JP2016534149A - 肝細胞増殖因子の2つ以上のアイソフォームを利用した筋萎縮性側索硬化症の予防又は治療用組成物 - Google Patents
肝細胞増殖因子の2つ以上のアイソフォームを利用した筋萎縮性側索硬化症の予防又は治療用組成物 Download PDFInfo
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Abstract
Description
(a)本発明は、筋萎縮性側索硬化症(Amyotrophic Lateral Sclerosis)の予防又は治療用薬学的組成物を提供する。
(b)本発明は、筋萎縮性側索硬化症(Amyotrophic Lateral Sclerosis)の予防又は治療方法を提供する。
(c)本発明の組成物又は方法を利用すると、胚神経細胞における神経突起発芽 、成長及び運動神経細胞の成長及び死滅抑制を通じて、筋萎縮性側索硬化症を予防又は治療することができる。
本発明のポリヌクレオチドを運搬する運搬体としてプラスミド(ベクター)が利用できる。ベクターに含まれるポリヌクレオチドは、適した発現カセット内に存在することが好ましい。前記発現カセットにおいて、ポリヌクレオチドは、プロモーターに作動的に連結されることが好ましい。
レトロウイルスは、自分の遺伝子を宿主のゲノムに挿入させて、大量の外来遺伝物質を運搬することができ、感染できる細胞のスペクトルが広いため、遺伝子伝達ベクターとしてよく利用されている。
アデノウイルスは、中間程度のゲノムサイズ、操作の便宜性、高いタイター、広範囲のターゲット細胞、及び優れた感染性から、遺伝子伝達ベクターとしてよく利用されている。ゲノムの両末端は、100〜200bpのITR(inverted terminal repeat)を含み、これは、DNA複製及びパッケージングに必須的なシスエレメントである。ゲノムのE1領域(E1A及びE1B)は、転写及び宿主細胞遺伝子の転写を調節する蛋白質をコードする。E2領域(E2A及びE2B)は、ウイルスDNA複製に関与する蛋白質をコードする。
アデノ関連ウイルス(AAV)は、非分裂細胞を感染させることができて、多様な種類の細胞に感染できる能力を有しているため、本発明の遺伝子伝達システムに好適である。AAVベクターの製造及び用途に対する詳細な説明は、米国特許第5,139,941号及び第4,797,368号に詳細に開示されている。
他のウイルスベクターも、本発明のポリヌクレオチド配列を生体内に運搬するに利用することができる。ワクシニアウイルス(Puhlmann M. et al., Human Gene Therapy 10:649−657(1999); Ridgeway, “Mammalian expression vectors,” In: Vectors: A survey of molecular cloning vectors and their uses. Rodriguez and Denhardt, eds. Stoneham: Butterworth, 467−492(1988); Baichwal and Sugden, “Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes,” In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, 117−148( 1986)及びCoupar et al., Gene, 68:1−10(1988))、レンチウイルス(Wang G. et al., J. Clin. Invest. 104(11):R55−62(1999))、又は単純ヘルペスウイルス(Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411−1415(1995))由来のベクターも、前記ポリヌクレオチドを細胞内に運搬できる運搬システムとして利用することができる。
リポソームは、水相に分散された燐脂質により自然に形成される。外来DNA分子をリポソームにより成功的に細胞内に運搬した例は、Nicolau及びSene, Biochim. Biophys. Acta, 721:185−190(1982)、及びNicolau et al., Methods Enzymol., 149:157−176(1987)に開示されている。本発明のポリヌクレオチド配列を内包したリポソームは、エンドサイトーシス、細胞表面への吸着又はプラズマ細胞膜との融合などのメカニズムを通じて細胞と相互作用し、細胞内にポリヌクレオチド配列を運搬する。
マウスの胚芽(embryo)から大脳皮質部分のみを取って単一細胞化した後、Ara−C 10μMを培養培地に入れて、神経細胞だけ培養した。ENCの成熟においてpCK−HGFX7が及ぼす影響を確認するために、2×104個の細胞を播種し、翌日細胞にpCK−HGFX7をトランスフェクションした293F細胞(Life technologies, USA)から得られたタンパク質1.25ng/mLを処理し、細胞の成熟で見られる神経突起発芽(neurite outgrowth)程度を確認した。細胞の神経突起発芽の程度は、神経細胞特異的に発現するチューブリン(tubulin)タンパク質のTUJ−1の発現を免疫細胞化学(immunocytochemistry)を通じて確認した。
