JP5877617B2 - 毒素産生性試験のための組成物および方法 - Google Patents
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Description
本発明の理解を促すため、いくつかの用語および語句を以下に定義する。
本明細書に記載されている通り、本発明の実施形態は、BoNTなどの病原因子の検出および分析において使用するための多能性由来幹細胞を提供する。いくつかの実施形態において、細胞はヒト(例えば、ヒト人工多能性幹由来細胞(hiPS,human induced pluripotent stem derived cells)由来神経細胞またはヒト胚性幹細胞)である。iPS細胞を生成する方法は、例えば、Yuら、Science.2009年5月8日;324(5928):797〜801頁.Epub 2009、WO2011056971およびWO2011025852に記載され、これらの各々は、その全体が参照により本明細書に組み込まれている。いくつかの実施形態において、iPS細胞は、適切な方法(例えば、米国特許出願US2010/0279403および米国特許出願US2010/0216181に記載されているもの(これらの各々は、その全体が参照により本明細書に組み込まれている))を用いて神経細胞に分化させる。
本発明の実施形態は、BoNTをアッセイするための組成物および方法を提供する。アッセイの、研究適用、臨床適用、診断適用および治療適用における使用が見出される。
なお、本願は特許請求の範囲に記載された発明に関するものであるが、他の態様として以下も包含し得る:
1.ボツリヌス菌(Clostridium botulinum)神経毒素(BoNT)を活性についてアッセイする方法であって、
a)ヒト人工多能性幹細胞(hiPS)由来神経細胞を、BoNTを含む組成物と接触させること;および
b)生物学的活性について前記BoNTをアッセイすること、
を含む方法。
2.前記BoNTが、A、B、C、Eおよび前記BoNTの改変バリアントからなる群から選択される血清型を有する、上記1の方法。
3.前記生物学的活性が、SNAP−25の切断、VAMP2の切断および神経伝達物質放出からなる群から選択される、上記1の方法。
4.前記アッセイが定性的である、上記1の方法。
5.前記アッセイが定量的である、上記1の方法。
6.前記BoNTが精製されている、上記1の方法。
7.前記BoNTが複合体中にある、上記1の方法。
8.前記hPS由来神経細胞と接触させる前に、前記BoNTを試験化合物と接触させるステップをさらに含む、上記1の方法。
9.前記試験化合物が抗体である、上記8の方法。
10.前記抗体が中和抗体である、上記9の方法。
11.前記中和抗体が、精製された抗体、血清および抗毒素からなる群から選択される試料中にある、上記10の方法。
12.ボツリヌス菌神経毒素(BoNT)を活性についてアッセイする方法であって
a)ヒト人工多能性幹(hiPS)細胞由来神経細胞を、i)BoNT、およびii)中和抗体を含む組成物と接触させること;および
b)生物学的活性について前記BoNTをアッセイすること、
を含む方法。
13.前記BoNTが、A、B、C、Eおよび前記BoNTの改変バリアントからなる群から選択される血清型を有する、上記12の方法。
14.前記生物学的活性が、SNAP−25の切断、VAMP2の切断および神経伝達物質放出からなる群から選択される、上記12の方法。
15.前記アッセイが定性的である、上記12の方法。
16.前記アッセイが定量的である、上記12の方法。
17.前記BoNTが精製されている、上記12の方法。
18.前記BoNTが複合体中にある、上記1の方法。
19.前記中和抗体が、精製された抗体、血清および抗毒素からなる群から選択される試料中にある、上記12の方法。
20.生物学的に活性なBoNTを含む調製物中の生物学的に活性なBoNTの量を測定する方法であって、以下のステップ:
(a)hiPS細胞由来神経細胞を、生物学的に活性なBoNTを含む調製物の試料と接触させること;および
(b)BoNTの生物学的活性について前記試料をアッセイすることにより、調製物中に存在する生物学的に活性なBoNTの量を測定すること、
を含む前記方法。
