Name: in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease
Uploaded: 2017-12-20T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

Les auteurs aimeraient remercier Mandy lenainquitousse et Tina Czajka excellent soutien technique. Nous remercions langue édition aide Flora Luo (Harvard Medical School, Boston, MA, é.-u.).

1. préparation du Mutant pcDNA3.1/GLA et pcDNA3.1/GAA constructions
Remarque : Les stratégies de clonage pour la GLA et GAA (cds) de séquences codantes ont été signalés plus tôt15,18.
<ol>
	<li>Mutagenèse dirigée à l’aide de mutagenèse
	<ol>
		<li>La référence des séquences NM_000169.2 et NM_000152.4 en tant que modèles pour la mutagénèse des gènes GLA et GAA ,...

<ol>
	<li>Ishii, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-galactosidase+A+enzymes+identified+in+Fabry+disease+patients+with+residual+enzyme+activity:+biochemical+characterization+and+restoration+of+normal+intracellular+processing+by+1-deoxygalactonojirimycin.">Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.</a> Biochem J. 406, 285-295 (2007).</li><li>Tajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biochemical+studies+on+Pompe+disease+and+a+&amp;#34;pseudodeficiency+of+acid+alpha-glucosidase&amp;#34.">Structural and biochemical studies on Pompe disease and a &amp;#34;pseudodeficiency of acid alpha-glucosidase&amp;#34.</a> J Hum Genet. 52, 898-906 (2007).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+Clinical+Consequences+of+Novel+&#945;-Galactosidase+A+Mutations+in+Fabry+Disease.">Functional and Clinical Consequences of Novel &#945;-Galactosidase A Mutations in Fabry Disease.</a> Hum Mutat. 37, 43-51 (2016).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+lysosomal+storage+diseases+with+pharmacological+chaperones:+from+concept+to+clinics.">Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics.</a> EMBO Mol Med. 1, 268-279 (2009).</li><li>Yasuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry+disease:+characterization+of+alpha-galactosidase+A+double+mutations+and+the+D313Y+plasma+enzyme+pseudodeficiency+allele.">Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.</a> Hum Mutat. 22, 486-492 (2003).</li><li>Shimotori, M., Maruyama, H., Nakamura, G., Suyama, T., Sakamoto, F., Itoh, M., Miyabayashi, S., Ohnishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mutations+of+the+GLA+gene+in+Japanese+patients+with+Fabry+disease+and+their+functional+characterization+by+active+site+specific+chaperone.">Novel mutations of the GLA gene in Japanese patients with Fabry disease and their functional characterization by active site specific chaperone.</a> Hum Mutat. 29, 331 (2008).</li><li>Andreotti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+of+Fabry+disease+with+pharmacological+chaperones:+from+in+silico+predictions+to+in+vitro+tests.">Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests.</a> Orphanet J Rare Dis. 6, 66 (2011).</li><li>Khanna, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacological+chaperone+AT2220+increases+the+specific+activity+and+lysosomal+delivery+of+mutant+acid+alpha-glucosidase,+and+promotes+glycogen+reduction+in+a+transgenic+mouse+model+of+Pompe+disease.">The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.</a> PLoS One. 9, e102092 (2014).</li><li>Siekierska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Galactosidase+aggregation+is+a+determinant+of+pharmacological+chaperone+efficacy+on+Fabry+disease+mutants.">&#945;-Galactosidase aggregation is a determinant of pharmacological chaperone efficacy on Fabry disease mutants.</a> J Biol Chem. 287, 28386-28397 (2012).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+enhancement+of+mutated+alpha-glucosidase+activity+in+fibroblasts+from+patients+with+Pompe+disease.">Pharmacological enhancement of mutated alpha-glucosidase activity in fibroblasts from patients with Pompe disease.</a> Mol Ther. 15, 508-514 (2007).</li><li>Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacogenetic+approach+to+identify+mutant+forms+of+&#945;-galactosidase+A+that+respond+to+a+pharmacological+chaperone+for+Fabry+disease.">A pharmacogenetic approach to identify mutant forms of &#945;-galactosidase A that respond to a pharmacological chaperone for Fabry disease.</a> Hum Mutat. 32, 965-977 (2011).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterisation+of+alpha-galactosidase+a+mutations+as+a+basis+for+a+new+classification+system+in+fabry+disease.">Functional characterisation of alpha-galactosidase a mutations as a basis for a new classification system in fabry disease.</a> PLoS Genet. 9, e1003632 (2013).