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Digital microfluidic platform for multiplexing LSD assays in newborn screening
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that this construct allowed not only sufficient MGMT expression for selection and metabolic-based enrichment of transduced cells, but also maintained a-gal A expression at appreciable levels. Future experiments involve assessment of transduction/transplantation experiments in Fabry mice. These results indicate that this MGMT-based drug selection is feasible and would represent a new therapeutic approach for Fabry disease. doi:10.1016/j.ymgme.2010.11.110
Lysosomal enhancement as a therapeutic strategy for Batten disease Michela Palmieri, Alberto di Ronza, Marco Sardiello, Baylor College of Medicine, Houston, Texas, USA Lysosomes represent one of the major degradative compartments of eukaryotic cells. They contain more than 60 acid hydrolases that in a concerted action are able to decompose simple and complex macromolecules and even membranes into their monomeric constituents, thereby allowing their recycling. Therefore, the lysosome plays an important role in cellular function and tissue homeostasis. Neuronal ceroid lipofuscinoses (NCL) are severe neurodegenerative disorders of childhood characterized by the intralysosomal accumulation of autofluorescent ceroid lipopigments, commonly referred to as lipofuscin. The most common form of NCL is the juvenile form (JNCL or Batten disease), which results from mutations in the CLN3 gene. There are no current therapeutic options for Batten disease and, differently from most other lysosomal storage disorders, enzyme replacement therapies or gene therapies based on supplying the deficient gene are technically difficult to conceive due to the membrane localization of the CLN3 protein. We recently demonstrated that the transcription factor EB, TFEB, is able to promote cellular clearance by enhancing lysosomal pathways. Here we obtained lysosomal enhancement by either direct supply of exogenous TFEB or treatment with a chemical able to activate endogenous TFEB. We evaluate in vitro and in vivo the effects of lysosomal enhancement on the clearance of lipofuscin in cells from patients and in a mouse model of Batten disease. doi:10.1016/j.ymgme.2010.11.111
Digital microfluidic platform for multiplexing LSD assays in newborn screening Vamsee Pamulaa, Rama Sistaa, Tong Wanga, Carrie Grahama, Allen Eckhardta, Theodore Wingera, Ning Wua, Yalin Xionga, Vijay Srinivasana, David Millingtonb, Adviye Tolunb, Deeksha Balib, a Advanced Liquid Logic, Research Triangle Park, NC, USA, bDuke University, Durham, USA Digital microfluidics is a flexible technique to manipulate discrete droplets independently and precisely under software control to perform biochemical assays on a disposable cartridge. We have recently applied the platform to perform high-throughput newborn screening tests using dried blood spots (DBS) for lysosomal storage disorders. These assays currently require expensive equipment to process and analyze samples and consume significant amounts of sample. A digital microfluidic platform can greatly reduce assay costs and specimen requirements with an inexpensive desktop instrument and test-specific disposable cartridges. Multiplexed and fully automated enzymatic assays for Pompe, Fabry, Hunter, Hurler, and Gaucher diseases were demonstrated on a disposable cartridge.
S33
Aqueous extracts from 48 DBS punches and 5 enzyme substrates were loaded on a cartridge which is programmed to dispense, transport, and mix droplets of 300 nL with an incubation time under 1 hour. Enzymes in the samples cleave fluorogenic substrates to release 4-methylumbelliferone (4-MU) which was measured using a custom-made fluorimeter. Each cartridge performs 240 distinct enzymatic assays which allows for 5 enzymatic assays on each of the 48 samples. The microfluidic method gives comparable results to a “gold standard” method performed at Duke using much larger sample volumes and longer incubation times. Currently, we are piloting LSD assays in the Illinois Department of Public Health. Using the same cartridges and the instrument, multiplex immunoassays, PCR, and DNA sequencing have also been demonstrated which eventually allows utilizing the same platform for hypothyroidism, cystic fibrosis, galactosemia and hemoglobinopathies, as well as planned expansion to SCID. doi:10.1016/j.ymgme.2010.11.112
