MOLECULAR-BASIS OF MAPLE-SYRUP-URINE-DISEASE AND STABLE CORRECTION BYRETROVIRAL GENE-TRANSFER

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is cau sed by a deficiency of the branched-chain alpha-keto acid dehydrogenas e (BCKAD) complex. This results in the accumulation of the branched-ch ain amino acids (BCAA) and branched-chain alpha-keto acids (BCKA), whi ch often produce severe neurological damage and mental retardation. Th e present studies focus on mutations in the E1 alpha gene of the BCKAD complex and their effects on the assembly of the E1 decarboxylase com ponent of the enzyme complex. We have developed an efficient histidine -tagged bacterial expression system that allows the folding and assemb ly of E1 alpha and E1 beta subunits into the E1 heterotetramer (alpha( 2) beta(2)) in the presence of overexpressed chaperonins GroEL and Gro ES. The results of pulse-chase experiments with this bacterial express ion system showed that a majority of the 15 known E1 alpha mutations, including the prevalent Y393N of Mennonite MSUD patients, decrease the rate of association of normal E1 beta with mutant E1 alpha. This resu lts in limited or no assembly of mutant E1. it is concluded that the c arboxy-terminal region of the E1 alpha subunit encoded by exons 7-9 is important for subunit interaction. To stably correct MSUD, we have de veloped a retroviral vector that contains a normal E1 alpha precursor complementary DNA. Transduction of cultured lymphoblasts from a Mennon ite MSUD patient with this recombinant retroviral vector completely re stored 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.