In vitro correction of medium chain acyl CoA dehydrogenase deficiency with a recombinant adenoviral vector

David B. Schowalter, Dietrich Matern, Jerry Vockley

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Defects of mitochondrial β-oxidation are a growing group of disorders with variable clinical presentations ranging from mild hypotonia to sudden infant death. Current therapy involves avoidance of fasting, dietary restrictions, and cofactor supplementation. Unfortunately, times of acute illness and noncompliance can interfere with these therapies and result in a rapid clinical decline. The development of a safe, durable, and effective gene delivery system remains an attractive alternative therapy for individuals with these disorders. To this end, a recombinant first-generation adenovirus vector (Ad/cmv-hMCAD) has been prepared that constitutively expresses the human medium chain acyl CoA dehydrogenase (MCAD) protein under the control of the CMV promoter and bovine polyadenylation signal. Characterization of human fibroblasts deficient in MCAD infected with Ad/cmv-hMCAD including Western analysis, immunohistological staining visualized with confocal microscopy, electron transfer protein (ETF) reduction assay, and palmitate loading studies was performed. Infection of MCAD deficient fibroblast with Ad/cmv-hmcad resulted in the production of a 55 kDa protein that co-localized in cells with a mitochondrial marker. Extracts prepared from Ad/cmv-hMCAD infected deficient fibroblasts demonstrated correction of the block seen in the MCAD catalyzed reduction of ETF in the presence of octanoyl CoA. Finally, MCAD deficient fibroblasts infected with increasing amounts of Ad/cmv-hMCAD showed a stepwise improvement of the abnormal acylcarnitine profile exhibited by the deficient cells. Together these studies demonstrate our ability to express and monitor the expression of MCAD in treated cells and support further in vivo murine studies to assess toxicity and duration of correction with this and other MCAD recombinant vectors.

Original languageEnglish (US)
Pages (from-to)88-95
Number of pages8
JournalMolecular Genetics and Metabolism
Volume85
Issue number2
DOIs
StatePublished - Jun 2005

Fingerprint

Acyl-CoA Dehydrogenase
Fibroblasts
Proteins
Electrons
Gene Transfer Techniques
Polyadenylation
Muscle Hypotonia
Aptitude
Sudden Infant Death
Confocal microscopy
Palmitates
Complementary Therapies
In Vitro Techniques
Medium chain acyl CoA dehydrogenase deficiency
Adenoviridae
Confocal Microscopy
Toxicity
Assays
Fasting
Genes

Keywords

  • Adenovirus
  • Co-focal microscopy
  • Human fibroblasts
  • Medium chain acyl CoA dehydrogenase
  • Tandem mass spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Endocrinology, Diabetes and Metabolism

Cite this

In vitro correction of medium chain acyl CoA dehydrogenase deficiency with a recombinant adenoviral vector. / Schowalter, David B.; Matern, Dietrich; Vockley, Jerry.

In: Molecular Genetics and Metabolism, Vol. 85, No. 2, 06.2005, p. 88-95.

Research output: Contribution to journalArticle

Schowalter, David B. ; Matern, Dietrich ; Vockley, Jerry. / In vitro correction of medium chain acyl CoA dehydrogenase deficiency with a recombinant adenoviral vector. In: Molecular Genetics and Metabolism. 2005 ; Vol. 85, No. 2. pp. 88-95.
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