</li><li>Andreotti, G., Citro, V., Correra, A., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermodynamic+assay+to+test+pharmacological+chaperones+for+Fabry+disease.">A thermodynamic assay to test pharmacological chaperones for Fabry disease.</a> Biochim Biophys Acta. 1840, 1214-1224 (2014).</li><li>. Available from: <a target="_blank" href="https://www.google.ch/patents/US9095584">https://www.google.ch/patents/US9095584</a> (2017)</li><li>Lukas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+enhancers+for+the+treatment+of+Fabry+and+Pompe+disease.">Enzyme enhancers for the treatment of Fabry and Pompe disease.</a> Mol Ther. 23, 456-4564 (2015).</li><li>Yam, G. H., Bosshard, N., Zuber, C., Steinmann, B., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+chaperone+corrects+lysosomal+storage+in+Fabry+disease+caused+by+trafficking-incompetent+variants.">Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants.</a> Am J Physiol Cell Physiol. 290, C1076-C1082 (2006).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+pharmacological+chaperones+in+Fabry+disease.">Screening for pharmacological chaperones in Fabry disease.</a> Biochem Biophys Res Commun. 359, 168-173 (2007).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+response+of+mutated+alpha-galactosidase+A+to+a+pharmacological+chaperone.">Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.</a> Pharmacogenet Genomics. 18, 773-7780 (2008).</li><li>Filoni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+of+new+GLA+gene+mutations+leading+to+conformational+Fabry+disease.">Functional studies of new GLA gene mutations leading to conformational Fabry disease.</a> Biochim Biophys Acta. 1802, 247-252 (2010).</li><li>Citro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Large+Phenotypic+Spectrum+of+Fabry+Disease+Requires+Graduated+Diagnosis+and+Personalized+Therapy:+A+Meta-Analysis+Can+Help+to+Differentiate+Missense+Mutations.">The Large Phenotypic Spectrum of Fabry Disease Requires Graduated Diagnosis and Personalized Therapy: A Meta-Analysis Can Help to Differentiate Missense Mutations.</a> Int J Mol Sci. 17, (2016).</li><li>Cammisa, M., Correra, A., Andreotti, G., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry_CEP:+a+tool+to+identify+Fabry+mutations+responsive+to+pharmacological+chaperones.">Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.</a> Orphanet J Rare Dis. 8, 111 (2013).</li><li>Leinekugel, P., Michel, S., Conzelmann, E., Sandhoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+correlation+between+the+residual+activity+of+beta-hexosaminidase+A+and+arylsulfatase+A+and+the+severity+of+the+resulting+lysosomal+storage+disease.">Quantitative correlation between the residual activity of beta-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease.</a> Hum Genet. 88, 513-523 (1992).</li><li>Germain, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+pharmacodynamic+effects+of+a+pharmacological+chaperone+on+&#945;-galactosidase+A+activity+and+globotriaosylceramide+clearance+in+Fabry+disease:+report+from+two+phase+2+clinical+studies.">Safety and pharmacodynamic effects of a pharmacological chaperone on &#945;-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies.</a> Orphanet J Rare Dis. 7, 91 (2012).</li><li>Hughes, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+pharmacological+chaperone+migalastat+compared+with+enzyme+replacement+therapy+in+Fabry+disease:+18-month+results+from+the+randomised+phase+III+ATTRACT+study.">Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.</a> J Med Genet. 54, 288-296 (2017).</li><li>Parenti, G., Andria, G., Valenzano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Chaperone+Therapy:+Preclinical+Development,+Clinical+Translation,+and+Prospects+for+the+Treatment+of+Lysosomal+Storage+Disorders.">Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.</a> Mol Ther. 23, 1138-1148 (2015).</li><li>Parenti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Chaperone+Enhances+Blood+&#945;-Glucosidase+Activity+in+Pompe+Disease+Patients+Treated+With+Enzyme+Replacement+Therapy.">A Chaperone Enhances Blood &#945;-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy.</a> Mol Ther. 22, 2004-2012 (2014).</li></ol>

Le protocole décrit ci-après fournit des résultats robustes pour l’évaluation des dommages enzyme dans les maladies lysosomales héréditaires du métabolisme. Ce manuscrit est qu'un amendement au protocole publiée plus tôt15. Les plus importantes modifications impliquent la rigueur (c'est-à-dire, dans le processus de préparation de construction de vecteur mutant), normalisation de la protocole de culture cellulaire (p. ex., conditions de maintenance et de transfection ...

