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.
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.
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 mutations1,2,3 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 disease, but cause the corresponding enzyme to fail to reach a thermodynamically favored folding state4. This results in ER retention and premature degradation of the otherwise functional enzyme. Similar conclusions have been drawn in other LSDs including Pompe disease5. Moreover, molecular characterization of enzyme variants can facilitate clinical interpretation of the mutations at the time of diagnosis6, suggesting that LSD progression is an individual process based on the nature of the mutation. Therefore, the conventional classification into typically 2 to 3 different clinical types should be reassessed in order to streamline clinical counselling and therapeutic decisions.
Enzyme Replacement Therapy (ERT) is available for both diseases. ERT, however, has limited efficacy in affected tissues/organs such as the brain and skeletal muscle. Furthermore, ERT can elicit an immunogenic response that jeopardizes its therapeutic benefits. Pharmacological Chaperones (PCs) are an attractive treatment alternative for patients with so-called responsive mutations. PCs serve as a molecular scaffold for correct protein folding and stabilization which in turn prevents endoplasmic reticulum (ER) retention and ER-associated degradation of the enzyme. Moreover, PCs can be administered orally and are potentially able to cross the blood brain barrier. Therefore, PCT might be a more viable option for treating patients with certain genotypes. For an extensive review on PC application in LSDs, refer to the excellent review by Parenti7.
The discovery of hundreds of disease causing mutant alleles challenges pre-clinical drug testing and necessitates a simple, fast, and highly standardized assessment of amenable patients for a personalized medicine approach. In order to assess the detrimental effects of LSD gene mutations and to test candidate mutations to predict amenable patients for PCT, a highly standardized over-expression system in HEK293H cells that allows for fast and reliable enzyme activity measurement was developed. Similar over-expression systems have been previously described for Fabry and Pompe disease using either COS-78,9,10,11, HeLa cells12, or HEK29313,14,15,16 cells for the glycosidase gene.
A very similar method has even been patented as a "Method to predict response to Pharmacological Chaperone treatment of diseases"17 indicating the relevance of a cell culture system capable of being integrated into clinical practice.
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 earlier15,18.
2. Cultivation of HEK293H Cells
3. pcDNA3.1/GLA and pcDNA3.1/GAA Plasmid Transfection and Treatment of HEK293H
4. Cell Harvest and α-galactosidase A or Acid α-glucosidase Activity Measurement
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 attempt.
Category 2: First mutagenesis PCR failed (no colonies on the plate, no inserted mutation); repetition using the same primer set by increasing the annealing temperature up to 68 °C was effective.
Category 3: More effort had to be undertaken to yield the desired clone (e.g., typically one or more new sets of primers were designed).
α-galactosidase A and acid α-glucosidase enzyme activity measurement
Enzyme activity of the different mutant enzymes was recorded after previous incubation of the transiently transfected cells in the presence or absence of a PC. Table 1 refers to the results for 3 α-galactosidase A and 3 acid α-glucosidase mutations. Data are displayed as (1) absolute values for the substrate turnover (nmol 4-MU* mg protein-1* h-1) and as (2) relative values normalized to the wild type enzyme. Both expressions are useful, since total substrate turnover illustrates the efficiency of the reaction and the sensitivity of the system, while the normalized values can give important hints for the likelihood of the malignancy of the mutation on the one hand and the efficiency of the applied PC treatment on the other hand. For the experimental phase, the work flow depicted in Figure 1 was set up, which scheduled a 60 h incubation period with the compound (due to technical reasons, e.g. fast HEK293H cell growth under the introduced conditions). Even though it has been stated that 10 µM was the approximate maximum achievable plasma concentration for DGJ14, the current protocol uses 20 µM as a reasonable concentration for the purpose of a screening for PC responsiveness as supported by numerous earlier works4,20,21,22,23. Moreover, it has been postulated that higher plasma levels can be reached24.
Figure 1: Work flow of the in vitro enzyme activity measurement. (A) The timeline of the experiment shows a 90 h cell culture effort with relatively little hands-on-time required at the indicated time points. (B) Representative plasmid vectors pcDNA3.1 containing the wild type cds of GLA and GAA are shown. GLA and GAA wild type plasmids and plasmids including the respective inserted mutation of interest were used to transiently transfect HEK293H cells plated in 24 well format. After a period to allow the cells to synthesize the respective gene products and allow for enzyme processing, lysosomal transport, and PC action, the cells were lysed and measured in a fluorescence plate reader and analyzed using the respective software. C: Cell morphology and growth status of the HEK293H cells are shown throughout the time course of the experiment. Scale bars = 100 µm Please click here to view a larger version of this figure.