Plant Cell Expressed Recombinant Glucocerebrosidase - taliglucerase alfa – as Therapy for Gaucher Disease in Patients Previously Treated with imiglucerase Gregory M Pastoresa, Paul M. Fernhoffb, Jeffrey Szerc, Milan Petakovd, Timothy M Coxe, Pilar Giraldof, Hanna Rosenbaumg, Dominick J Amatoh, Eugen Mengeli, Raul Chertkoffj, Einat Almon-Brillj, Ari Zimranj, aNew York University School of Medicine, New York, NY, USA, b Emory University School of Medicine, Atlanta, GA, USA, cRoyal Melbourne Hospital, Victoria, Australia, dInstitute of Endocrinology, Diabetes and Diseases of Metabolism, Belgrade, Serbia, eDepartment of Medicine, Addenbrooke's Hospital, Cambridge, UK, fHospital Universitario "Miguel Servet", Instituto Aragonés de Ciencias de la Salud, Zaragoza, Spain, g Gaucher clinic, Shaare Zedek Medical Center, Jerusalem, Israel, hMount Sinai Hospital, Toronto, Ontario, Canada, iUniversity Children's Hospital, Mainz, Germany, jProtalix Biotherapeutics, Carmiel, Israel taliglucerase alfa is a carrot-cell-expressed recombinant human beta-glucocerebrosidase formulation offered as a treatment for Gaucher disease, which is currently being evaluated in a Phase 3 worldwide multicentre, open-label, switch-over trial in patients with Gaucher disease (GD) treated previously with imiglucerase. Study objective: assessment of the safety and efficacy of taliglucerase alfa in GD patients, 2 years or older, who have been receiving imiglucerase ERT for at least 2 years and on a stable maintenance regimen.Patients were evaluated for disease stability and enrolled in a 12-week stability evaluation period, to further establish stability of their hematological parameters. For patients whose imiglucerase regimen was changed due to drug shortage, eligibility was based on historical data of disease stability. Eligible patients switched from imiglucerase to taliglucerase alfa received a total of 20 infusions, given every two weeks on a dose equal to the patient's previous imiglucerase dose. Safety endpoints were adverse events, clinical laboratory tests, electrocardiogram, echocardiography, pulmonary function test and antibody formation. Efficacy endpoints were spleen and liver volume, and platelets and haemoglobin levels. The control for this study is the patient's previous historical clinical and laboratory status while on imiglucerase. Patients were to be discontinued from taliglucerase treatment if they demonstrate a clinically relevant deterioration of the predefined parameters. An interim analysis of the first 15 patients, all of whom have completed the 9 month protocol, was performed and this dataset will be presented. doi:10.1016/j.ymgme.2010.11.113
benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that this construct allowed not only sufficient MGMT expression for selection and metabolic-based enrichment of transduced cells, but also maintained a-gal A expression at appreciable levels. Future experiments involve assessment of transduction/transplantation experiments in Fabry mice. These results indicate that this MGMT-based drug selection is feasible and would represent a new therapeutic approach for Fabry disease. doi:10.1016/j.ymgme.2010.11.110
Lysosomal enhancement as a therapeutic strategy for Batten disease Michela Palmieri, Alberto di Ronza, Marco Sardiello, Baylor College of Medicine, Houston, Texas, USA Lysosomes represent one of the major degradative compartments of eukaryotic cells. They contain more than 60 acid hydrolases that in a concerted action are able to decompose simple and complex macromolecules and even membranes into their monomeric constituents, thereby allowing their recycling. Therefore, the lysosome plays an important role in cellular function and tissue homeostasis. Neuronal ceroid lipofuscinoses (NCL) are severe neurodegenerative disorders of childhood characterized by the intralysosomal accumulation of autofluorescent ceroid lipopigments, commonly referred to as lipofuscin. The most common form of NCL is the juvenile form (JNCL or Batten disease), which results from mutations in the CLN3 gene. There are no current therapeutic options for Batten disease and, differently from most other lysosomal storage disorders, enzyme replacement therapies or gene therapies based on supplying the deficient gene are technically difficult to conceive due to the membrane localization of the CLN3 protein. We recently demonstrated that the transcription factor EB, TFEB, is able to promote cellular clearance by enhancing lysosomal pathways. Here we obtained lysosomal enhancement by either direct supply of exogenous TFEB or treatment with a chemical able to activate endogenous TFEB. We evaluate in vitro and in vivo the effects of lysosomal enhancement on the clearance of lipofuscin in cells from patients and in a mouse model of Batten disease. doi:10.1016/j.ymgme.2010.11.111
Digital microfluidic platform for multiplexing LSD assays in newborn screening Vamsee Pamulaa, Rama Sistaa, Tong Wanga, Carrie Grahama, Allen Eckhardta, Theodore Wingera, Ning Wua, Yalin Xionga, Vijay Srinivasana, David Millingtonb, Adviye Tolunb, Deeksha Balib, a Advanced Liquid Logic, Research Triangle Park, NC, USA, bDuke University, Durham, USA Digital microfluidics is a flexible technique to manipulate discrete droplets independently and precisely under software control to perform biochemical assays on a disposable cartridge. We have recently applied the platform to perform high-throughput newborn screening tests using dried blood spots (DBS) for lysosomal storage disorders. These assays currently require expensive equipment to process and analyze samples and consume significant amounts of sample. A digital microfluidic platform can greatly reduce assay costs and specimen requirements with an inexpensive desktop instrument and test-specific disposable cartridges. Multiplexed and fully automated enzymatic assays for Pompe, Fabry, Hunter, Hurler, and Gaucher diseases were demonstrated on a disposable cartridge.
