AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice

Abstract

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa−/−) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.

The Respiratory Phenotype of Pompe Disease Mouse Models

“…As in the phrenic motor neurons, the hypoglossal motor neurons have extensive pathology [42,44,66]. The hypoglossal motor neurons of Gaa −/− mice lack GAA protein [42,47] and have PAS+ staining [42,47,48,66]. Additionally, the hypoglossal motor neurons are also morphologically abnormal with swollen somas and glycogen-filled vacuoles [42,44,47] (Figure 2).…”

“…The hypoglossal nerves, which innervate the genioglossus, transmit an impulse to open the airway immediately before inspiration when the phrenic nerve innervates the diaphragm [66]. All Gaa −/− mice models have pathology in the tongue, particularly in the genioglossus muscle [39,42,47,51]. No detectable GAA activity is present in Gaa −/− mice tongues [47,51], which is accompanied by excessive glycogen buildup [39,42,47,51,64].…”

“…All Gaa −/− mice models have pathology in the tongue, particularly in the genioglossus muscle [39,42,47,51]. No detectable GAA activity is present in Gaa −/− mice tongues [47,51], which is accompanied by excessive glycogen buildup [39,42,47,51,64]. As in the phrenic motor neurons, the hypoglossal motor neurons have extensive pathology [42,44,66].…”

“…The hypoglossal motor neurons of Gaa −/− mice lack GAA protein [42,47] and have PAS+ staining [42,47,48,66]. Additionally, the hypoglossal motor neurons are also morphologically abnormal with swollen somas and glycogen-filled vacuoles [42,44,47] (Figure 2). PAS+ staining is also present in ependymal cells around the central canal as well as astrocytes in the hypoglossal motor pool [66].…”

“…Similarly, the model developed by Raben et al, in which exon 6 is disrupted, has progressive accumulation of lysosomal glycogen in the muscles and motor neurons but does not display obvious muscle wasting and weakness until 8-9 months of age [40]. Several researchers have back-crossed this Gaa −/− to pure 129SVE [41][42][43][44][45][46][47][48]. The Gaa −/− mouse on the pure 129SVE background is thought to have less severe respiratory deficits as compared to the Gaa −/− mouse on the mixed B6/129 background [42,45,49].…”