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 deficiency of branched-chain keto acid dehydrogenase, a mitochondrial multienzyme complex responsible for the decarboxylation of leucine, isoleucine and valine. The complex consists of three subunits (E₁, E₂, and E₃) and mutations in any subunit result in MSUD. No satisfactory treatment for MSUD is currently available. Here we report the successful use of retroviral gene transfer to restore leucine decarboxylation activity in fibroblasts derived from a MSUD patient containing a mutation in the E₂ subunit. A full-length human E₂ cDNA was inserted into a retrieval vector (MFG) and a stable CRIP producer line was generated. The amphotropic virus was then used to transduce mutant human fibroblasts. In untransduced mutant cells, 1-¹⁴C leucine decarboxylation activity was less than 2% that of the wild-type cells. Decarboxylation of 1-¹⁴C leucine in transduced mutant cells was restored to 93% of the wild-type level. Correct targeting of the expressed wild-type E₂ protein to mitochondria was demonstrated by comparing the immunofluorescent pattern of E₂ and a mitochondrial marker protein. Stable expression of enzyme activity has been obtained for at least 7 weeks. In contrast to most previous gene therapy attempts, which replace a single enzyme defect, the present results demonstrate complementation of a phenotype resulting from a gene defect whose product is a part of multienzyme complex. Based on these results, studies can now be undertaken to investigate the feasibility of gene therapy to correct MSUD.