JP2019534018A - Rabエスコートタンパク質の力価アッセイ - Google Patents
Rabエスコートタンパク質の力価アッセイ Download PDFInfo
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- JP2019534018A JP2019534018A JP2019523685A JP2019523685A JP2019534018A JP 2019534018 A JP2019534018 A JP 2019534018A JP 2019523685 A JP2019523685 A JP 2019523685A JP 2019523685 A JP2019523685 A JP 2019523685A JP 2019534018 A JP2019534018 A JP 2019534018A
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Abstract
Description
[0074]簡単なウェスタンアッセイは、適切化された定量的アッセイの例である(WES)。再現性、定量、結果までの時間及び全データの信頼性に影響する使用される手動のプロセスの変動性は排除される。
[0078]ビオチン組み込みは、DyLight800を用いてストレプトアビジンコンジュゲートを使用することにより分析した。REP−1発現は、一次抗REP1−抗体、クローン2F1(MABN52)、及びDyLight680とコンジュゲートさせた二次ヤギ抗マウスIgG(H+L)抗体を用いて検出した。プレニル化されていないRabだけが、インビトロプレニル化アッセイで標識されて、分子量約20〜25kDaのバンドとして可視化された。イムノブロットをOdyssey赤外撮像装置(LiCOR)を使用して走査した。
[0084]RPE細胞中のRep−1及びRabタンパク質について本明細書で例示したプレニル化の力価アッセイは、RPE及び他のタイプの細胞において、プレニル化プロセスで決定的に重要な他のタンパク質(例えばRep−2)、又はプレニル化のための主要な基質(記載された約70種のRabタンパク質)についても使用することができる。例えば、網膜における他の対応する遺伝子の突然変異が、コーン−ロッドジストロフィー(CRD)、先天性停止性夜盲症(CSNB)、ジュベール症候群(JS)、レーバー先天性黒内障(LCA)、メバロン酸キナーゼ欠損症(MKD)、網膜疾患(RD)及び網膜色素変性症(RP)の原因となることが発見された。
[0001]本出願は、2016年11月6日出願の米国特許仮出願第62/418,637号に対する優先権を主張する。前述の出願の全内容は、本文、表、配列表及び図面の全てを含めて参照により本明細書に組み込まれる。
Claims (37)
- REP−1又は活性を測定するための方法であって、
(a)内因性機能的REP−1タンパク質を発現しない細胞を、REP−1タンパク質をコードするCHM遺伝子を含むアデノ随伴ウイルス(AAV)ベクターと、細胞への形質導入を可能にする条件下で接触させるステップと、
(b)形質導入された細胞を、コードされた前記REP−1タンパク質の発現を可能にする条件下でインキュベートするステップと、
(c)前記形質導入された細胞を溶解して、前記コードされたREP−1タンパク質及びRab低分子GTPアーゼ(Rab)を含む抽出物を産生するステップと、
(d)前記抽出物とRab基質とを、前記Rabのプレニル化を可能にする期間及び条件下でインキュベートして、それによりプレニル化Rabを形成するステップと、
(e)前記プレニル化Rabを検出及び/又は定量するステップであり、プレニル化Rabの量がREP−1活性を反映し、それによりREP−1活性を測定するステップと
を含む方法。 - 前記REP−1タンパク質が哺乳動物のものである、請求項1に記載の方法。
- 前記REP−1タンパク質がヒトREP−1を含む、請求項1に記載の方法。
- 前記細胞がREP−1陰性細胞(CHMneg)を含む、請求項1〜3のいずれか一項に記載の方法。
- 前記細胞がREP−1陰性RPE細胞(CHMneg)を含む、請求項1〜4のいずれか一項に記載の方法。
- 前記細胞がヒトREP−1陰性細胞(CHMneg)を含む、請求項1〜5のいずれか一項に記載の方法。
- 前記細胞がヒトREP−1陰性RPE細胞(CHMneg)を含む、請求項1〜6のいずれか一項に記載の方法。
- 前記細胞が、REP−1を安定的又は一過性に発現する、請求項1〜7のいずれか一項に記載の方法。
- 前記Rab基質がゲラニル部分を含む、請求項1〜8のいずれか一項に記載の方法。
- 前記Rab基質が、検出可能に標識されている、請求項1〜9のいずれか一項に記載の方法。
