Systemic dual-gene therapy reverses biochemical intoxication in the central metabolic compartment of Bckdha-/- mice

Branched-chain 2-ketoacid dehydrogenase (BCKDH) deficiency (maple syrup urine disease; MSUD) causes lethal encephalopathy by disrupting cerebral metabolism, a process imperfectly reflected by circulating biomarkers. Diet and liver transplantation stabilize peripheral metabolites but fail to restore brain neurochemistry, demarcating the central nervous system as the decisive therapeutic compartment. To define the pathogenesis of intoxication and its therapeutic response, we performed paired serum-brain metabolomics in Bckdha-/- mice treated with a systemic AAV9 dual-gene vector encoding human BCKDHA and BCKDHB (A-BiP-B). Untreated neonates exhibited a 9-fold elevation of brain 2-ketoisocaproate accompanied by cerebral depletion of glutamate and glutamine, as well as shifts in tricarboxylic acid cycle and ketone body metabolism. These disturbances originated from reversal of branched-chain aminotransferase 2 flux and destabilization of glutamate-2-ketoglutarate mass balance, producing divergent metabolic endophenotypes in blood versus brain. A single intravenous injection of A-BiP-B rescued mice from fatal encephalopathy, partially restored cerebral BCKDHA mRNA expression, and brought core brain neurochemical endpoints within wild-type range despite persistent elevation of serum 2-ketoacids. These findings expose limitations of current MSUD management and establish systemic dual-gene therapy as a means of restoring neurochemical homeostasis while enabling survival on unrestricted protein intake.