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

Summary

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

Abstract

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

Introduction

There are over a dozen lysosomal storage disorders (LSDs) related to glycosidase dysfunction as a result of primary gene mutations. In Fabry (OMIM #301500) and Pompe (OMIM #232300) disease, more than 500 and 200 missense mutations^1,2,3 have been reported, respectively, which corresponds to about 60% of the total mutation count. Numerous new gene variants are still being identified, many of which have unknown significance. Extensive biochemical studies revealed that certain genotypes do not lead to a complete loss-of-function of the GLA gene (OMIM *300644) in Fabry di....

Protocol

1. Preparation of Mutant pcDNA3.1/GLA and pcDNA3.1/GAA Constructs

NOTE: The cloning strategies for the GLA and GAA coding sequences (cds) have been reported earlier^15,18.

1. Site-directed Mutagenesis Using Site-Directed Mutagenesis
   1. Use the reference sequences NM_000169.2 and NM_000152.4 as templates for the mutagenesis of GLA and GAA genes, respectively. Have a set of high purity salt free primers (25-37-mers) synthesized by a commercial provider, with sense and antisense primers carrying one of the respective....

Results

The mutagenesis procedure
To assess the efficiency of GLA gene mutagenesis, the mutations were classified into one of the following categories. This approach to generate mutations revealed that about 66.5% of the GLA mutations were obtained in the first attempt. A further 25% could be obtained after a slightly modified second PCR.

Category 1: The mutagenesis PCR was effective at first attem.......

Discussion

The protocol described herein delivers robust results for enzyme damage assessment in hereditary lysosomal diseases of metabolism. This manuscript is an amendment to the protocol published earlier¹⁵. The most crucial modifications involve stringency (i.e., in the process of mutant vector construct preparation), standardization of the cell culture protocol (i.e., HEK293H cell maintenance and transfection conditions), and the high number of experimental repetitions (at least 5), wh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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