Molecular basis of maple syrup urine disease and stable correction by retroviral gene transfer

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency of the branched-chain α-keto acid dehydrogenase (BCKAD) complex. This results in the accumulation of the branched-chain amino acids (BCAA) and branched-chain α-keto acids (BCKA), which often produce severe neurological damage and mental retardation. The present studies focus on mutations in the E1α gene of the BCKAD complex and their effects on the assembly of the E1 decarboxylase component of the enzyme complex. We have developed an efficient histidine-tagged bacterial expression system that allows the folding and assembly of E1α and E1β subunits into the E1 heterotetramer (α₂β₂) in the presence of overexpressed chaperonins GroEL and GroES. The results of pulse-chase experiments with this bacterial expression system showed that a majority of the 15 known E1α mutations, including the prevalent Y393N of Mennonite MSUD patients, decrease the rate of association of normal E1β with mutant E1α. This results in limited or no assembly of mutant E1. It is concluded that the carboxy-terminal region of the E1α subunit encoded by exons 7-9 is important for subunit interaction. To stably correct MSUD, we have developed a retroviral vector that contains a normal E1α precursor complementary DNA. Transduction of cultured lymphoblasts from a Mennonite MSUD patient with this recombinant retroviral vector completely restored the rate of decarboxylation of BCKA. The normal decarboxylation activity in transduced MSUD cells remained stable without antibiotic selection during the 14-week study. The results provide a paradigm for the development of somatic gene therapy for MSUD.