Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA mutase: Implications for somatic gene therapy

Abstract

Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit > 10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

“…Other authors have demonstrated that hepatocytes possess an increased basal capacity for propionate metabolism[23,35] as our results also suggest. Furthermore, when individual murine tissues were examined for methylmalonyl-CoA mutase activity, the liver possessed a large amount of active enzyme, with an total activity of > 1400 nmol succinate formed/h per mg of protein[31].…”

Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

“…In sheep and rats, the highest levels of MCM activity were reported in the kidney and liver followed by brain, intestinal mucosa and skeletal muscle [4][5][6]51]. Our [21,26], and that overexpression of MCM in fibroblasts and hepatoma cells does not increase propionate flux above normal [33].…”

“…Promoter activity was assessed by counting the number of X-gal-stained cells in ten random high-power fields from triplicate samples. Electroporation efficiency did not vary significantly in replicate control experiments performed simultaneously with identical conditions [26,33]. The number of cells exhibiting visible X-gal staining reflects the fraction of cells with expression above the threshold of visual detection and is a valid screening test for the level of gene expression.…”

Adenovirus-Mediated Gene Delivery Rescues a Neonatal Lethal Murine Model of mut0 Methylmalonic Acidemia

“…The studies described here prove that virus-mediated rescue of neonatal lethality in Mut Ϫ/Ϫ mice, and by extension, other lethal organic acidemia murine models, can be successfully accomplished. These results are surprising when the pleiotropic nature of the enzyme defect in MMA is considered (Wilkemeyer et al, 1993) and suggest that metabolic cooperativity of propionate metabolism (Wilkemeyer et al, 1992;Stankovics and Ledley, 1993) may contribute to the biological effects observed by transient restoration of metabolism in the hepatocyte. The rapidity and severity of symptom onset in this model differ when compared with other inborn error of metabolism models that have been treated successfully with integration viruses and transposons as gene delivery agents to achieve permanent correction, such as lysosomal storage disorders (Ponder et al, 2002), aminoacidopathies (Chen and Woo, 2005), and bilirubin metabolic defects (Nguyen et al, 2005).…”