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Background <p>Pompe disease (PD), caused by a deficiency in acid α-glucosidase (GAA), is a lysosomal storage disorder. Existing Gaa knockout and point mutation mouse models have substantially advanced mechanistic understanding, but most are homozygous for single mutations and do not faithfully model the compound‑heterozygous GAA genotypes common in patients. A more clinically relevant in vivo model is still needed to enable systematic evaluation and faithful recapitulation of multi‑organ pathology. We previously reported an infantile-onset PD (IOPD) family carrying compound heterozygous <i>GAA</i> mutation (<i>GAA</i> c.1822&#xa0;C &gt; T/2662G &gt; T; R608*/E888*). We here generated a novel compound heterozygous <i>Gaa</i> mouse model (<i>Gaa</i> c.1822&#xa0;C &gt; T/2665A &gt; T; R608*/K889*), corresponding to human R608*/E888*), corresponding to human <i>GAA</i> identified in the IOPD family.</p> Methods <p>CRISPR/Cas9-mediated knock-in introduced R608* (exon 14) and K889* (exon 18) mutations into C57BL/6 zygotes. Compound heterozygotes (CHet) were bred, validated via Sanger sequencing, and phenotyped using GAA activity assays, glycogen quantification, histopathology, transmission electron microscopy (TEM), and echocardiography. Parameters included cardiac structure/function, skeletal muscle integrity, hepatic glycogenosis, and diaphragmatic pathology.</p> Results <p>CHet mice exhibited reduced GAA activity and elevated plasma glycogen levels, recapitulating the metabolic hallmark of human PD. Systemic pathophysiological characterization revealed multi-organ dysfunction: the heart showed pronounced hypertrophy with structural remodeling, evidenced by thickened ventricular walls and sarcolemmal disarray, despite preserved compensatory function. Skeletal muscle pathology was marked by vacuolated myofibers, lysosomal glycogen accumulation, and mitochondrial abnormalities, reflecting impaired autophagic flux. Hepatic tissues displayed prominent glycogen storage, hepatocyte swelling, and disrupted cellular architecture. Diaphragmatic dysfunction, a critical determinant of respiratory failure in PD, was characterized by vacuolation, nuclear centralization, and inflammatory infiltration, with ultrastructural evidence of lysosomal glycogen deposition and mitochondrial damage across all tissues.</p> Conclusions <p>To our knowledge, this is among the first compound heterozygous <i>Gaa</i> mouse models integrating East Asian-specific <i>GAA</i> mutations (R608*/E888*) and demonstrating multi-organ pathophysiology similar to human PD. By preserving residual GAA activity (~ 18%) and recapitulating cardiac, skeletal, hepatic, and respiratory defects, our model provides an important complementary platform for elucidating mutation-specific mechanisms, optimizing enzyme replacement therapy (ERT), and advancing gene-editing strategies. Its alignment with patient-derived iPSC findings enhances the translational relevance of research in PD and lysosomal disorders.</p>

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Generation and characterization of a novel Gaa compound heterozygous mouse model recapitulating human Pompe disease

  • 期刊论文

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  • 发表日期：

    2026年2月25日

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  • Wenjun Huang,
  • Jie Wang,
  • Yafei Zhou,
  • Hongyu Xiao,
  • Kaichong Jiang,
  • Jiale Cui,
  • Yanmin Zhang,
  • Rui Zhou

摘要

Background

Pompe disease (PD), caused by a deficiency in acid α-glucosidase (GAA), is a lysosomal storage disorder. Existing Gaa knockout and point mutation mouse models have substantially advanced mechanistic understanding, but most are homozygous for single mutations and do not faithfully model the compound‑heterozygous GAA genotypes common in patients. A more clinically relevant in vivo model is still needed to enable systematic evaluation and faithful recapitulation of multi‑organ pathology. We previously reported an infantile-onset PD (IOPD) family carrying compound heterozygous GAA mutation (GAA c.1822 C > T/2662G > T; R608*/E888*). We here generated a novel compound heterozygous Gaa mouse model (Gaa c.1822 C > T/2665A > T; R608*/K889*), corresponding to human R608*/E888*), corresponding to human GAA identified in the IOPD family.

Methods

CRISPR/Cas9-mediated knock-in introduced R608* (exon 14) and K889* (exon 18) mutations into C57BL/6 zygotes. Compound heterozygotes (CHet) were bred, validated via Sanger sequencing, and phenotyped using GAA activity assays, glycogen quantification, histopathology, transmission electron microscopy (TEM), and echocardiography. Parameters included cardiac structure/function, skeletal muscle integrity, hepatic glycogenosis, and diaphragmatic pathology.

Results

CHet mice exhibited reduced GAA activity and elevated plasma glycogen levels, recapitulating the metabolic hallmark of human PD. Systemic pathophysiological characterization revealed multi-organ dysfunction: the heart showed pronounced hypertrophy with structural remodeling, evidenced by thickened ventricular walls and sarcolemmal disarray, despite preserved compensatory function. Skeletal muscle pathology was marked by vacuolated myofibers, lysosomal glycogen accumulation, and mitochondrial abnormalities, reflecting impaired autophagic flux. Hepatic tissues displayed prominent glycogen storage, hepatocyte swelling, and disrupted cellular architecture. Diaphragmatic dysfunction, a critical determinant of respiratory failure in PD, was characterized by vacuolation, nuclear centralization, and inflammatory infiltration, with ultrastructural evidence of lysosomal glycogen deposition and mitochondrial damage across all tissues.

Conclusions

To our knowledge, this is among the first compound heterozygous Gaa mouse models integrating East Asian-specific GAA mutations (R608*/E888*) and demonstrating multi-organ pathophysiology similar to human PD. By preserving residual GAA activity (~ 18%) and recapitulating cardiac, skeletal, hepatic, and respiratory defects, our model provides an important complementary platform for elucidating mutation-specific mechanisms, optimizing enzyme replacement therapy (ERT), and advancing gene-editing strategies. Its alignment with patient-derived iPSC findings enhances the translational relevance of research in PD and lysosomal disorders.