Adenine base editing rescues disrupted BCKDH function and reduces BCAAs toxic accumulation in maple syrup urine disease patient iPSC-hepatic organoids

Sep 27, 2025Stem cell research & therapy

Correcting enzyme problems with adenine base editing lowers toxic amino acid buildup in liver-like cells from maple syrup urine disease patients

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Abstract

The levels of BCKDHA and BCKDHB were dramatically decreased in -HOs compared with control-HOs (P < 0.01).

Maple syrup urine disease is characterized by the toxic buildup of branched-chain amino acids due to a deficiency in the BCKDH complex.
Patient-derived liver organoids exhibited high expression of hepatoblast-specific proteins and reproduced hepatocyte-like functions.
Correcting the BCKDHB mutation in these organoids resulted in restored BCKDH enzymatic function and reduced levels of branched-chain amino acids.
Transcriptome analysis revealed that the BCKDHB mutation impairs metabolic processes related to liver mitochondrial function, which were rescued following gene correction.
Gene editing with adenine base editor (ABE8e) demonstrated high on-target efficiency with no detectable off-target effects.

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BACKGROUND: (MSUD) is an inherited metabolic disorder caused by a deficiency in the activity of the hepatic branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which leads to the toxic accumulation of three branched-chain amino acids (BCAAs) and their respective α-ketoacid, resulting in severe neurotoxicity, coma and even death without effective therapeutic measures.

METHODS: In this study, we established the patient induced pluripotent stem cells (iPSC)-derived hepatic organoids (HOs), analyzed the characteristics, and applied adenine base editor (ABE8e) to correct a mutation (T322I) of the BCKDHB (branched chain keto acid dehydrogenase E1, beta polypeptide) gene in patient induced pluripotent stem cells (iPSC)-derived hepatic organoids (HOs). qRT-PCR and western blot analysis were performed to assess the expression level of BCKDHA (branched chain keto acid dehydrogenase E1, alpha polypeptide) and BCKDHB. The effects of base editing were comprehensively analyzed using both bulk RNA sequencing and single-cell RNA sequencing (scRNA-Seq).

RESULTS: Immunofluorescence and RT-PCR arrayed the high expression of hepatoblast specific proteins in HOs, such as α-1-anti-trypsin (A1AT), hepatocyte nuclear factor-4-alpha (HNF4A), cytokeratin18 (CK18), albumin (ALB), cytochrome P450 family 3 subfamily A member 4 (CYP3A4) and cytochrome P450 family 3 subfamily A member 7(CYP3A7). Functional experiments indicated that these HOs recapitulated characteristics of hepatocytes like glycogen accumulation, low-density lipoprotein (LDL) uptake, indocyanine green (ICG) uptake and release as well as quantitation of ALB and urea from HOs. The levels of BCKDHA and BCKDHB were dramatically decreased in MSUD-HOs compared with control-HOs (P < 0.01) detected by qRT-PCR, western blot and immunofluorescence. Deep sequencing and whole genome sequencing (WGS) demonstrated that the correction of BCKDHB mutation in patient iPSC-derived HOs caused high on-target gene editing without any detectable off-target effects. Moreover, the corrected MSUD-HOs exhibited restored BCKDH enzymatic function and reduced BCAAs level. The transcriptome analysis indicates that the MSUD-HOs with BCKDHB mutation reduced mRNA level of regulating metabolism associated with liver mitochondrial function, while the corrected MSUD-HOs rescued those processes after ABE8e correction. The scRNA-Seq analysis further validated the rescue effects of BCKDH function after gene editing.

CONCLUSION: Our study provides reliable evidence that ABE8e is highly efficient and safe in correcting patient-derived HOs from MSUD, indicating the feasibility to be a transformative treatment for genetic hepatic diseases like MSUD.

Key numbers

100%

Editing Efficiency

Editing efficiency of the mutation in one organoid clone.

P < 0.01

Increased and Levels

Comparison of and expression levels in corrected vs. untreated organoids.

P < 0.05

Decreased Levels

Comparison of levels in corrected vs. untreated organoids.

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Adenine base editing rescues disrupted BCKDH function and reduces BCAAs toxic accumulation in maple syrup urine disease patient iPSC-hepatic organoids

Abstract

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