Il existe plus d’une douzaine lysosomales (DSL) associés au dysfonctionnement de glycosidase par suite de mutations géniques primaires. Fabry (OMIM #301500) et la maladie de Pompe (OMIM #232300), plus de 500 et 200 mutations faux-sens ont1,2,3 , respectivement, ce qui correspond à environ 60 % du nombre total de mutation. Nombreuses nouvelles variantes du gène sont toujours identifiés, beaucoup dont ayez inconnu signification. Des études biochimiques ont révélé que certains génotypes ne conduisent pas à une perte de fonction complète du gène GLA (OMIM * 300644) dans la maladie de Fabry, mais provoquer l’enzyme correspondante à l’échec atteindre un État thermodynamiquement favorisé pliant4 . Cela se traduit par la rétention de l’ER et la dégradation prématurée de l’enzyme fonctionnelle dans le cas contraire. Des conclusions similaires ont été tirées dans autres DSL, y compris la maladie de Pompe5. De plus, la caractérisation moléculaire des variantes de l’enzyme peut faciliter l’interprétation clinique des mutations au moment du diagnostic6, ce qui suggère que la progression de LSD est un processus individualisé, basé sur la nature de la mutation. Par conséquent, la classification conventionnelle en généralement de 2 à 3 différents types cliniques devrait être réévaluée afin de rationaliser les conseils cliniques et les décisions thérapeutiques.
Enzymatique substitutive (ERT) est disponible pour ces deux maladies. ERT, cependant, a une efficacité limitée en tissus/organes touchés comme le cerveau et les muscles squelettiques. En outre, ERT peut provoquer une réaction d’immunogène qui met en péril ses bienfaits thérapeutiques. Chaperons pharmacologiques (PCs) sont une alternative séduisante de traitement pour les patients présentant des mutations réactives ce qu’on appelle. SCP service un échafaudage moléculaire pour le repliement des protéines correcte et de stabilisation, qui à son tour empêche la rétention de réticulum endoplasmique (re) et ER-associés de la dégradation de l’enzyme. En outre, PCs peuvent être administrés par voie orale et sont potentiellement capables de traverser la barrière hémato-encéphalique. Par conséquent, le PCT peut être une option plus viable pour le traitement des patients atteints de certains génotypes. Pour une étude approfondie sur l’application de PC en DSL, veuillez consulter l’excellente revue par Parenti7.
La découverte de centaines d’allèles mutants pathogène défie le dépistage des drogues précliniques et nécessite une évaluation simple, rapide et hautement normalisée des patients se prêtent à une approche de la médecine personnalisée. Afin d’évaluer les effets néfastes des mutations du gène LSD et de tester des mutations de candidat pour prédire les patients se prêtent pour le PCT, un système très standardisé surexpression dans les cellules HEK293H qui permet de mesurer l’activité enzymatique rapide et fiable a été mis au point. Des systèmes similaires de surexpression ont été décrits précédemment pour la maladie de Fabry et Pompe utilisant COS-78,9,10,11, HeLa cellules12ou HEK29313 ,14,15,16 cellules pour le gène glycosidase.
Une méthode très similaire a même été brevetée comme « Méthode pour prédire la réponse au traitement pharmacologique chaperon des maladies »17 indiquant la pertinence d’une cellule culture système capable de s’intégrer dans la pratique clinique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL Site-Directed Mutagenesis Kit&#160;</td>
			<td>Stratagene, La Jolla, CA, USA</td>
			<td>#200522</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-frw: 5&#180;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-rev: 5&#180;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-rev: 5&#180;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A143T]-rev: 5&#180;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-frw: 5&#180;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-rev: 5&#180;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-frw: 5&#180;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[L552P]-frw: 5&#180;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer T7: 5&#180;-TAA TAC GAC TCA CTA TAG GG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer BGHrev: 5&#180;-TAG AAG GCA CAG TCG AGG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>103,753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,049,360,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,058,330,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,083,422,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,980,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>3908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X863.3</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,065,860,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,062,640,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>ZymoResearch, Freiburg, Germany</td>
			<td>D4020</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAfilter Plasmid Midi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12245</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium&#160;</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum&#160;</td>
			<td>GE Healthcare, South Logan, Utah, USA</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td>
			<td>VWR International GmbH, Hannover, Germany</td>
			<td>82050-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (24 well)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>831,836</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 96 well plate</td>
			<td>Greiner bio one, Frickenhausen, Germany</td>
			<td>655185</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1,5mL&#160;</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9641</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Toronto Research Chemicals, Toronto, Canada</td>
			<td>D236500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxynojirimycin&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9305</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Calcium, w/o Magnesium</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-galactopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M7633</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-glucopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M9766</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incubator type T6</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminous Plate Gr. 2 E</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems, Darmstadt, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GFL-3032 bacterial shaker</td>
			<td>GFL, Burgwedel, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-25 centrifuge</td>
			<td>Beckman/Coulter, Krefeld, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraspec 3100 pro Spectrophotometer</td>
			<td>Amersham Biosciences, Buckinghamshire, United Kingdom</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>water-jacket incubator&#160;</td>
			<td>Binder, Tuttlingen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 1 touch mixer</td>
			<td>Scientific Industries, Bohemia, NY, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Z 233 MK-2 refrigerated microtube centrifuge</td>
			<td>Hermle, Tuttlingen, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboStar ultrapure water device</td>
			<td>Siemens, Berlin, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker PST-60HL-4 orbital shaker</td>
			<td>Biosan, Riga, Latvia</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>HI 223 Microprocessor pH Meter</td>
			<td>HANNA Instruments, Arvore, Portugal</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY 
			Cell Counter + Analyser System 
			Model TT</td>
			<td>Innovatis AG, Cham/Zug, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.04</td>
			<td>GraphPad Software, Inc., La Jolla, CA; USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>BioEdit Alignment Editor, V7.0.1</td>
			<td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbreviations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>frw = forward; rev = reverse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n.a. = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

