Randy J. Chandler

Mitochondrial dysfunction in mut methylmalonic acidemia

Methylmalonic acidemia is an autosomal recessive inborn error of metabolism caused by defective activity of methylmalonyl‐CoA mutase (MUT) that exhibits multiorgan system pathology. To examine whether mitochondrial dysfunction is a feature of this organic acidemia, a background‐modified Mut‐knockout mouse model was constructed and used to examine mitochondrial ultrastructure and respiratory chain function in the tissues that manifest pathology in humans. In parallel, the liver from a patient with mut methylmalonic acidemia was studied in a similar fashion. Megamitochondria formed early in life in the hepatocytes of the Mut−/− animals and progressively enlarged. Liver extracts prepared from the mutants at multiple time points displayed respiratory chain dysfunction, with diminished cytochrome c oxidase activity and reduced intracellular glutathione compared to control littermates. Over time, the exocrine pancreas and proximal tubules of the kidney also exhibited megamitochondria, and older mutant mice eventually developed tubulointerstitial renal disease. The patient liver displayed similar morphological and enzymatic findings as observed in the murine tissues. These murine and human studies establish that megamitochondria formation with respiratory chain dysfunction occur in a tissue‐specific fashion in methylmalonic acidemia and suggest treatment approaches based on improving mitochondrial function and ameliorating the effects of oxidative stress.—Chandler, R. J., Zerfas, P. M., Shanske, S., Sloan, J., Hoffmann, V., DiMauro, S., Venditti, C. P. Mitochondrial dysfunction in mut methylmalonic acidemia. FASEB J. 23, 1252–1261 (2009)

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

Methylmalonic acidemia is a severe metabolic disorder caused by a deficiency of the ubiquitously expressed mitochondrial enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation has been used to treat a small number of patients with variable success, and whether liver-directed gene therapy might be employed in such a pleiotropic metabolic disorder is uncertain. In this study, we examined the therapeutic effects of hepatocyte-directed delivery of the Mut gene to mice with a severe form of methylmalonic acidemia. We show that a single intrahepatic injection of recombinant adeno-associated virus serotype 8 expressing the Mut gene under the control of the liver-specific thyroxine-binding globulin (TBG) promoter is sufficient to rescue Mut(-/-) mice from neonatal lethality and provide long-term phenotypic correction. Treated Mut(-/-) mice lived beyond 1 year of age, had improved growth, lower plasma methylmalonic acid levels, and an increased capacity to oxidize [1-(13)C]propionate in vivo. The older treated mice showed increased Mut transcription, presumably mediated by upregulation of the TBG promoter during senescence. The results indicate that the stable transduction of a small number of hepatocytes with the Mut gene can be efficacious in the phenotypic correction of an inborn error of organic acid metabolism and support the rapid translation of liver-directed gene therapy vectors already optimized for human subjects to patients with methylmalonic acidemia.

Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia

Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (nu2009=u200935), AAV8 (nu2009=u200941), and AAV9 (nu2009=u200942) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (pu2009<u20090.05) and AAV8 (pu2009<u20090.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (pu2009<u20090.05 all serotypes) or had received solid organ transplantation (pu2009<u20090.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (nu2009=u200924)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA.

168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)

Methylmalonic acidemia (MMA) is an inborn error of metabolism most commonly caused by deficient methylmalonyl-CoA mutase (MUT) activity. The disorder can have multiple clinical manifestations, including metabolic instability, stroke of the basal ganglia, pancreatitis, end-stage renal failure, growth impairment, osteoporosis and developmental delay. Unfortunately, current non-invasive therapies fail to chronically manage the disease, and patients still suffer from increased morbidity and early mortality. Solid organ transplantation, including elective liver, combined liver-kidney and isolated kidney transplantation, has been used to provide sustained benefit to patients, but the procedures come with substantial risks as well as the post-operative requirement for life-long immunosuppression. To address the large and unmet need for new therapies for patients with MMA, we have developed an effective adeno-associated viral (AAV) gene therapy that has been previously validated in a neonatal lethal mouse model of Mut deficiency (Mut−/−). The current project compares how two distinct AAV8 vectors that express the human MUT gene under the control of either the liver specific, human alpha 1-antitrypsin (hAAT) promoter, or the ubiquitous CMV-enhanced chicken β-actin (CBA) promoter differentially affect metabolite levels following systemic delivery to adult mice. The animals used in this study (Mut−/−;TgINS-MCK-Mut) express wild-type Mut in a muscle-specific fashion via a stable germline transgene and completely lack transgene expression in the liver. Mut−/−;TgINS-MCK-Mut) mice accurately model the hepatorenal manifestations of MMA, but afford an opportunity to assess gene therapy vectors at or after weaning because mice are rescued from neonatal lethality, yet experience massive elevations of the characteristic metabolites (methylmalonic and 2-methylcitric acid) because of the lack of hepatic Mut activity. Disease related metabolites were measured in plasma samples derived from Mut−/−; TglNS-MCK-Mut mice (n=7) prior to AAV gene delivery. The mice were then injected via the retro-orbital route with 1.5X1011 GC of either AAV8-hAAT-MUT or AAV8-CBA-MUT. At 10 days and 30 days post-treatment, mice treated with either vector showed a significant reduction in methylcitrate and methymalonic acid levels, with AAV8-CBA-MUT (n=2) treated mice manifesting methylcitrate and methylmalonic acid levels that trended lower than those measured in mice injected with AAV8-hAAT-MUT (n=4) at both time points. Further studies will be needed to precisely compare the differences between the hAAT and CBA promoters in MMA mouse models, but our preliminary results demonstrate that the Mut−/−; TgINS-MCK-Mut mice can be used to easily ascertain hepatic correction of Mut deficiency, which should help inform the selection of regulatory elements that will provide maximal therapeutic efficacy to treat patients with MMA.