21.BoNTを活性についてアッセイするための、ヒト人工多能性幹(hiPS)細胞由来神経細胞の使用。
以下の例は、本発明の特定の好ましい実施形態および態様を実証し、さらに例示するために提供されるものであり、本発明の範囲を限定するものとして解釈すべきではない。
A.方法
神経細胞:ヒトiPS由来神経細胞は、Cellular Dynamics Inc.(マジソン、WI)により凍結して供給された。神経細胞をCellular Dynamics指示書に従って解凍し、生細胞をトリパンブルー排除アッセイによりカウントした。細胞は、特に指示のない限り、0.01%ポリ−L−オルニチン(SIGMA)および8.3μg/cm2マトリゲル(BD Biosciences)でコートした96ウェルディッシュ(TPP、MidSci)に1ウェル当たり40,000細胞密度で播種し、37℃、約5%CO2下で、指示された成熟時間、神経培地(B27およびグルタマックスを補充したNeurobasal、全てInvitrogen製でCDIにより供給された)でインキュベートした。播種24時間後、培地を完全に交換し、その後は培地の半分を2〜3日ごとに交換した。
iPS神経細胞はBoNT中毒に重要な受容体を発現する:ヒトiPS由来神経細胞がBoNT活性を検出するのに使用できるかどうかを判定するため、BoNT細胞侵入および触媒活性に必要な受容体および酵素標的の発現を、それぞれウエスタンブロットおよび定量的PCR(qPCR)により分析した(図1)。ウエスタンブロットは、SV2Aに対するシグナル、SV2B、シナプトタグミン1、シンタキシン、SNAP−25、VAMP2、およびベータアクチンに対するかすかなバンドを生じ、これらは細胞播種後21日間にわたり変化しなかった(図1A)。VAMP2はVAMP2特異的抗体により検出されたが、3つのVAMPアイソフォーム全てを認識する抗体はシグナルが得られず、VAMP2がiPS神経細胞における優勢なVAMPアイソフォームであることを示している。初代ラット脊髄細胞溶解物を抗体検出の陽性対照として使用し、iPS神経細胞対RSC細胞のバンドにおける強度の違いは、抗体による特異的認識または異なる発現レベルに起因している可能性がある。qPCRによる同じタンパク質のmRNAレベルの分析は、ウエスタンブロットにより検出されなかったSV2BおよびCアイソフォーム、シナプトタグミン2、ならびにVAMP1および3を含む、分析した全てのタンパク質の発現を示した(図2B)。しかし、これらのアイソフォームのmRNAレベルは、ウエスタンブロットにより検出されたアイソフォーム(SV2A、シナプトタグミン1、およびVAMP2)のmRNAレベルより少なくとも200倍低かった。故に、qPCRデータはウエスタンブロットデータを裏付けるものであり、iPS神経細胞がこれらのタンパク質のSV2A、シナプトタグミン1、およびVAMP2アイソフォーム(前脳神経細胞を代表する神経細胞(Janz R & Sudhof TC(1999)Neuroscience 94: 1279〜1290頁)と一致する)を主に発現することを示している。全てのタンパク質の発現レベルは試験期間を通じて変化せず、細胞が播種後4日目で十分に成熟し、少なくとも21日間安定したままであることを示している。
Claims (23)
- ボツリヌス菌(Clostridium botulinum)神経毒素(BoNT)を活性についてアッセイする方法であって、
a)ヒト人工多能性幹細胞(hiPS)由来神経細胞を、BoNTを含む組成物と接触させること;および
b)生物学的活性について前記BoNTをアッセイすること、
を含む方法。 - 前記BoNTが、A、B、C、Eおよび前記BoNTの改変バリアントからなる群から選択される血清型を有する、請求項1の方法。
- 前記生物学的活性が、SNAP−25の切断、VAMP2の切断および神経伝達物質放出からなる群から選択される、請求項1の方法。
- 前記アッセイが定性的である、請求項1の方法。
- 前記アッセイが定量的である、請求項1の方法。
- 前記BoNTが精製されている、請求項1の方法。
- 前記BoNTが複合体中にない、請求項1の方法。
- 前記BoNTが複合体中にある、請求項1の方法。
- 前記hiPS由来神経細胞と接触させる前に、前記BoNTを試験化合物と接触させるステップをさらに含む、請求項1の方法。
