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<p>The IVS4+919G&gt;A mutation in the <i>GLA</i> gene, prevalent in East Asian populations, causes cardiac-type Fabry disease by creating an abnormal splice site. This results in the insertion of a 57-nucleotide segment between exon 4 and exon 5, introducing a premature stop codon and leading to a truncated, non-functional α-Gal A protein. We evaluated whether adenine base editing (ABEmax) can modulate this allele-induced cryptic splice event in patient-derived fibroblasts in vitro as a proof-of-concept. Two ABEmax/sgRNA constructs targeting intron 4 (ABEmax-sgRNA1 and ABEmax-sgRNA2) were tested; both induced on-target +919 A → G conversion with frequent bystander edits at +918/+920. Edited bulk populations and single-cell–derived clones showed restoration of correctly spliced <i>GLA</i> mRNA with reduced aberrant transcripts, increased GLA protein, higher α-Gal A activity (approaching wild-type levels in some clones), and reduced intracellular Gb3 signal. A focused next-generation sequencing panel identified a low-frequency intronic change at one predicted off-target locus without predicted coding consequences. These findings demonstrate in vitro splice rescue of a deep intronic, cardiac-type Fabry disease variant by adenine base editing and suggest that bystander edits in non-coding sequence can further enhance correction by suppressing cryptic splicing, with concordant improvements in α-Gal A activity and Gb3 signals.</p>

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Beneficial bystander-enhanced cryptic splice rescue of cardiac-type Fabry GLA IVS4+919G>A by adenine base editing in patient fibroblasts

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月18日

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  • Hua-Chuan Chao,
  • Yu-Ying Lu,
  • Yu-Ting Chiang,
  • Yun-Ru Chen,
  • Ching-Tzu Yen,
  • Chun-Ying Huang,
  • Sheng-Kai Chang,
  • Yen-Fu Cheng,
  • Yung-Hsiu Lu,
  • Dau-Ming Niu

摘要

The IVS4+919G>A mutation in the GLA gene, prevalent in East Asian populations, causes cardiac-type Fabry disease by creating an abnormal splice site. This results in the insertion of a 57-nucleotide segment between exon 4 and exon 5, introducing a premature stop codon and leading to a truncated, non-functional α-Gal A protein. We evaluated whether adenine base editing (ABEmax) can modulate this allele-induced cryptic splice event in patient-derived fibroblasts in vitro as a proof-of-concept. Two ABEmax/sgRNA constructs targeting intron 4 (ABEmax-sgRNA1 and ABEmax-sgRNA2) were tested; both induced on-target +919 A → G conversion with frequent bystander edits at +918/+920. Edited bulk populations and single-cell–derived clones showed restoration of correctly spliced GLA mRNA with reduced aberrant transcripts, increased GLA protein, higher α-Gal A activity (approaching wild-type levels in some clones), and reduced intracellular Gb3 signal. A focused next-generation sequencing panel identified a low-frequency intronic change at one predicted off-target locus without predicted coding consequences. These findings demonstrate in vitro splice rescue of a deep intronic, cardiac-type Fabry disease variant by adenine base editing and suggest that bystander edits in non-coding sequence can further enhance correction by suppressing cryptic splicing, with concordant improvements in α-Gal A activity and Gb3 signals.