- 前記Rab基質が、非放射性に標識されている、請求項1〜9のいずれか一項に記載の方法。
- 前記Rab基質が、放射性に標識されている、請求項1〜11のいずれか一項に記載の方法。
- 前記Rab基質が、GGPPである、請求項1〜12のいずれか一項に記載の方法。
- 前記Rab基質が、ビオチニル化ゲラニル部分を含む、請求項1〜13のいずれか一項に記載の方法。
- 前記Rab基質がBGPPを含む、請求項1〜14のいずれか一項に記載の方法。
- ステップ(c)が、前記抽出物からRabを単離又は精製することをさらに含む、請求項1〜15のいずれか一項に記載の方法。
- ステップ(d)又は(e)が、前記プレニル化Rabを単離又は精製することをさらに含む、請求項1〜16のいずれか一項に記載の方法。
- 前記プレニル化Rabが、検出可能な標識により検出及び/又は定量される、請求項1〜17のいずれか一項に記載の方法。
- 前記プレニル化Rabが、非放射性の検出可能な標識により検出及び/又は定量される、請求項1〜18のいずれか一項に記載の方法。
- 前記プレニル化Rabが、ELISAにより検出及び/又は定量される、請求項1〜17のいずれか一項に記載の方法。
- 前記プレニル化Rabが、ウェスタンブロット又はウェスタンポリペプチドサイズに基づく(WES)アッセイにより検出及び/又は定量される、請求項1〜17のいずれか一項に記載の方法。
- 前記プレニル化Rabが、質量分析法により検出及び/又は定量される、請求項1〜17のいずれか一項に記載の方法。
- 前記プレニル化Rabが、放射性の検出可能な標識により検出及び/又は定量される、請求項1〜17のいずれか一項に記載の方法。
- 前記REP−1活性を、対照のREP−1陽性細胞(CHMpos)のREP−1活性と比較するステップをさらに含む、請求項1〜23のいずれか一項に記載の方法。
- 前記REP−1活性を、対照のREP−1陽性RPE細胞(CHMpos)のREP−1活性と比較するステップをさらに含む、請求項1〜24のいずれか一項に記載の方法。
- 前記REP−1陰性細胞(CHMneg)が、コロイデレミアを有する患者に由来するか又は前記患者から得られる、請求項1〜24のいずれか一項に記載の方法。
- 前記REP−1陰性細胞(CHMneg)が、コロイデレミアを有する患者から得られるiPS細胞から誘導される、請求項1〜24のいずれか一項に記載の方法。
- 前記対照のREP−1陽性細胞(CHMpos)が、コロイデレミアを有しない対象から得られるか又は前記対象に由来する、請求項24又は25に記載の方法。
- 前記対照のREP−1陽性細胞(CHMpos)が、コロイデレミアを有しない対象から得られるiPS細胞から誘導される、請求項24又は25に記載の方法。
- 前記対照のREP−1陽性細胞(CHMpos)が、コロイデレミアを有する対象から得られるか又は前記対象に由来し、前記対照細胞に、REP−1をコードするCHM遺伝子が安定的又は一過性に形質導入されている、請求項24又は25に記載の方法。
- 前記対照のREP−1陽性細胞(CHMpos)が、コロイデレミアを有する対象から得られるiPS細胞から誘導され、前記対照細胞又はiPS細胞に、REP−1をコードするCHM遺伝子が安定的又は一過性に形質導入されている、請求項24又は25に記載の方法。
- 前記REP−1陰性細胞(CHMneg)及び/又は前記REP−1陽性(CHMpos)細胞がRPE細胞を含む、請求項25〜31のいずれか一項に記載の方法。
- 前記RPE細胞が、iPS細胞から誘導されるか又は分化したものである、請求項32に記載の方法。
- 前記REP−1陰性RPE細胞が、iPSC−RPE−CHMneg細胞を含む、請求項5、7、26、27、30及び32のいずれか一項に記載の方法。
- 前記REP−1陽性RPE細胞が、iPSC−RPE−CHMpos細胞を含む、請求項24、25及び28〜32のいずれか一項に記載の方法。
- 前記アデノ随伴ウイルス(AAV)ベクターが、AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、及びAAV10から選択される任意の血清型のカプシドタンパク質配列又は逆位末端反復配列に対して70%以上の配列同一性を有するカプシドタンパク質配列又は逆位末端反復配列を含む、請求項1〜35のいずれか一項に記載の方法。
- 前記アデノ随伴ウイルス(AAV)ベクターが、AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10から選択される任意の血清型のカプシドタンパク質又は逆位末端反復を含む、請求項1〜35のいずれか一項に記載の方法。
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