- 前記試験化合物が抗体である、請求項9の方法。
- 前記抗体が中和抗体である、請求項10の方法。
- 前記中和抗体が、精製された抗体、血清および抗毒素からなる群から選択される試料中にある、請求項11の方法。
- ボツリヌス菌神経毒素(BoNT)を活性についてアッセイする方法であって
a)ヒト人工多能性幹(hiPS)細胞由来神経細胞を、i)BoNT、およびii)中和抗体を含む組成物と接触させること;および
b)生物学的活性について前記BoNTをアッセイすること、
を含む方法。 - 前記BoNTが、A、B、C、Eおよび前記BoNTの改変バリアントからなる群から選択される血清型を有する、請求項13の方法。
- 前記生物学的活性が、SNAP−25の切断、VAMP2の切断および神経伝達物質放出からなる群から選択される、請求項13の方法。
- 前記アッセイが定性的である、請求項13の方法。
- 前記アッセイが定量的である、請求項13の方法。
- 前記BoNTが精製されている、請求項13の方法。
- 前記BoNTが複合体中にない、請求項13の方法。
- 前記BoNTが複合体中にある、請求項1の方法。
- 前記中和抗体が、精製された抗体、血清および抗毒素からなる群から選択される試料中にある、請求項13の方法。
- 生物学的に活性なBoNTを含む調製物中の生物学的に活性なBoNTの量を測定する方法であって、以下のステップ:
(a)hiPS細胞由来神経細胞を、生物学的に活性なBoNTを含む調製物の試料と接触させること;および
(b)BoNTの生物学的活性について前記試料をアッセイすることにより、調製物中に存在する生物学的に活性なBoNTの量を測定すること、
を含む前記方法。 - BoNTを活性についてアッセイするための、ヒト人工多能性幹(hiPS)細胞由来神経細胞の使用。
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AU2012315783A1 (en) | 2014-04-17 |
IL231768B (en) | 2019-06-30 |
CN103958747A (zh) | 2014-07-30 |
MX2014003874A (es) | 2014-08-18 |
KR20140086981A (ko) | 2014-07-08 |
EP2761065A4 (en) | 2015-05-06 |
RU2014117161A (ru) | 2015-11-10 |
EP2761065B1 (en) | 2017-12-06 |
SG11201401007SA (en) | 2014-04-28 |
IL231768A0 (en) | 2014-05-28 |
RU2616281C2 (ru) | 2017-04-13 |
HRP20171937T1 (hr) | 2018-02-09 |
MX348188B (es) | 2017-06-01 |
EP2761065A1 (en) | 2014-08-06 |
SI2761065T1 (en) | 2018-02-28 |
WO2013049508A1 (en) | 2013-04-04 |
HK1199911A1 (en) | 2015-07-24 |
US9217172B2 (en) | 2015-12-22 |
PL2761065T3 (pl) | 2018-02-28 |
JP2014528724A (ja) | 2014-10-30 |
KR101640694B1 (ko) | 2016-07-18 |
BR112014007717B1 (pt) | 2021-07-20 |
CA2850531A1 (en) | 2013-04-04 |
LT2761065T (lt) | 2018-01-10 |
AU2012315783B2 (en) | 2015-08-20 |
US20140234857A1 (en) | 2014-08-21 |
BR112014007717A2 (pt) | 2017-04-25 |
HUE037509T2 (hu) | 2018-09-28 |
CN103958747B (zh) | 2017-03-01 |
CA2850531C (en) | 2016-06-07 |
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