体外酶法检测药物陪护与庞贝氏症的反应
Summary
有一个要求, 使临床前测试的一个新的类 “孤儿” 药物称为药理伴侣可再生, 快速, 高效。我们开发了一个简单, 高度标准化, 多功能细胞文化检测, 以筛选合格的病人以及新的药理陪护药物。
Abstract
使用个性化药物治疗罕见的基因疾病, 如溶酶体存储紊乱 (LSDs) 是挑战的复杂的临床试验设计, 高成本, 和低的病人人数。在大多数的 LSDs 中, 有数百个变种等位基因被牵连。根据病情轻重, 这些疾病通常分为2到3种不同的临床类型。此外, 基因型的分子特性可以帮助预测临床结果和告知病人护理。因此, 我们开发了一个简单的细胞培养方法的基础上 HEK293H 细胞 heterologously 发酵的突变发现的法布里和庞贝氏病。最近还引入了类似的检测方法作为临床前检查, 以确定在法布里病中药物陪护治疗 (PCT) 的易变异。这份手稿描述了一个修订的细胞培养法, 使位变异的快速表型评估, 以确定符合标准的患者, 并可能有助于发展新的 pharmacochaperones。
Introduction
有十几个溶酶体存储紊乱 (LSDs) 与糖苷功能障碍相关的主要基因突变的结果。在法布里 (OMIM #301500) 和庞贝氏病 (OMIM #232300) 疾病, 超过500和200义突变1,2,3分别报告, 这相当于大约60% 的总变异计数。许多新的基因变种仍在被识别, 其中很多是未知的意义。广泛的生物化学研究表明, 某些基因型不会导致一个完整的功能的玻璃基因 (OMIM * 300644) 在法布里病, 但导致相应的酶未能达到一个热力青睐的折叠状态 4.这导致 ER 保留和过早退化, 否则功能酶。类似的结论也在其他 LSDs 包括庞贝氏病5中得出。此外, 分子特性的酶变体可以促进临床解释的突变在诊断时的6, 这表明 LSD 进展是一个个体的过程中, 基于突变的性质。因此, 为了简化临床咨询和治疗决策, 应重新评估传统的2到3种不同临床类型的分类。
两种疾病都有酶替代疗法。然而, 在受影响的组织/器官, 如大脑和骨骼肌肉的功效有限。此外, 专家审评可以引起免疫的反应, 危及其治疗效益。药理陪护 (PCs) 是一种有吸引力的治疗方案, so-called 反应性突变患者。pc 作为正确的蛋白质折叠和稳定的分子支架, 从而防止内质网 (er) 保留和 er 相关的酶降解。此外, 个人电脑可以口服和潜在的能够跨越血脑屏障。因此, PCT 可能是一个更可行的选择, 以治疗患者的某些基因型。有关 LSDs 中 PC 应用程序的广泛审查, 请参阅 Parenti7的优秀评审。
发现数以百计的疾病, 导致突变等位基因挑战前临床药物测试, 并要求一个简单, 快速, 和高度标准化的评估, 以适合病人的个性化药物的方法。为了评估 LSD 基因突变的不利影响, 并测试候选突变, 以预测适合的患者 PCT, 一个高度标准化的表达系统的 HEK293H 细胞, 允许快速和可靠的酶活性测量开发.类似的表达系统曾被描述为法布里和庞贝氏病, 使用 COS-78,9,10,11, HeLa 细胞12, 或 HEK29313 ,14,15,16糖苷基因的单元格。
一个非常类似的方法甚至被授予专利作为 “预测反应的方法, 药理陪护治疗疾病”17表明的相关性, 细胞培养系统能够融入临床实践。
Protocol
1. 制备突变 pcDNA3.1/pcDNA3.1/GAA 结构 注意: 玻璃和GAA编码序列 (cd) 的克隆策略已在较早的15、18中报告。 用定点诱变的定点诱变 分别使用参照序列 NM_000169.2 和 NM_000152.4 作为玻璃化和 GAA基因突变的模板。有一套高纯度无盐底漆 (25-37-市面) 由一个商业供应商合成, 与意义和反义引…
Representative Results
诱变过程为了评估玻璃化基因突变的效率, 突变被归类为以下类别之一. 这种产生突变的方法显示, 在第一次尝试中, 大约有66.5% 的玻璃化突变得到了. 另有25% 可在稍加修饰的第二 PCR 后获得。 1类: 突变 PCR 在第一次尝试中是有效的。 类别 2 : 第一诱变 PCR…
Discussion
本文所述的协议为遗传性溶酶体代谢疾病的酵素损伤评估提供了健壮的结果。本手稿是对先前发布的15协议的修正。最关键的修改涉及严格 (即, 在突变体的载体构造准备过程中), 细胞培养协议 (即, HEK293H 细胞维护和转染条件) 的标准化, 以及高实验重复次数 (至少 5), 对结果的重现性有很高的贡献。总之, 两个目标基因都很容易获得诱变和多样性的突变可以并行组装。…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者想感谢曼迪 Loebert 和蒂娜 Czajka 的优秀技术支持。我们感谢弗洛拉罗 (哈佛医学院, 波士顿, 马萨诸塞州, 美国) 的语言编辑帮助。
Materials
| Material | |||
| QuikChange II XL Site-Directed Mutagenesis Kit | Stratagene, La Jolla, CA, USA | #200522 | |
| Primer GLA[R301Q]-frw: 5´- CTA ATG ACC TCC AAC ACA TCA GCC C-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GLA[R301Q]-rev: 5´- GGG CTG ATG TGT TGG AGG TCA TTA G-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GLA[A156V]-frw: 5´-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GLA[A156V]-rev: 5´-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GLA[A156V]-frw: 5´-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GLA[A143T]-rev: 5´-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[F455Y]-frw: 5´-CTG