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.

Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are often inadequate, and the tongue is a readily accessible target for therapeutic gene delivery. In this regard, gene therapy specifically targeting the tongue motor system offers two general strategies for treating lingual disorders. First, correcting tongue myofiber and/or hypoglossal (XII) motoneuron pathology in genetic neuromuscular disorders may be readily achieved by intralingual delivery of viral vectors. The retrograde movement of viral vectors such as adeno-associated virus (AAV) enables targeted distribution to XII motoneurons via intralingual viral delivery. Second, conditions with impaired or reduced tongue muscle activation can potentially be treated using viral-driven chemo- or optogenetic approaches to activate or inhibit XII motoneurons and/or tongue myofibers. Further considerations that are highly relevant to lingual gene therapy include (1) the diversity of the motoneurons which control the tongue, (2) the patterns of XII nerve branching, and (3) the complexity of tongue muscle anatomy and biomechanics. Preclinical studies show considerable promise for lingual directed gene therapy in neuromuscular disease, but the potential of such approaches is largely untapped.

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Previous work by our group shows that AAV9 can robustly transduce XII MNs following direct intralingual injection and subsequent retrograde transport along the XII nerve. The current experiments compared the effectiveness of this approach in commonly studied strains of mouse and rat. In the adult C57BL/6 mouse, delivery of AAV9‐CBA‐GFP (1.20e10vg/μL, 8.33μL) via a unilateral or bilateral injection to the base of the tongue both produced a robust transgene expression in XII MNs as determined by immunohistochemistry (tissues harvested 6 weeks post‐injection). The extent of XII MN transduction was similar with uni‐ or bilateral tongue injection, presumably owing to the small volume of the mouse tongue. In contrast to the murine data, following intralingual delivery of AAV9‐CMV‐mCherry in adult Sprague‐Dawley (SD) rats (1.42e10vg/μL, 1.5 μL or 7.0μL), transgene expression in XII MNs was not detected using either fluorescence microscopy or antibodies against mCherry. Intralingual delivery was repeated in 3 separate cohorts (N=28 total) and MN transduction was never detected. We next tried a direct unilateral injection of AAV9‐mCherry into the main trunk of the XII nerve of adult SD rats (1.42e10vg/μL, 1.5μL). The nerve was injected in the neck, proximal to the point where it bifurcates into medial and lateral branches. At 6 wks post‐injection, we observed clear expression of mCherry in XII MNs detected using immunochemistry. Unexpectedly, MN transgene expression was seen in both XII motor nuclei, despite the unilateral nature of the AAV delivery. We are unaware of any neuroanatomical evidence to suggest that retrograde transport along a single XII nerve would result in bilateral targeting of XII motoneurons, and ongoing experiments are aimed at determining the mechanisms underlying this result. Collectively, these results indicate that retrograde transport of AAV9 following intramuscular delivery may show species variability. Problems with intralingual delivery in the rat were circumvented by direct delivery of AAV9 to the XII nerve.Support or Funding InformationFunding: 1R01HL139708‐01A1 (DDF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.