S33
Aqueous extracts from 48 DBS punches and 5 enzyme substrates were loaded on a cartridge which is programmed to dispense, transport, and mix droplets of 300 nL with an incubation time under 1 hour. Enzymes in the samples cleave fluorogenic substrates to release 4-methylumbelliferone (4-MU) which was measured using a custom-made fluorimeter. Each cartridge performs 240 distinct enzymatic assays which allows for 5 enzymatic assays on each of the 48 samples. The microfluidic method gives comparable results to a “gold standard” method performed at Duke using much larger sample volumes and longer incubation times. Currently, we are piloting LSD assays in the Illinois Department of Public Health. Using the same cartridges and the instrument, multiplex immunoassays, PCR, and DNA sequencing have also been demonstrated which eventually allows utilizing the same platform for hypothyroidism, cystic fibrosis, galactosemia and hemoglobinopathies, as well as planned expansion to SCID. doi:10.1016/j.ymgme.2010.11.112
Plant Cell Expressed Recombinant Glucocerebrosidase - taliglucerase alfa – as Therapy for Gaucher Disease in Patients Previously Treated with imiglucerase Gregory M Pastoresa, Paul M. Fernhoffb, Jeffrey Szerc, Milan Petakovd, Timothy M Coxe, Pilar Giraldof, Hanna Rosenbaumg, Dominick J Amatoh, Eugen Mengeli, Raul Chertkoffj, Einat Almon-Brillj, Ari Zimranj, aNew York University School of Medicine, New York, NY, USA, b Emory University School of Medicine, Atlanta, GA, USA, cRoyal Melbourne Hospital, Victoria, Australia, dInstitute of Endocrinology, Diabetes and Diseases of Metabolism, Belgrade, Serbia, eDepartment of Medicine, Addenbrooke's Hospital, Cambridge, UK, fHospital Universitario "Miguel Servet", Instituto Aragonés de Ciencias de la Salud, Zaragoza, Spain, g Gaucher clinic, Shaare Zedek Medical Center, Jerusalem, Israel, hMount Sinai Hospital, Toronto, Ontario, Canada, iUniversity Children's Hospital, Mainz, Germany, jProtalix Biotherapeutics, Carmiel, Israel taliglucerase alfa is a carrot-cell-expressed recombinant human beta-glucocerebrosidase formulation offered as a treatment for Gaucher disease, which is currently being evaluated in a Phase 3 worldwide multicentre, open-label, switch-over trial in patients with Gaucher disease (GD) treated previously with imiglucerase. Study objective: assessment of the safety and efficacy of taliglucerase alfa in GD patients, 2 years or older, who have been receiving imiglucerase ERT for at least 2 years and on a stable maintenance regimen.Patients were evaluated for disease stability and enrolled in a 12-week stability evaluation period, to further establish stability of their hematological parameters. For patients whose imiglucerase regimen was changed due to drug shortage, eligibility was based on historical data of disease stability. Eligible patients switched from imiglucerase to taliglucerase alfa received a total of 20 infusions, given every two weeks on a dose equal to the patient's previous imiglucerase dose. Safety endpoints were adverse events, clinical laboratory tests, electrocardiogram, echocardiography, pulmonary function test and antibody formation. Efficacy endpoints were spleen and liver volume, and platelets and haemoglobin levels. The control for this study is the patient's previous historical clinical and laboratory status while on imiglucerase. Patients were to be discontinued from taliglucerase treatment if they demonstrate a clinically relevant deterioration of the predefined parameters. An interim analysis of the first 15 patients, all of whom have completed the 9 month protocol, was performed and this dataset will be presented. doi:10.1016/j.ymgme.2010.11.113