Gene delivery to the hypoglossal motor system: preclinical studies and translational potential.

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.

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

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.

Oxymetazoline Applied Topically to the Nasal Mucosa Decreases Trans-Mucosal Nitrous Oxide Exchange for the Middle Ear.

OBJECTIVE: Determine if the middle ear (ME) trans-mucosal nitrous oxide (N2O) gas exchange rate can be pharmacologically modulated by the nasal application of a vasoconstrictor. METHODS: In a randomized, double-blind, crossover study, 20 adults received a nasal spray challenge containing either oxymetazoline or saline (placebo). At each session, subjects were fitted with a non-rebreathing mask and breathed room air for 20 minutes, 50% N2O:50% O2 for 20 minutes, and 100% O2 for 10 minutes. Throughout, heart rate, blood pressure (BP), and blood O2 saturation were monitored, and bilateral ME pressure was recorded by tympanometry every minute. The primary outcome measure was the slope of the ME pressure-time function for the experimental period, a direct measure of the transMEM N2O exchange constant. The effects of treatment, session, and period on the measured vital signs and of treatment, session, disease history, and ear on the ME pressure-time slopes were evaluated for statistical significance using repeated measures ANOVAs. RESULTS: The analysis documented a significant effect of period on O2 saturation (N2O > room air, P = .03) and of treatment on blood pressure (oxymetazoline > placebo, P < .02) and the ME pressure-time slope (placebo > oxymetazoline, P = .05). CONCLUSION: The exchange rate across the ME mucosa of inert gases can be decreased by topical treatment of the nasal mucosa with oxymetazoline.

Oral pseudoephedrine decreases the rate of transmucosal nitrous oxide exchange for the middle ear.

OBJECTIVES/HYPOTHESIS: Determine if oral treatment with a vasoconstrictor decreases the blood to middle ear exchange rate of the perfusion-limited gas, nitrous oxide (N2O). STUDY DESIGN: Randomized, double-blind, crossover study. METHODS: Ten adult subjects with and 10 without past middle ear disease completed paired experimental sessions, identical except for oral treatment with either pseudoephedrine hydrochloride or lactose placebo. At each session, subjects were fitted with a nonrebreathing mask and breathed room air for 20 minutes (acclimation period), 50% N2O:50% O2 for 20 minutes (experimental period), and 100% O2 for 10 minutes (recovery period). Throughout, heart rate, blood pressure, and O2 saturation were monitored, and bilateral middle ear pressures were recorded by tympanometry every minute. The primary outcome was the slope of the middle ear pressure-time function for the experimental period, which estimates the volume N2O exchange rate. Using repeated measures analysis of variance, the effects of group (disease history), treatment (active vs. placebo), and period (1 vs. 2) on the recorded vital signs, and of group, treatment, and ear (left/right) on the middle ear pressure-time slope were evaluated for statistical significance. RESULTS: Statistically significant effects of period on O2 saturation (period 2 > period 1) and of treatment on heart rate (active > placebo) were documented. Only treatment was statistically significant for the middle ear pressure-time slope, with a shallower slope characterizing the active treatment session. CONCLUSIONS: The volume exchange rate across the middle ear mucosa of perfusion-limited gases can be modulated pharmacologically. Theoretically, similar drugs can be used to reduce the requisite eustachian tube opening efficiency for adequate middle ear pressure regulation. LEVEL OF EVIDENCE: 1b.

Role of the mastoid in middle ear pressure regulation.

OBJECTIVES/HYPOTHESIS: Determine the role of mastoid volume in middle ear pressure (MEP) regulation. The hypothesis was that inert gas exchange between blood and middle ear (ME) is slower for larger mastoid volumes. STUDY DESIGN: Prospective. METHODS: For 21 enrolled subjects, the bilateral surface areas and volumes of the mastoid and tympanum were measured from computed tomography scans in 20 subjects with a wide range of mastoid volumes. Then, 19 subjects were reclined in a chair, fitted with a non-rebreathing mask and breathed room air for 20 minutes (acclimation), a gas composition of 25% N(2)O, 20% O(2), balance N(2) for 30 minutes (experiment), and room air for 30 minutes (recovery). Bilateral MEPs were recorded by tympanometry every 2 minutes. The slopes of the MEP-time functions during N(2)O breathing were calculated to the first observation of eustachian tube opening and divided by the estimated blood-ME N(2)O gradient to yield a N(2)O time constant. Sufficient data were available for 16 right and 11 left MEs to calculate the time constant. RESULTS: MEP did not change during the baseline period, but within 10 minutes of breathing the N(2)O mixture showed a progressive increase. The right-left correlation for the time constant was 0.87 (n = 10 ears, P = .001). Regression of the time constants on ME volume showed an inverse relationship (n = 23 ears, r = -41, P = .05). A better data fit was the curvilinear relationship predicted by a mathematical model of the mastoid acting as a ME ear gas reserve. CONCLUSIONS: These results support the tested hypothesis that the mastoid could serve as ME gas reserve.

Surface area-volume relationships for the mastoid air cell system and tympanum in adult humans: Implications for mastoid function.

CONCLUSIONS: The middle ear (ME) surface area/volume ratio (SA/V) is greater than that of the tympanum. The rate of ME pressure decrease is proportional to the ME SA/V. The mastoid air-cell system (MACS) will not function as an ME gas reserve unless the blood perfusion/surface area is much greater for the tympanum than the MACS and decreases as MACS volume increases. OBJECTIVE: To measure the MACS and tympanum surface areas and volumes and ascertain whether the MACS could function as an ME gas reserve. METHODS: Twenty adult subjects with a wide range of MACS volumes underwent CT of their MEs. The left and right surface areas and volumes of the tympanum and MACS were reconstructed and entered into a simple perfusion-limited model of transmucosal gas exchange. In this model the MACS would be a gas reserve if ME SA/V was less than the tympanum SA/V or equivalently, if their ratio was less than a critical value of 1. RESULTS: Both MACS and tympanum SA were linearly related to their volumes. MACS SA/V and the ME SA/V were significantly greater than those for the tympanum. Inputting the measured values into the model yielded a critical value of 1.4, which was significantly greater than 1.