Biochemical correction of very long-chain acyl-CoA dehydrogenase deficiency following adeno-associated virus gene therapy.

We report the development of a gene replacement strategy for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD is a mitochondrial enzyme involved in fatty acid beta-oxidation, a key step in energy production during times of fasting or stress. Deficiency of VLCAD classically presents as hepatic dysfunction, hypoglycemia, cardiomyopathy, rhabdomyolysis, and/or sudden death. While dietary therapy for VLCAD deficiency has proven beneficial in preventing some symptoms, a risk of metabolic catastrophic decompensation remains throughout life during times of increased energy demand. We designed a recombinant adeno-associated virus (AAV) expressing the human VLCAD gene (AAV8-hVLCAD). To demonstrate its in vivo activity, AAV8-hVLCAD was administered via the tail vein to VLCAD-knockout mice. A reduction in accumulated serum long-chain acylcarnitines and increased fasting tolerance judged on blood glucose concentrations were observed as of 11 days postinjections through >100 days. Western analysis of liver, skeletal muscle, and heart extracts using PEP1 anti-hVLCAD antibody revealed short-term hVLCAD expression in the liver and muscle and longer-term expression in heart. This demonstrates the ability of human VLCAD to correct the biochemical phenotype of VLCAD-deficient mice.

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

Defects of mitochondrial beta-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 55kDa 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.