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1.
Mol Genet Metab ; 138(1): 106982, 2023 01.
Article in English | MEDLINE | ID: mdl-36580829

ABSTRACT

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid ß-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.


Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain , Lipid Metabolism, Inborn Errors , Humans , Animals , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Disease Models, Animal , Fibroblasts/metabolism , Lipid Metabolism, Inborn Errors/genetics , Lipid Metabolism, Inborn Errors/therapy
2.
Mol Genet Metab ; 134(1-2): 29-36, 2021.
Article in English | MEDLINE | ID: mdl-34535384

ABSTRACT

INTRODUCTION: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS. METHODS: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity. RESULTS: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants. CONCLUSIONS: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.


Subject(s)
Amino Acid Metabolism, Inborn Errors/diagnosis , Genetic Variation , Isovaleryl-CoA Dehydrogenase/deficiency , Isovaleryl-CoA Dehydrogenase/genetics , Mutation , Neonatal Screening/methods , HEK293 Cells , Humans , In Vitro Techniques , Infant, Newborn , Isovaleryl-CoA Dehydrogenase/classification , Models, Biological , Molecular Diagnostic Techniques , Neonatal Screening/standards , Tandem Mass Spectrometry
3.
Anal Biochem ; 581: 113332, 2019 09 15.
Article in English | MEDLINE | ID: mdl-31194945

ABSTRACT

Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro. Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity. The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.


Subject(s)
Acyl-CoA Dehydrogenases/chemistry , Electron-Transferring Flavoproteins/chemistry , Acyl-CoA Dehydrogenases/genetics , Animals , Electron-Transferring Flavoproteins/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Fatty Acids/chemistry , Fatty Acids/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Swine
4.
Hum Mol Genet ; 28(6): 928-941, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30445591

ABSTRACT

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common defect of mitochondrial long-chain fatty acid ß-oxidation. Patients present with heterogeneous clinical phenotypes affecting heart, liver and skeletal muscle predominantly. The full pathophysiology of the disease is unclear and patient response to current therapeutic regimens is incomplete. To identify additional cellular alterations and explore more effective therapies, mitochondrial bioenergetics and redox homeostasis were assessed in VLCAD-deficient fibroblasts, and several protective compounds were evaluated. The results revealed cellular and tissue changes, including decreased respiratory chain (RC) function, increased reactive oxygen species (ROS) production and altered mitochondrial function and signaling pathways in a variety of VLCAD-deficient fibroblasts. The mitochondrially enriched electron and free radical scavengers JP4-039 and XJB-5-131 improved RC function and decreased ROS production significantly, suggesting that they are viable candidate compounds to further develop to treat VLCAD-deficient patients.


Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain/deficiency , Antioxidants/pharmacology , Congenital Bone Marrow Failure Syndromes/metabolism , Electron Transport/drug effects , Energy Metabolism/drug effects , Lipid Metabolism, Inborn Errors/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Muscular Diseases/metabolism , Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , Cell Survival/drug effects , Congenital Bone Marrow Failure Syndromes/etiology , Endoplasmic Reticulum/metabolism , Lipid Metabolism, Inborn Errors/etiology , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Diseases/etiology , Mitochondrial Dynamics/drug effects , Muscular Diseases/etiology , Oxidation-Reduction/drug effects , Oxygen Consumption , Reactive Oxygen Species/metabolism , Signal Transduction
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