892. Neural Deficits Contribute to Respiratory Insufficiency in Pompe Disease: A Therapeutic Approach with AAV1
Authors: Lara R. DeRuisseau, Cathryn Mah, David D. Fuller, Barry J. Byrne
Journal: Molecular Therapy (2006)

Pompe disease is a lysosomal storage disorder resulting from deficiency of the enzyme acid alpha glucosidase (GAA). The lack of GAA is associated with glycogen accumulation in all cells. Skeletal muscle is particularly affected and diaphragm dysfunction is often life-threatening and thus necessitates mechanical ventilation. Accordingly, the first purpose of this study was to determine if breathing is affected in a mouse model of Pompe disease (the Gaa−/− knockout mouse). Using barometric plethysmography, minute ventilation (MV; mL/min), breathing frequency (F; breaths/min) and tidal volume (TV; mL/min) were measured in conscious control and Gaa−/− mice. During quiet breathing (inspired O2 = 21%; balanced N2), MV (control vs. Gaa−/−: 60 ± 6 vs. 43 ± 4), F (234 ± 11 vs. 195 ± 8), and TV (0.26 ± 0.01 vs. 0.22 ± 0.02) were attenuated in Gaa−/− mice (p<0.01). Ventilation deficits in Pompe disease patients have been attributed to respiratory muscle weakness, however, several case reports (from autopsy) suggest that the central nervous system is also affected. Therefore, we tested the hypothesis that the neural control of the respiratory muscles is impaired in Gaa−/− mice. Initially, we quantified glycogen (μg/mg wet wt) in the spinal cord of control (14.3 ±1.8) and Gaa−/− (30.0 ± 5.0, p<0.01) mice to verify that glycogen accumulation was present in this model of Pompe disease. To directly test that a neural deficit was contributing to ventilation impairments, phrenic motor output (the final output to the diaphragm) was quantified in Gaa−/− (5.2 ± 1.2 mV) and control (49.7 ± 13.9 mV, p<0.01) mice. Our data suggest that ventilatory deficits in Pompe disease may reflect both muscular and neural mechanisms. Importantly, therapies aimed to correct breathing deficits in Pompe disease should target both the diaphragm and central nervous system. For this reason, we have initiated experiments to identify the ability of AAV serotype 1 to transduce both the diaphragm and the phrenic motoneuron pool (cervical segments C3–C5) after a single injection. AAV1-CMV-LacZ was administered via an intrathoracic injection (4.0 × 1011 particles); spinal cord (C3–C5) and diaphragm were harvested 4 weeks post injection. PCR was performed on isolated DNA using primers specific to the LacZ gene. Mice injected with AAV1-CMV-LacZ were positive for LacZ compared to negative controls in both tissues. The protein product of LacZ (betagalactosidase; rlu/mg protein above sham injected control tissue) was present in both the diaphragm (18914 ± 9346) and cervical spinal segments C3–C5 (2380 ± 792). We conclude that AAV1-CMV-LacZ is transported to the spinal cord after an intrathoracic injection. Ongoing experiments will identify if AAV1 is co-localized with retrogradely labled phrenic motoneurons. These studies contribute to our long-term goal of developing a clinically relevant method for transgene delivery to both the diaphragm and phrenic motoneuron pool for correction of respiratory insufficiency in Pompe disease.