COMPLEMENTATION OF DEFECTIVE LEUCINE DECARBOXYLATION IN FIBROBLASTS FROM A MAPLE-SYRUP-URINE-DISEASE PATIENT BY RETROVIRUS-MEDIATED GENE-TRANSFER

Maple syrup urine disease (MSUD) is a genetic disease caused by a defi ciency of branched-chain keto acid dehydrogenase, a mitochondrial mult ienzyme complex responsible for the decarboxylation of leucine, isoleu cine and valine. The complex consists of three subunits (E(1), E(2), a nd E(3)) and mutations in any subunit result in MSUD. No satisfactory treatment for MSUD is currently available. Here we report the successf ul use the retroviral gene transfer to restore leucine decarboxylation activity in fibroblasts derived from a MSUD patient containing a muta tion in the E(2) subunit. A full-length human E(2) cDNA was inserted i nto a retroviral vector (MFG) and a stable CRIP producer lien was gene rated. The amphotropic virus was then used to transduce mutant human f ibroblasts. In untransduced mutant cells, 1-C-14 leucine decarboxylati on activity was less than 2% that of the wild-type cells. Decarboxylat ion of 1-C-14 leucine in transduced mutant cells was restored to 93% o f the wild-type level. Correct targeting of the expressed wild-type E( 2) protein to mitochondria was demonstrated by comparing the immunoflu orescent pattern of E(2) and a mitochondrial marker protein. Stable ex pression of enzyme activity has been obtained for at least 7 weeks. In contrast to most previous gene therapy attempts, which replace a sing le enzyme defect, the present results demonstrate complementation of a phenotype resulting from a gene defect whose product is a part of a m ultienzyme complex. Based on these results, studies can now be underta ken to investigate the feasibility of gene therapy to correct MSUD.