BACKGROUND: Very long chain acyl coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is a relatively common mitochondrial beta-oxidation disorder. The most severe form of VLCAD deficiency presents with neonatal cardiomyopathy and hepatic failure and is generally fatal within the first year of life. Mice deficient for long chain acyl CoA dehydrogenase (LCAD) closely resemble the clinical syndrome observed in VLCAD-deficient humans. Recombinant adeno-associated viral (rAAV) vectors with pseudotype capsids were investigated for their potential towards correcting the phenotype observed in mice heterozygous (+/-) for LCAD (i.e. liver and muscle steatosis). METHODS: rAAV containing the mouse LCAD cDNA (mLCAD) under the transcriptional control of the CMV/chicken beta-actin hybrid promoter were injected intramuscularly into the tibialis anterior (TA) muscle of LCAD(+/-) mice or injected into the portal vein to transduce hepatocytes. RESULTS: Ten weeks post-injection of rAAV1-mLCAD into the TA muscle, significantly increased levels of mLCAD within mitochondria were demonstrated by immunostaining of TA sections, immunoblotting of mitochondrial isolates and by the electron transfer flavoprotein (ETF) fluorescence reduction enzyme activity assay. Magnetic resonance spectroscopy of vector-injected TA muscle demonstrated a reduction in the lipid content compared to phosphate-buffered saline-injected mice, whereas a systemic effect was observed as a reduction in liver macrosteatosis. Eight weeks after portal vein injection of rAAV8-mLCAD into LCAD(+/-) mice, increased levels of mLCAD within hepatocyte mitochondria were demonstrated by immunostaining and also by the ETF assay. Scoring of the hepatosteatosis observed in partially deficient LCAD mice indicated a reduction in the lipid content within livers of vector-treated mice. CONCLUSIONS: These studies show that rAAV-mediated delivery of mLCAD was efficient and led to an amelioration of local and systemic pathologies observed in partially deficient LCAD mice.

Recombinant adeno-associated viral vectors pseudotyped with serotype 5 and 8 capsids (AAV5 and AAV8) have been shown to be efficient gene transfer reagents for the liver. We have produced AAV5 and AAV8 vectors that express mouse short-chain acyl-CoA dehydrogenase (mSCAD) cDNA under the transcriptional control of the cytomegalovirus-chicken beta-actin hybrid promoter. We hypothesized that these vectors would produce sufficient hepatocyte transduction (after administration via the portal vein) and thus sufficient SCAD enzyme to correct the phenotype observed in the SCAD-deficient (BALB/cByJ) mouse, which includes elevated blood butyrylcarnitine and hepatic steatosis. Ten weeks after portal vein injection into 8-week-old mice, AAV8-treated livers contained acyl-CoA dehydrogenase activity (14.3 mU/mg) toward butyryl-CoA, compared with 7.6 mU/mg in mice that received phosphate-buffered saline. Immunohistochemistry showed expression of mSCAD within rAAV8-mSCAD-transduced hepatocytes, as seen by light microscopy. A significant reduction of circulating butyrylcarnitine was seen in AAV5-mSCAD- and AAV8-mSCAD-injected mice. Magnetic resonance spectroscopy of fasted mice demonstrated a significant reduction in relative lipid content within the livers of AAV8-mSCAD-treated mice. These results demonstrate biochemical correction of SCAD deficiency after AAV8-mediated SCAD gene delivery.