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

Abstract

Background: Methylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition.

Liver-Directed Recombinant Adeno-Associated Viral Gene Delivery Rescues a Lethal Mouse Model of Methylmalonic Acidemia and Provides Long-Term Phenotypic Correction

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done with 35 mg of clarified liver extract and transferred to nylon membranes. Western blot analysis was performed with a rabbit polyclonal antibody against murine Mut at a 1:500 dilution (Chandler et al, 2007b) and mouse monoclonal anti-OxPhos (oxidative phosphorylation) complex III core II antibody (A-11143; Invitrogen, Carlsbad, CA) at a 1:4000 dilution; incubation was for 3 hr. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1858415; Pierce Protein Research Products/Thermo Scientific) at a 1:10,000 dilution or HRP-conjugated goat anti-mouse antibody (1858413; Pierce Protein Research Products/Thermo Scientific) at a 1:30,000 dilution was incubated for 1 hr.…”

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

“…This exon encodes the putative substrate-binding pocket in the methylmalonyl-CoA mutase enzyme. The mutant allele does not produce detectable RNA, protein, or enzymatic activity (Chandler et al, 2007). Homozygous knockout animals on a mixed (C57BL/6 ϫ 129Sv/Ev) background display a neonatal lethal phenotype and perish within the early neonatal period.…”

“…Previous studies have demonstrated that the cytomegalovirus (CMV)-Mut bifunctional adenovirus (Ad-Mut-GFP) used in these experiments expressed enhanced green fluorescent protein (eGFP) in vivo and could correct the propionate metabolic defect in Mut Ϫ/Ϫ murine embryonic fibroblasts (MEFs) and primary hepatocytes derived from a mut 0 patient (Chandler et al, 2007). Another adenovirus, carrying only the Rous sarcoma virus (RSV) eGFP expression cassette in the E3 region (Ad-GFP), was used as an injection control.…”

“…Moreover, the liver is recognized to play a pivotal role in methylmalonyl-CoA metabolism (Frenkel and Kitchens, 1975). In addition, the demonstration that adenovirus-mediated gene transfer to primary human mut 0 hepatocytes completely corrected the biochemical defect in vitro provides the foundation for a study of in vivo correction in an animal model (Chandler et al, 2007).…”

Inborn Errors of Metabolism and Brain Involvement - 5 Years Experience from a Tertiary Care Center in South India

“…Patients with a mut(0) phenotype had a severe phenotype and early and more severe CRF than patients with mut-/cblA phenotype. Chandler et al (2007) reports successful use of adenoviral mediated gene therapy for methylmalonic aciduria in murine models and human patients with MUT gene mutation. Yang et al (2006) and other authors report that in China methylmalonic acidurias is more commonly associated with homocysteinemia.…”