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. 2012 Jan;20(1):21-7.
doi: 10.1038/mt.2011.214. Epub 2011 Oct 18.

Spinal delivery of AAV vector restores enzyme activity and increases ventilation in Pompe mice

Kai Qiu  1 , Darin J Falk, Paul J Reier, Barry J Byrne, David D Fuller
Affiliations

Spinal delivery of AAV vector restores enzyme activity and increases ventilation in Pompe mice

Kai Qiu et al. Mol Ther. 2012 Jan.

Abstract

Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.

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Figures

Figure 1
Figure 1
AAV distribution following spinal cord injection. (a) Quantitative real-time PCR was used to examine the distribution of AAV at 4 weeks postspinal cord injection. A robust number AAV vector genome copies was detected in the cervical and high thoracic (T1–T5) spinal cord. A smaller number of genomes were detected in the medulla and caudal thoracic cord region. Importantly, AAV could not be detected in the diaphragm muscle. (b) Robust GFP expression in a neuronal cluster in the C4 spinal cord in the approximate location of the phrenic motor nucleus, and (c) GFP expression throughout the ventral horn and also in C4 ventral root axons. The area indicated by the box in the left image is presented at a higher magnification in the image immediately to the right. The dashed line indicates the approximate outline of the ventral horn. Bars: 100 µm (lower power) and 50 µm (higher power).
Figure 2
Figure 2
GAA activity in the spinal cord is restored following AAV5-GAA injection. (a) At 4 weeks postspinal injection, homogenates from the cervical (C3–C5) or thoracic spinal cord (T5–T8) were assayed for GAA enzyme activity. Following intraspinal AAV5-GAA injection, Gaa−/− mice showed cervical GAA activity comparable to what was observed in B6/129 mice. However, Gaa−/− mice receiving intraspinal AAV5-GFP had negligible cervical GAA activity. (b) Immunohistochemistry experiments revealed positive GAA staining in putative phrenic motoneurons (box) and cervical interneurons (arrows). (c) GAA immunostaining was absent in Gaa−/− mice receiving AAV5-GFP. In b and c, the area indicated by the box in the left image is presented at a higher magnification in the image immediately to the right. *P < 0.05 vs. corresponding AAV-GFP data point; **P < 0.05 vs. corresponding AAV-GFP and AAV5-GAA data points; Bars: 100 µm (lower power) and 50 µm (higher power).
Figure 3
Figure 3
Histological evidence for glycogen clearance following spinal cord injection with AAV5-GAA. (a–d) 40 µm paraffin-embedded and PAS stained spinal cord sections from an adult Gaa−/− mouse obtained 1 month following cervical spinal injection with AAV5-GAA. The area indicated by the box in the left column is presented at a higher magnification in the image immediately to the right; arrows indicate neuronal cell bodies. Neuronal PAS staining is minimal at or near the site of AAV5-GAA injection (mid-cervical spinal cord, a,b). However, at sites more distant from the injection (mid-thoracic spinal cord, c,d) neuronal PAS staining and neuropathology is evident throughout the ventral horn. The dashed lines indicate the approximate outline of the cervical ventral horn. Bars: 100 µm (lower power) and 50 µm (higher power).
Figure 4
Figure 4
Ventilation in Gaa−/− mice after AAV injection. Inspiratory frequency (breaths/min), tidal volume (ml/breath), and inspired minute ventilation (ml/min) were measured at intervals following spinal AAV injection in unanesthetized mice using whole-body plethysmography. (a) Representative airflow traces during quiet breathing (baseline conditions of 21% O2 with balance of N2) and a hypercapnic respiratory challenge (7% inspired CO2) are shown. Expanded traces in the lower portion of a are provided to illustrate individual breaths. Bars on the airflow traces are in ml/sec. (b) Group average ventilation data during baseline (left panels) and hypercapnic respiratory challenge (right panels) in Gaa−/− mice injected with AAV5-GAA (dashed lines) or AAV-GFP (solid lines), and B6/129 control mice (square symbol). Small but statistically significant differences in tidal volume and minute ventilation between AAV5-GAA vs. AAV5-GFP injected Gaa−/− mice were noted at baseline. More robust differences between these groups were seen during the hypercapnic respiratory challenge. *Indicates a significant overall difference between AAV5-GFP and AAV5-GAA injected Gaa−/− mice (two-way analysis of variance); ‡Indicates that the data point is significantly greater than corresponding AAV5-GFP data point.
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