CCG GGA GCT TCA GGC CCT ACG A-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[F455Y]-rev: 5´-TCG TAG GGC CTG AAG CTC CCG GCA G-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[P545L]-frw: 5´-CAC CCT ACG TGC TTG GGG TGG TTG G-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[P545L]-rev: 5´-CCA ACC ACC CCA AGC ACG TAG GGT G-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[L552P]-frw: 5´-TTG GGG GGA CCC CCC AGG CGG CCA C-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer GAA[P545L]-rev: 5´-GTG GCC GCC TGG GGG GTC CCC CCA A-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer T7: 5´-TAA TAC GAC TCA CTA TAG GG-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Primer BGHrev: 5´-TAG AAG GCA CAG TCG AGG-3´ | MWG Eurofins Operon, Ebersberg, Germany | n.a. | |
| Tryptone | Carl Roth, Karlsruhe, Germany | 8952.1 | |
| Yeast Extract | Merck, Darmstadt, Germany | 103,753 | |
| Sodium chloride | Merck, Darmstadt, Germany | 1,064,041,000 | |
| Potasium chloride | Merck, Darmstadt, Germany | 1,049,360,500 | |
| MgCl2 x 6H2O | Merck, Darmstadt, Germany | 1,058,330,250 | |
| D (+)-Glucose monohydrate | Merck, Darmstadt, Germany | 1,083,422,500 | |
| Sodium Hydroxide | Merck, Darmstadt, Germany | 1,064,980,500 | |
| Glycine | Carl Roth, Karlsruhe, Germany | 3908.2 | |
| Citric acid | Carl Roth, Karlsruhe, Germany | X863.3 | |
| disodium hydrogen phosphate | Merck, Darmstadt, Germany | 1,065,860,500 | |
| Sodium acetate | Merck, Darmstadt, Germany | 1,062,640,050 | |
| Glacial acetic acid | Sigma Aldrich, Munich, Germany | A6283 | |
| LB Agar | Carl Roth, Karlsruhe, Germany | X969.2 | |
| LB Medium | Carl Roth, Karlsruhe, Germany | X968.2 | |
| Zyppy Plasmid Miniprep Kit | ZymoResearch, Freiburg, Germany | D4020 | |
| QIAfilter Plasmid Midi Kit | Qiagen, Hilden, Germany | 12245 | |
| HEK293H cell line | Invitrogen, Karlsruhe, Germany | 11631-017 | |
| Dulbecco´s Modified Eagle Medium | Invitrogen, Karlsruhe, Germany | 31966-047 | |
| HyClone fetal bovine serum | GE Healthcare, South Logan, Utah, USA | SV30160.03 | |
| Penicillin/streptomycin | Invitrogen, Karlsruhe, Germany | 15140-122 | |
| CELLSTAR Standard Cell Culture Flasks, Greiner Bio One | VWR International GmbH, Hannover, Germany | 82050-854 | |
| Cell culture plates (24 well) | Sarstedt, Nümbrecht, Germany | 831,836 | |
| Cellstar 96 well plate | Greiner bio one, Frickenhausen, Germany | 655185 | |
| SafeSeal reaction tubes, 1,5mL | Sarstedt, Nümbrecht, Germany | 72,706 | |
| Lipofectamine 2000 | Invitrogen, Karlsruhe, Germany | 11668-019 | |
| 1-Deoxygalactonojirimycin Hydrochloride | Sigma Aldrich, Munich, Germany | D9641 | |
| 1-Deoxygalactonojirimycin Hydrochloride | Toronto Research Chemicals, Toronto, Canada | D236500 | |
