Volume 6, Issue 5, Pages 601-608 (November 2002) Glycogen Stored in Skeletal but Not in Cardiac Muscle in Acid α-Glucosidase Mutant (Pompe) Mice Is Highly Resistant to Transgene-Encoded Human Enzyme  Nina Raben, Tejas Jatkar, Alicia Lee, Nina Lu, Sunita Dwivedi, Kanneboyina Nagaraju, Paul H. Plotz  Molecular Therapy  Volume 6, Issue 5, Pages 601-608 (November 2002) DOI: 10.1006/mthe.2002.0716 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Immunodetection of GAA and GFP proteins using specific antibodies. Low levels of GAA protein (but not GFP protein) were detected in heart (H) and muscle (M) in F.3 single-transgenic GAA/Gaa–/– mice. The absence of GFP expression confirms that GAA is expressed at extremely low levels in GAA/Gaa–/– mice. Double-transgenic Mck-T-GAA/Gaa–/– mice derived from the F.3 line were used as positive controls (left lane). Note that the gel was loaded with 10 μg protein for Mck-T-GAA/Gaa–/– and 100 μg protein for GAA/Gaa–/–. Molecular Therapy 2002 6, 601-608DOI: (10.1006/mthe.2002.0716) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 PAS-stained section of cardiac muscle from 4- to 4.5-month-old mice. (A) Control Mck-T/Gaa–/– single-transgenic mutant mouse. (B, C, and D) Single-transgenic mutant mice (GAA/Gaa–/–; F.3) showing glycogen accumulation ranging from 0 to 0.5 to 1.6%, respectively. Original magnification, ×2.5. Molecular Therapy 2002 6, 601-608DOI: (10.1006/mthe.2002.0716) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 PAS-stained section of skeletal muscle from 4- to 4.5-month-old mice. (A) Control Mck-T/Gaa–/– single-transgenic mutant mouse. (B) Single-transgenic mutant mouse (GAA/Gaa–/–; F.3) showing glycogen accumulation similar to that in the control (3.3%). (C) Single-transgenic mutant mouse (GAA/Gaa–/–; F.3) showing reduced glycogen load (1.9%). Original magnification, ×10. Molecular Therapy 2002 6, 601-608DOI: (10.1006/mthe.2002.0716) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 PAS-stained section of cardiac muscle after somatic induction of GAA expression in Gca–/– mice. (A) Control Mck-T-GAA/Gaa–/– on dox for 3 months. (B and C) Induction in 2-month-old animals (2 months gene off/1 month on) resulting in near-complete glycogen clearance with the levels of GAA activity of 24 and 30% wild type, respectively. (D) Control Mck-T-GAA/Gaa–/– on dox for 9.5 months. (E and F) Induction in 6-month-old animals (6 months gene off/4 months on) resulting in only ≈ 50% glycogen reduction with the levels of GAA activity of 40 and 200% of wild type, respectively. Original magnification, ×2.5. Molecular Therapy 2002 6, 601-608DOI: (10.1006/mthe.2002.0716) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 PAS-stained section of skeletal muscle after somatic induction of GAA expression in 2-month-old Gaa–/– mice. (A) Control Mck-T-GAA/Gaa–/– on dox for 3 months; (B and C) 2 months gene off/1 month on, resulting in partial glycogen clearance with levels of GAA activity of 22 and 38% wild type, respectively. (D) Control Mck-T-GAA/Gaa–/– on dox for 5 months; (E and F) 2 months gene off/3 months on, resulting in ≈ 80% glycogen reduction even with the levels of GAA activity of ≈ 2 times (mouse 3-7) and 8 times (mouse 3-9) above normal, respectively. Original magnification, × 10. Molecular Therapy 2002 6, 601-608DOI: (10.1006/mthe.2002.0716) Copyright © 2002 American Society for Gene Therapy Terms and Conditions