Background: The GAA gene encodes lysosomal acid alpha-glucosidase, an enzyme critical for breaking down glycogen into glucose in lysosomes. Mutations cause Pompe disease (glycogen storage disease type II), an autosomal recessive disorder with infantile (severe cardiomyopathy) and late-onset (progressive muscle weakness) forms. Approved treatment is enzyme replacement therapy (ERT); in-advance strategies include AAV gene therapy (e.g., AT845), antisense oligonucleotides, and chaperone therapies to boost residual enzyme activity.

Targeting strategy:

Gene targeting strategy for B-hGAA*c.-32-13T>G mice.

The exons 1-20 of mouse Gaa gene that encode whole protein domains are replaced by human GAA gene counterparts in B-hGAA*c.-32-13T>G mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. c.-32-13T>G mutation was introduced into human GAA gene.

Gene targeting strategy for B-hGAA mice.

The exons 1-20 of mouse Gaa gene that encode whole protein domains are replaced by human GAA gene counterparts in B-hGAA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts.

Validation:

Human GAA mRNA was exclusively detectable in homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice. Abnormal splicing of GAA mRNA (lower band) was only detected in homozygous B-hGAA*c.-32-13T>G mice but not in B-hGAA mice, because of c.-32-13T>G mutation.

Human GAA protein was exclusively detectable in homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice.GAA protein expression levels in B-hGAA*c.-32-13T>G mice were lower than those in homozygous B-hGAA mice, which is consistent with the abnormal splicing results of GAA mRNA in B-hGAA*c.-32-13T>G mice.

Application: B-hGAA*c.-32-13T>G mice can serve as a faithful preclinical model of Pompe disease to evaluate the therapeutic efficacy of ERT, gene therapy, and other drugs. B-hGAA mice can be used as a control model without onset of Pompe disease.

Gene targeting strategy for B-hGAA*c.-32-13T>G mice.

The exons 1-20 of mouse Gaa gene that encode whole protein domains are replaced by human GAA gene counterparts in B-hGAA*c.-32-13T>G mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. c.-32-13T>G mutation was introduced into human GAA gene. The human GAA expression is driven by human GAA promoter, while mouse Gaa gene transcription and translation will be disrupted.

Gene targeting strategy for B-hGAA mice.

The exons 1-20 of mouse Gaa gene that encode whole protein domains are replaced by human GAA gene counterparts in B-hGAA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. The human GAA expression is driven by human GAA promoter, while mouse Gaa gene transcription and translation will be disrupted.

Strain specific analysis of GAA mRNA expression in wild-type C57BL/6JNifdc mice, homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice by RT-PCR. Various tissues RNA was isolated from wild-type C57BL/6JNifdc mice (+/+), homozygous B-hGAA mice (H/H), and homozygous B-hGAA*c.-32-13T>G mice (Mut/Mut), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GAA primers. Mouse Gaa was only detected in wild-type C57BL/6JNifdc mice. Human GAA mRNA was exclusively detectable in homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice. Abnormal splicing of GAA mRNA (lower band) was only detected in homozygous B-hGAA*c.-32-13T>G mice but not in B-hGAA mice, because of c.-32-13T>G mutation.

Western blot analysis of GAA protein expression in homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+), homozygous B-hGAA mice (H/H) and homozygous B-hGAA*c.-32-13T>G mice (Mut/Mut), and then analyzed by western blot with anti-human GAA antibody (Abcam, ab137068). 40 μg total proteins were loaded for western blotting analysis. Human GAA protein was exclusively detectable in homozygous B-hGAA mice and homozygous B-hGAA*c.-32-13T>G mice.GAA protein expression levels in B-hGAA*c.-32-13T>G mice were lower than those in homozygous B-hGAA mice, which is consistent with the abnormal splicing results of GAA mRNA in B-hGAA*c.-32-13T>G mice.