Gene therapy for methylmalonic aciduria.

Abstract

Methylmalonic aciduria (MMAuria) most commonly results from a deficiency of methylmalonyl coenzyme A mutase (MCM). Current treatments for MMAuria remain unsatisfactory and research on novel therapies remains a high priority. A lentiviral (LV) vector was developed to treat in vitro and in vivo models of MMAuria. The overall aim of this project was to examine the therapeutic effect of a LV vector that expresses human MCM transgene in MCM knockout fibroblasts and a MMA affected, mut -/- muth2, murine model. In the first study, a self-inactivating LV vector that expressed human MCM, HIV- 1SDmEF1αhMCM, was constructed and transduced into MCM knockout fibroblasts. Normal cells and untransduced MCM knockout fibroblasts served as controls. Real-time PCR showed a high level of vector copy number, 8 ± 2 copies/cell in LV-treated MCM-knockout fibroblasts, resulting in correction of both the MCM enzyme activity and propionate metabolism in MCM-knockout fibroblasts. The HIV-1SDmEF1αhMCM was then delivered intravenously into mut -/- muth2 mice (n=2). Untreated mut -/- muth2 mice (n=2) and normal mice (n=5) were used as controls. Vector was detected at a copy number of 0.19 ± 0.04 copies/cell in liver. Nevertheless, the MCM enzyme analysis showed only a modest restoration of enzyme activity in the treated mice, resulting in a mild reduction of plasma and urine MMA levels in the treated animals. These data suggest success in targeting the liver with the intravenous gene delivery approach. Nevertheless, it was required to improve the human MCM transgene expression in order to enhance the level of restoration of MCM enzyme activity to further reduce the MMA levels. In the second study, a LV vector that expresses a codon-optimised human MCM transgene, HIV-1SDmEF1αmurSigHutMCM, was produced and transduced into MCM-knockout fibroblasts. High levels of vector, 20 ± 0.8 copies/cell, were detected in LV-treated MCM- knockout fibroblasts. Western blot analysis and MCM enzyme activity analysis by HPLC demonstrated a high level of MCM expression in the treated fibroblasts, resulting in the correction of MCM enzyme activity, with the formation of a significant level of succinyl coenzyme A (179 ± 19 nM/min/μg of total cell protein). The HIV-1SDmEF1αmurSigHutMCM was then injected intravenously into mut -/- muth2 mice (n=5). Untreated mut -/- muth2 (n=6) and normal mice (n=6) were used as controls. The HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for sex. The western blot analysis demonstrated significant MCM enzyme expression in the liver of treated mice, with the measurement of high level of enzyme activity (66 ± 21 nM/min/μg of total cell protein). Biochemical analyses demonstrated that the normalization of MCM enzyme activity in the treated group was associated with a reduction in plasma and urine MMA levels. Furthermore, that a significantly lower MMA concentration, 133± 20 μM/g tissue, was measured in the liver compared to the untreated mice, 1003 ± 124 μM/g tissue. These results confirm that HIV-1SDmEF1αmurSigHutMCM provides significant, if incomplete, biochemical correction for the treatment of this disease, suggesting that gene therapy is a potential treatment for MMAuria.