ENCの成熟後、pCK−HGFX7が細胞成長に及ぼす影響を確認した。そのために、5×104個のENCを播種し、6日間成熟過程を進行した。6日後、pCK−HGFX7をトランスフェクションした293F細胞から得られたタンパク質1.25ng/mLを処理し、pCK−HGFX7が細胞の成長に及ぼす影響を確認した。pCK−HGFX7を処理した3日後、MTTアッセイを行って、細胞成長を測定した。
3−1.細胞株及び細胞培養
本実験に使用されたNSC−34細胞(Cellution Biosystem, Vancouver, CA)は、マウス由来の運動神経細胞である。NSC−34細胞は、胚芽期マウスの脊髄神経由来の運動神経細胞と、神経母細胞腫細胞とが混合された細胞株であって、運動神経に係る研究に広く使用される細胞株である。前記細胞は、37℃, 5% CO2条件で培養し、10%牛胎児血清(Gibco BRL, USA)及び抗生物質(Gibco BRL, USA)が含まれたダルベッコ・フォークト変法イーグル最小必須培地(DMEM, Sigma)内で培養した。細胞培養媒質、試薬及び血清は、GibcoとSigma aldrich社から購入した。
HGFタンパク質を発現する上澄み液を生産するために、DNAトランスフェクション法を利用した。トランスフェクションは、FuGene HDトランスフェクションシステム(Promega, USA)を利用して、製造者のプロトコールにしたがって行った。1×106個の293T細胞を播種した後、翌日pCK、pCK−HGF728(PCT/KR03/000548のpCK−cHGF)、pCK−HGF723(PCT/KR03/000548のpCK−dHGF)及びpCK−HGFX7 DNA 3μgをそれぞれトランスフェクションした。48時間培養後、それぞれの上澄み液を全部収集して、0.22μmフィルターを利用してろ過した。ヒトHGF免疫分析法を利用し、それぞれの上澄み液に含まれたHGFタンパク質の発現量を測定した。それぞれの上澄み液を再び1μg/mLで希釈して、実験に使用した。ヒトHGF免疫分析法に使用された組換えヒトHGFタンパク質は、R&D社(R&D system, Inc., USA)の製品を購入して使用した。
pCK−HGF7が運動神経細胞の成長に及ぼす効果を確認するために、NSC−34細胞にpCK−HGF7を処理した後、細胞の増殖程度を評価した。細胞は、10%牛胎児血清を含む培養培地で培養した後、実験に使用するために、1%牛胎児血清を含むダルベッコ・フォークト変法イーグル最小必須培地を利用して懸濁した。1%の血清を含む培地2mLに3×104個の細胞が含まれるように懸濁して、6ウェルプレートに播種した。播種2時間後、pCK−HGF728、pCK−HGF723、pCK−HGFX7をトランスフェクションした293T細胞から得た各上澄み液を、HGFタンパク質の濃度が50ng/mLになるように、6ウェルプレートにウェル当たり100μLずつそれぞれ添加した。pCKベクターを293T細胞にトランスフェクションして得た上澄み液を対照群として使用した。48時間培養後、6ウェルプレートの培地を入れ替えた。それぞれのウェルに1%の牛胎児血清を含む培地2mLを添加した後、pCK−HGF728、pCK−HGF723、pCK−HGFX7をトランスフェクションした293T細胞で得た各上澄み液をHGFタンパク質の濃度が50ng/mLになるように添加した後、48時間培養した。pCKベクターを293T細胞にトランスフェクションして得た上澄み液を対照群として使用した。培養した細胞を収集して細胞を計数した。pCKベクターを対照群として使用した。
NSC−34細胞を1%牛胎児血清を含むダルベッコ・フォークト変法イーグル最小必須培地で3×104個で懸濁して、6ウェルプレートに播種した。細胞播種後、2時間細胞を安定化させた後、pCK−HGF728、pCK−HGF723、pCK−HGFX7をトランスフェクションした293T細胞から得た各上澄み液を、HGFタンパク質の濃度50ng/mLとして添加した。pCKベクターを293T細胞にトランスフェクションして得た上澄み液を対照群として使用した。細胞は、2〜3日間隔で培地と上記の各上澄み液を入れ替え、5日間培養した。
4−1.酸化的ストレスによるNSC−34細胞の細胞死滅を誘導する過酸化水素水の濃度選定
過酸化水素水により誘導されるNSC−34細胞の細胞死滅にpCK−HGFX7が及ぼす影響を確認する前に、NSC−34細胞の死滅を確認するに適切な細胞の播種濃度及び細胞死滅を誘導する過酸化水素水の濃度を選定した。
NSC−34細胞は、10%牛胎児血清を含む培養培地で培養した後、細胞死滅抑制実験に使用するために、1%牛胎児血清を含むダルベッコ・フォークト変法イーグル最小必須培地を利用して懸濁した。1%の血清を含む培地2mLに3×105個の細胞が含まれるように懸濁して、6ウェルプレートに播種した。翌日、以前の実験で選定された過酸化水素水30μMを各ウェルに処理した後、pCK−HGF728、pCK−HGF723、pCK−HGFX7をトランスフェクションした293T細胞から得た各上澄み液を、HGFタンパク質の濃度が50ng/mLになるように処理した。実験対照群として、pCKベクターをトランスフェクションして得られた培養培地を利用した。7日間細胞を培養しながら細胞死滅程度を観察した。細胞播種7日後、細胞を収集して細胞を計数した。細胞計数結果、pCK、pCK−HGF728、pCK−HGF723を処理した実験群の細胞は、最初播種した細胞数に比べ、約60〜70%の細胞のみが生存したが、pCK−HGFX7を処理したNSC−34細胞は、約92%程度の生存を示し、他の実験物質処理群に比べ、細胞死滅が抑制されることを確認した。この結果を通じて、HGFX7タンパク質が過酸化水素水による酸化的ストレスで誘導される運動神経の細胞死滅を効果的に抑制することを確認することができた(参照:図5)。
