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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01169

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Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease

Powell Gene Therapy Center, and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610, USA

View larger version (159K): [in a new window]   Fig. 1. Large glycogen inclusions are observed in 1-month-old Gaa-/- tissues. (A-D) Histological sections of 1-month-old Gaa-/- heart (A), skeletal muscle (B), diaphragm (C) and liver (D) were stained for glycogen with PAS. Similar tissues from age-matched normal C57B6/129-SvJ animals were included as controls (E-H). (I-P) Electron micrographs of single cells from Gaa-/- (I-L) and normal (M-P) heart (I,M), skeletal muscle (J,N), diaphragm (K,O) and liver (L,P). Scale bars: 20 µm (A-H); 500 µm (I-P).

View larger version (11K): [in a new window]   Fig. 2. Luciferase expression detected in the diaphragm and liver after hepatic in utero delivery of rAAV2-CBA-Luc. At 15 days gestation, Gaa-/- fetuses were injected in the liver with 3x107 infectious particles of rAAV2-CBA-Luc. Animals were sacrificed at 1 month and assayed for luciferase expression. The highest levels of luciferase activity were detected in diaphragm (A) and liver (B). Black circles reflect values of individual samples; white and gray bars represent averages from saline-treated and rAAV2-treated samples, respectively. Diaphragm expression is plotted on a log scale.

View larger version (25K): [in a new window]   Fig. 3. Normal levels of GAA expression are achieved in Gaa-/- diaphragm after in utero rAAV2-hGAA transduction. 15 day pc Gaa-/- fetuses were injected with saline (n=4), 3x107 infectious particles (i.p.) of rAAV2-CBA-Luc (n=4), 2x108 i.p. rAAV2-CBA-hGAA (n=8) or 1x109 i.p. rAAV2-CMV-hGAA (n=4). (A) GAA expression was assayed from 1-month-old diaphragms of normal C57B6/129-SvJ mice and in utero-treated Gaa-/- mice. Black circles represent individual values and bars show averaged values within experimental groups (C57B6/129-SvJ, white bar; rAAV2-CBA-hGAA, light gray bar; rAAV2-CMV-hGAA, dark gray bar). Animals within treated groups are assigned numbers to simplify explanations in the text. (B,C) Protein extracts from same animals as shown in A were separated by SDS-PAGE and the blots probed with a polyclonal antibody against human placental GAA. Normal C57B6/129-SvJ (B,C; lane 2), untreated Gaa-/- (B,C; lane 3), rAAV2-CBA-hGAA-treated Gaa-/- (B, lanes 4-11) and rAAV2-CMV-hGAA-treated Gaa-/- (C, lanes 4-7) diaphragms are shown. GAA purified from human placenta was included as a positive control showing predominant isoforms of 95 kDa precursor, and 76 and 67 kDa processed forms (B,C; lane 1). Arrows indicate the 76 kDa mature form. The asterisk designates a cross-reacting protein of unknown origin.

View larger version (68K): [in a new window]   Fig. 4. Higher than normal levels of GAA expression with serotype 1. Diaphragms harvested from 1-month-old Gaa-/- mice treated in utero with 8.14x1010 particles of rAAV1-CMV-hGAA (1-8) were assayed for GAA expression by: (A) X-Gluc staining, (B) GAA activity assay and (C) western analysis. Expression levels ranged from nothing to vastly greater than normal. In the western analysis, human placental GAA was included as a positive control, showing predominant isoforms of 95 kDa precursor, and 76 and 67 kDa processed forms, with the arrow indicating the 76 kDa mature form. The unknown species detected at the bottom of the blot serves as a loading control (asterisk).

View larger version (162K): [in a new window]   Fig. 5. Gaa-/- diaphragms transduced with rAAV1-CMV-hGAA are free of glycogen deposits. Epon-embedded diaphragms from normal C57B6/129-SvJ (A), untreated Gaa-/- (B) and Gaa-/- mice injected with 8.14x1010 particles of rAAV1-CMV-hGAA in the liver in utero (C) were sectioned and stained with PAS to determine intracellular glycogen content. Electron micrographs were taken of similar tissues from normal (D), untreated (E) and in utero-treated (F) diaphragms to identify glycogen deposits at the cellular level. Representative micrographs from three different vector-treated diaphragms are pictured in C and F. Heart tissue from the rAAV1-CMV-hGAA-treated animal whose diaphragm is pictured in C was stained with PAS to show that other deficient tissues did not benefit from transduction of the diaphragm (I). Heart tissue from normal C57B6/129-SvJ (G) and untreated Gaa-/- (H) mice was included as controls. Scale bars: 20 µm (A-C,G-I); 500 µm (D-F).

View larger version (22K): [in a new window]   Fig. 6. Vector is detected in in utero rAAV1-CMV-hGAA-transduced diaphragms by QC-PCR. Quantitative-competitive PCR was performed to determine vector genome copies in in utero vector-treated and untreated diaphragm samples that were previously analyzed in Fig. 4. PCR was performed after mixing increasing amounts of competitor DNA (100 to 108 copies) and 200 ng of total DNA isolated from the diaphragms of Gaa-/- untreated mice or Gaa-/- mice in utero-treated with 8.14x1010 particles of rAAV1-CMV-hGAA. Representative samples (3 of the 8 assayed) are listed, with their respective values representing estimated vector copies per diploid genome and average GAA activity. Raw QC-PCR data from each diaphragm is shown on the far right.

View larger version (17K): [in a new window]   Fig. 7. In utero delivery of rAAV2-CBA-hGAA preserved diaphragm muscle contractile force in Gaa-/- mice. A dose of 2x108 infectious particles of rAAV2-CBA-hGAA was delivered in utero to Gaa-/- mice (black triangles; n=3). Animals were sacrificed and diaphragm muscle strips were prepared 6 months postpartum. In vitro isometric force-frequency relationships were obtained and compared with those of age-matched, untreated normal C57B6/129-SvJ (black squares; n=3) and Gaa-/-(white circles; n=6) mice.

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