STABLE CORRECTION OF MAPLE-SYRUP-URINE-DISEASE IN CELLS FROM A MENNONITE PATIENT BY RETROVIRAL-MEDIATED GENE-TRANSFER

We have successfully used retroviral gene transfer to correct the deficiency of the branched-chain alpha-oxo acid dehydrogenase complex in lymphoblasts from a homozygous Mennonite maple syrup urine disease (MSUD) patient. The mutation in Mennonites is a Tyr-393 to Asn substitution in the branched-chain alpha-oxo acid decarboxylase (E1)alpha subunit of the enzyme complex. This promotes improper assembly of mutant E1alpha with E1beta subunits, leading to degradation of both polypeptides. For transduction studies, a full-length human E1alpha cDNA was inserted into the retroviral vector LXSN to produce the recombinant LSN-E1alpha. High-titre [6 x 10(5) colony-forming units/ml] amphotropic retroviral preparations free of helper viruses were obtained by co-cultivation of infected GP + E86 with PA317 cells. Transduction of MSUD lymphoblasts from the Mennonite patient with LSN-E1alpha viruses restored the decarboxylation of alpha-oxo[1-C-14]isovalerate to the normal level. The normal decarboxylation activity in transduced MSUD cells remained stable without G418 selection during the 14 weeks studied. Southern-blot analysis indicated that the recombinant E1alpha cDNA was integrated into the host genome. Northern and Western blotting showed that both the normal E1alpha mRNA and the subunit were properly expressed in transduced MSUD cells. However, the level of E1beta subunits is lower than that of normal cells, suggesting competition of the recombinant E1alpha with the mutant form for assembly with E1beta. The results provide a paradigm for the development of somatic gene therapy for disorders involving mitochondrial multienzyme complexes.