L’utilisation de la médecine personnalisée pour traiter les maladies monogéniques rares comme lysosomales (DSL) est contestée par des conceptions du procès cliniques complexes, des coûts élevés et faible nombre de patients. Des centaines d’allèles mutants sont impliqués dans la plupart de la DSL. Les maladies sont généralement classés en 2 ou 3 types cliniques différentes selon la gravité. De plus, la caractérisation moléculaire du génotype peut aider à prédire les résultats cliniques et informer des soins aux patients. Donc, nous avons mis au point un essai de culture de cellule simple basé sur des cellules de HEK293H façon hétérologue surexprimant les mutations identifiées dans la maladie de Fabry et Pompe. Un test semblable a récemment été introduit comme un test préclinique pour identifier les mutations se prête pour la thérapie pharmacologique de chaperon (PCT) la maladie de Fabry. Ce manuscrit décrit un essai de culture de cellule modifiée qui permet une évaluation rapide de phénotypique des variants alléliques dans la maladie de Fabry et Pompe pour identifier les patients éligibles pour le PCT et peut aider au développement de nouveaux pharmacochaperones.

La procédure de mutagenèse Afin d’évaluer l’efficacité de la mutagénèse gène GLA , les mutations ont été classées dans une des catégories suivantes. Cette approche pour générer des mutations a révélé qu’environ 66,5 % des mutations GLA ont été obtenus dans la première tentative. Un autre 25 % pourrait être obtenu après une PCR deuxième légèrement modifiée.
Cat?...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Il existe une demande pour faire précliniques Essais pour une nouvelle classe de médicaments « orphelins » appelées chaperons pharmacologiques reproductible, rapide et efficace. Nous avons développé un test culture cellule simple, très standardisé et polyvalent à écran pour patients admissibles ainsi que des médicaments nouveaux chaperons pharmacologiques.

In Vitro Mesure d’enzyme à Test pharmacologique chaperon réactivité Fabry et la maladie de Pompe

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

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