NSC−34細胞を1%牛胎児血清を含むダルベッコ・フォークト変法イーグル最小必須培地で3×105個で懸濁して、6ウェルプレートに播種した。翌日、30μM過酸化水素水を各ウェルに処理した後、pCK−HGF728、pCK−HGF723、pCK−HGFX7をトランスフェクションした293T細胞から得た各上澄み液を、HGFタンパク質の濃度が50ng/mLになるように処理して7日間培養した。実験対照群として、pCKベクターをトランスフェクションして得られた上澄み液を利用した。
ALSの原因の1つと知られたSOD1(Superoxide dismutase 1)のG93A突然変異型を利用したin vitro試験法が多くの研究者によって開発された。特に、運動神経細胞の研究で広く使用するNSC−34細胞にhSOD1のG93A突然変異型を伝達した時、細胞の死滅を誘導することができるという報告が知られている(Cheema et al.,2005)。したがって、本試験では、human SOD1野生型遺伝子(wildtype; WT) (NM_000454)とhuman SOD1遺伝子の93番目アミノ酸配列をグリシンからアラニンに変更した遺伝子をそれぞれpCKベクターのBamHI部位(site)に挿入し、pCK−hSOD1−野生型とpCK−hSOD1−G93Aを制作した。制作したプラスミドを利用し、hSOD1−G93Aを伝達したNSC−34細胞においてpCK−HGFX7の効能を確認するために、下記のような実験を進行した。
配列番号11:GGC AGA CAG TGA CCA TCT TT
配列番号12:AGT GGA CCT GAG GTT TAT TG
配列番号13:CCA TCA ATC AAA GCC AAG CA
配列番号14:AGC CTT CAC GCA AGT TCA GG
配列番号15:CCA TCA CTG CCA CTC AGA AGA C
配列番号16:TCA TAC TTG GCA GGT TTC TCC
ALSの原因の1つとしてSOD1(Superoxide dismutase 1)の突然変異が明かされたことにより、この遺伝子を利用したALSマウスモデルが開発されて、現在全世界のALS研究者たちがこの動物モデルを利用して多様な研究を行っている(Weydt P et al., 2003)。その中、広く使われるB65JL−Tg(SOD1*G93A)1Gur/J(002726)を選定して実験に使用した。ALSマウスの制作は、ウジョンBSC(Korea)に依頼し、genotyping過程を経て突然変異型のSOD1遺伝子を発現するかどうかを確認した後、本実験に使用した。
Claims (12)
- HGF(Hepatocyte Growth Factor)の2つ以上のアイソフォーム又は前記アイソフォームをコードするポリヌクレオチドを有効成分として含む筋萎縮性側索硬化症(Amyotrophic Lateral Sclerosis)の予防又は治療用薬学的組成物。
- 前記HGFの2つ以上のアイソフォームは、全長HGF(full length HGF; flHGF)及び欠損型HGF(deleted variant HGF; dHGF)を含むことを特徴とする請求項1に記載の組成物。
- 前記全長HGFは、配列番号1のアミノ酸配列を含み、前記欠損型HGFは、配列番号2のアミノ酸配列を含むことを特徴とする請求項2に記載の組成物。
- 前記HGFの2つ以上のアイソフォームは、別途のヌクレオチド配列によりコードされることを特徴とする請求項1に記載の組成物。
- 前記HGFの2つ以上のアイソフォームは、単一のヌクレオチド配列によりコードされることを特徴とする請求項1に記載の組成物。
- 前記ポリヌクレオチドは、ネイキッドDNA(naked DNA)であるか、あるいは、遺伝子運搬体に含まれていることを特徴とする請求項1に記載の組成物。
- 前記遺伝子運搬体は、ベクターであることを特徴とする請求項6に記載の組成物。
- 前記ベクターは、プラスミドであることを特徴とする請求項7に記載の組成物。
- 前記プラスミドは、pCKであることを特徴とする請求項8に記載の組成物。
- HGF(Hepatocyte Growth Factor)の2つ以上のアイソフォーム又は前記アイソフォームをコードするポリヌクレオチドを有効成分として含む組成物を哺乳動物に投与する段階を含む、筋萎縮性側索硬化症(Amyotrophic Lateral Sclerosis)の予防又は治療方法。
- 前記HGFの2つ以上のアイソフォームは、全長HGF(full length HGF; flHGF)及び欠損型HGF(deleted variant HGF; dHGF)を含むことを特徴とする請求項10に記載の方法。
- 前記全長HGFは、配列番号1のアミノ酸配列を含み、前記欠損型HGFは、配列番号2のアミノ酸配列を含むことを特徴とする請求項11に記載の方法。
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