請求項1〜9のいずれか一項に記載の化合物。
- 前記TLRリガンドがTLR−2に結合する、請求項1〜10のいずれか一項に記載の化合物。
- 核酸及び請求項1〜11のいずれか一項に記載の化合物を含む複合体であって、前記核酸と前記正荷電基との間の静電相互作用によって前記核酸が前記化合物と会合している、複合体。
- 少なくとも1個のTLRを発現する細胞を、請求項12に記載の複合体に接触させる段階を含む、トランスフェクション方法。
- 抗原に対する免疫応答を生じさせる方法であって、請求項12に記載の複合体を対象に投与する段階を含み、前記核酸が前記抗原又はそのエピトープをコードする、方法。
- 抗原に対する免疫応答を生じさせる方法であって、請求項12に記載の複合体をトランスフェクションされた細胞を対象に投与する段階を含み、前記核酸が前記抗原又はそのエピトープをコードする方法。
- TLRを発現する細胞で遺伝子の発現を抑圧する方法であって、請求項12に記載の複合体を対象に投与する段階を含み、前記核酸が、siRNA、shRNA、siRNAをコードするDNA、及びshRNAをコードするDNAからなる群より選択され、かつ前記siRNA又はshRNAが前記遺伝子をターゲットとする、方法。
- 対象における免疫応答の誘導のためのワクチンの製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 遺伝子異常又は遺伝子欠損をもつ対象の処置のための医薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 遺伝子の異常な発現又は望ましくない発現を患っている対象の処置のための医薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 細胞のトランスフェクションのための試薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
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US20100310595A1 (en) | 2010-12-09 |
EP2200638B1 (en) | 2016-03-16 |
EP2200638A1 (en) | 2010-06-30 |
AU2008310313A1 (en) | 2009-04-16 |
CA2702154C (en) | 2017-05-16 |
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