in vitro enzyme activity | |||||
GLA | native | 20 µM DGJ | |||
mutation (AA) | nmol 4-MUG-gal /mg/h (mean) | % mean (±SD, N) | nmol 4-MUG-gal /mg/h (mean) | % mean (±SD, N) | significance |
p.A143T | 2384.7 | 29.2 (±2.6, 5) | 4223.0 | 52.0 (±4.4, 5) | *** |
p.A156V | 379.2 | 4.3 (±.8, 5) | 1437.5 | 16.3 (±1.3, 5) | **** |
p.R301Q | 777.4 | 8.8 (±1.1, 5) | 3002.6 | 44.2 (±3.6, 5) | **** |
GAA | native | 20 µM DNJ | |||
mutation (AA) | nmol 4-MUG-glu /mg/h (mean) | % mean (±SD, N) | nmol 4-MUG-glu /mg/h (mean) | % mean (±SD, N) | significance |
p.Y455F | 41.1 | 5.4 (±.4, 5) | 246.6 | 31.4 (±2.8, 5) | *** |
p.P545L | 65.7 | 6.6 (±.4, 5) | 166.5 | 16.7 (±1.5, 5) | ** |
p.L552P | 0.0 | 0.0 (±.2, 5) | 104.3 | 10.4 (±1.4, 5) | *** |
Table 1: Enzyme activity of α-galactosidase A and acid α-glucosidase. The table shows representative results for 3 α-galactosidase A and 3 acid α-glucosidase mutants with and without the PCs DGJ or DNJ. Absolute enzyme activity data was corrected for endogenous enzyme activity of the HEK293H cells. To evaluate endogenous enzyme activity, the cells were transfected with a pcDNA3.1 empty vector. The values were 97.5 nmol 4-MU* mg protein-1* h-1 for α-galactosidase A and 41.6 nmol 4-MU* mg protein-1* h-1 for acid α-glucosidase, respectively; enzyme activity values were normalized to wild type enzyme from corresponding experiments, which explains the deviations of relative values between the different mutants. After 5 independent cell culture experiments (N = 5), each carried out in technical duplicates, the standard deviation was not allowed to be >15% of the mean. Ratio T-Tests were used to calculate the difference between untreated and PC-treated enzyme.
*p <0.05, **p <0.01, ***p <0.005, ****p <0.001
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), which highly contributed to the reproducibility of the results. Altogether, both target genes have been easily accessible for mutagenesis and a multiplicity of mutations can be assembled in parallel. A relatively high signal/noise ratio was achieved considering that endogenous enzyme activity within the HEK293H cells was a negligible 1/50 (α-galactosidase A) and 1/20 (acid α-glucosidase) of the over-expressed wild type gene. Thus, there is an excellent resolution between wild type and mutant enzyme. It should be noted that a different amount of total protein was applied for the activity assay of both enzymes (0.5 and 5 µg, respectively), because acid α-glucosidase activity was an order of magnitude lower than that of α-galactosidase A.
As stated above, assay parameters are often controversial, since many laboratories have developed their own assays to investigate lysosomal glycosidases. The systems can differ in cell type, plasmid-vector design, cultivation conditions, and period of drug exposure just to name a few of the numerous parameters. A recently published meta-analysis for Fabry disease demonstrated that enzyme activity recordings (with and without PC) of transfected cell models (e.g., HEK293, COS-7) was largely in accordance with the results obtained from patient-derived cells (mostly lymphocytes or fibroblasts) with regard to the question, whether a mutation is responsive to the PC24. Earlier reports directly comparing patient-derived cells and transfection models demonstrated that over-expression systems reflect the situation in patient cells without compromising the conclusion13,14. It can therefore be concluded that, regardless of the particular protocol, the results obtained by different authors was not changed by the different cellular systems, even though responder definitions can be divergent. The current definition for a responding mutation is 20% relative enzyme activity increase and 5% absolute enzyme activity increase compared to the wild type enzyme after incubation with 20 µM DGJ for 60 h. A comprehensive database, FabryCEP, permits the comparison of results obtained by different experimental approaches for hundreds of α-galactosidase mutants25.
Whether these criteria are sufficient to predict clinical benefits of DGJ and DNJ remains unclear, since the level of activity necessary to prevent symptomatic disease is controversial. A former study suggested that 10 to 15% of residual activity might be sufficient for the system to work properly26. However, in a recently published report from a phase 2 clinical study for DGJ, a 1% activity increase was deemed potentially beneficial for the patient, when the baseline activity was less than 1% of normal27. Recent clinical results suggest that patients predicted to respond to PCs by the responder definition of ≥20% relative increase and ≥3% absolute increase of wild type α-galactosidase A activity in HEK293H cells after incubation with 10 µM DGJ actually derived clinical benefit with disease stabilization or even improvement by renal and cardiac parameters28. Whereas DGJ has already been approved by the FDA and the European Commission as a monotherapy for Fabry disease, the course of PCs such as DNJ and the derivative N-Butyl-DNJ in Pompe disease appears to be different. A monotherapeutical approach with DNJ has been terminated during a Phase II clinical trial due to severe adverse events in some of the patients29. However, PC treatment in combination with approved ERT showed significantly improved results with regards to disease substrate (glycogen) reduction compared to the ERT monotherapy30.
We suggest that the presented approach can be extended to other LSDs and other PCs such as Gaucher, Krabbe, Tay-Sachs/Sandhoff, and GM1 gangliosidosis, but this might not work in specific cases, where for example the signal/noise ratio of the system is insufficient. Care should be taken to select a suitable cell disruption method to prepare a cell lysate for enzyme activity determination, because the addition of detergents into the lysis buffer might be indicative of membrane-bound enzymes such as glucocerebrosidase in Gaucher disease while similar amounts of detergent may harm other enzymes. For α-galactosidase A, a sonication-based cell disruption method should be avoided.
The authors have nothing to disclose.
The authors would like to acknowledge Mandy Loebert and Tina Czajka for excellent technical support. We thank Flora Luo (Harvard Medical School, Boston, MA, USA) for language editing help.
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 |