Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice.

Cystathionine β-synthase (CBS) deficiency causes classical homocystinuria with severe hyperhomocysteinemia (HHcy) that is inadequately controlled by current therapies. We tested whether liver-targeted CBS gene therapy provides durable biochemical and phenotypic rescue. Using a Cre-inducible adult mouse model of whole-body CBS loss, a single intravenous dose of AAV-DJ-hCBS (3 × 1012 or 3 × 1013 vg/kg) was administered, and the animals were followed for 12 months. Vector biodistribution showed ~100-fold hepatic enrichment over the kidney and spleen. Both doses rapidly normalized plasma homocysteine (<8 µM), maintaining correction throughout the study while preventing alopecia, weight loss, and loss of adiposity. Liver histology showed resolution of inflammation, and only 2 of 19 mice developed anti-hCBS antibodies. Liver proteomics (3998 proteins quantified) revealed CBS deficiency-associated suppression of tRNA aminoacylation and dysregulation of lipid and carbon metabolism with an HNF4A transcriptional signature, all normalized by therapy. Liver metabolomics demonstrated accumulation of S-adenosylmethionine and S-adenosylhomocysteine and disruption of phosphatidylcholine synthesis, also corrected by treatment. Plasma metabolomics revealed systemic disturbances fully normalized by hepatic CBS restoration. These findings identify the liver as the central metabolic control point in CBS deficiency and support liver-targeted gene therapy as a durable corrective strategy.