| 1-Deoxynojirimycin | Sigma Aldrich, Munich, Germany | D9305 | |
| PBS Dulbecco w/o Calcium, w/o Magnesium | Biochrom, Berlin, Germany | L 1825 | |
| Trypsin-EDTA (0.05%), phenol red | Thermo Scientific, Braunschweig, Germany | 25300054 | |
| Pierce BCA Protein Assay Kit | Thermo Scientific, Braunschweig, Germany | 23225 | |
| 4-Methylumbelliferone | Sigma Aldrich, Munich, Germany | M1381 | |
| 4-Methylumbelliferyl α-D-galactopyranoside | Sigma Aldrich, Munich, Germany | M7633 | |
| 4-Methylumbelliferyl α-D-glucopyranoside | Sigma Aldrich, Munich, Germany | M9766 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| incubator type T6 | Heraeus Instruments, Hanau, Germany | n.a. | |
| Luminous Plate Gr. 2 E | Carl Roth, Karlsruhe, Germany | P265.1 | |
| 3130 xl Genetic Analyzer | Applied Biosystems, Darmstadt, Germany | n.a. | |
| GFL-3032 bacterial shaker | GFL, Burgwedel, Germany | n.a. | |
| Avanti J-25 centrifuge | Beckman/Coulter, Krefeld, Germany | n.a. | |
| Ultraspec 3100 pro Spectrophotometer | Amersham Biosciences, Buckinghamshire, United Kingdom | n.a. | |
| water-jacket incubator | Binder, Tuttlingen, Germany | n.a. | |
| Vortex Genie 1 touch mixer | Scientific Industries, Bohemia, NY, USA | n.a. | |
| Z 233 MK-2 refrigerated microtube centrifuge | Hermle, Tuttlingen, Germany | n.a. | |
| LaboStar ultrapure water device | Siemens, Berlin, Germany | n.a. | |
| Thermo Shaker PST-60HL-4 orbital shaker | Biosan, Riga, Latvia | n.a. | |
| Tecan GENios Reader | Tecan, Männedorf, Switzerland | n.a. | |
| HI 223 Microprocessor pH Meter | HANNA Instruments, Arvore, Portugal | n.a. | |
| CASY Cell Counter + Analyser System Model TT |
Innovatis AG, Cham/Zug, Switzerland | ||
| Name | Company | Catalog Number | Comments |
| Software | |||
| Magellan data analysis software v6.6 | Tecan, Männedorf, Switzerland | n.a. | |
| GraphPad Prism 5.04 | GraphPad Software, Inc., La Jolla, CA; USA | n.a. | |
| Microsoft Office 2010 | Microsoft Corporation, Redmond, WA, USA | n.a. | |
| BioEdit Alignment Editor, V7.0.1 | http://www.mbio.ncsu.edu/bioedit/bioedit.html | n.a. | |
| Abbreviations | |||
| frw = forward; rev = reverse | |||
| n.a. = not applicable |
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Tags
In VitroEnzyme MeasurementPharmacological ChaperoneFabry DiseasePompe DiseasePhenotypic AssessmentAllelic VarianceLysosomal Storage DiseasesPrognostic OutcomesTherapeutic DecisionsSite-directed MutagenesisGLA GeneGAA GenePrimer DesignPCRDpn1E.coliPlasmid PurificationSpectrophotometerHEK293H CellsCell Transfection
Citazione di questo articolo
Lukas, J., Knospe, A., Seemann, S., Citro, V., Cubellis, M. V., Rolfs, A. In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease. J. Vis. Exp. (130), e56550, doi:10.3791/56550 (2017).