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Molecular Basis of Maple Syrup Urine Disease and Stable Correction by Retroviral Gene Transfer^1,2,

Department of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency of the branched-chain -keto acid dehydrogenase (BCKAD) complex. This results in the accumulation of the branched-chain amino acids (BCAA) and branched-chain -keto acids (BCKA), which often produce severe neurological damage and mental retardation. The present studies focus on mutations in the E1 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 E1 and E1ß subunits into the E1 heterotetramer (₂ß₂) 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 E1 mutations, including the prevalent Y393N of Mennonite MSUD patients, decrease the rate of association of normal E1ß with mutant E1. This results in limited or no assembly of mutant E1. It is concluded that the carboxy-terminal region of the E1 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 E1 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.

KEY WORDS: • maple syrup urine disease • branched-chain -keto acid dehydrogenase • protein assembly defect • retroviral gene transfer

¹ Presented as part of the symposium "Alpha-Keto Acid Dehydrogenase Complexes: Nutrient Control, Gene Regulation and Genetic Defects" given at the Experimental Biology '94 meeting, Anaheim, CA, on April 27, 1994. This symposium was sponsored by the American Institute of Nutrition. Guest editors for this symposium were Mulchand S. Patel, State University of New York at Buffalo, Buffalo, NY and Robert A. Harris, Indiana University School of Medicine, Indianapolis, IN.

² Supported by Grants DK-26758 and DK-37373 from the National Institutes of Health and Grant 1-1149 from the March of Dimes Birth Defects Foundation.

³ To whom correspondence should be addressed: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9038.

⁴ Medical Scientist Trainee supported by Grant 5-P32 GM-08014 from the National Institutes of Health and by the Perot Family Foundation.

⁵ Present address: Department of Biochemistry, Toyama Medical and Pharmaceutical University School of Medicine, Toyama, Japan.