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

Abstract

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.

Fig. 1. Schematic diagram of the hypoglossal (XII) motor system emphasizing potential sites for viral injection.

The XII motor nucleus innervates the tongue via cranial nerve XII. The intrinsic muscles form the body of the tongue and the extrinsic muscles insert into the body of the tongue. The main XII nerve trunk bifurcates with the lateral branch innervating extrinsic retractors and the medial branch innervating extrinsic protrusors. Both branches also innervate intrinsic tongue muscles. The star symbols show potential injection sites for targeted gene delivery directly to the main body of the tongue. The yellow oval on the genioglossus represents a potential site of viral injection if the goal is selective activation of this muscle (or associated XII motoneurons) using opto- or chemogenetics. Potential sites for direct XII nerve injection include the medial branch (blue asterisk), lateral branch (green asterisk) and main trunk (white asterisk). Direct stereotaxic viral delivery to the XII nucleus is also possible in preclinical studies. Inset panels: a coronal section of the tongue illustrating organization of extrinsic and intrinsic muscles; b horizontal section of the medulla highlighting somatotopic organization of XII motoneurons.

Fig. 2. Bilateral XII motoneuron transgene expression following intralingual injection of AAV9-GFP.

The right panel shows a higher power view of both XII nuclei. XII motoneuron staining can be seen to extend beyond the soma and with extensive axonal/dendritic branching. Scale bars: left panel, 500 µm; right panel, 100 µm.

Fig. 3. Motoneuron transgene expression following direct delivery of AAV9-GFP to the XII nerve.

A shows GFP expression visualized using a fluorescent secondary antibody. B GFP fluorescence in XII motoneuron soma. In both panels, the boxes show the approximate locations of the left and right XII nucleus and the * indicates the central canal. The images confirm XII motoneuron transgene expression is present ipsilateral to the AAV9 injection to the XII nerve. IV vent = fourth ventricle. Scale bars: A left panel, 500 µm; right panel, 100 µm. B 250 µm.

Fig. 4. Effect of JHU37160 dihydrochloride (J60) on genioglossal muscle activity in adult mice treated with intralingual injection of AAV9 encoding an inhibitory DREADD.

Data were collected 8 weeks after intralingual injection with AAV9-HA-hM4D-mCherry. A Representative genioglossus muscle electromyography (EMG) activity recorded at baseline (left) and after administration of the DREADD ligand J60 (right). J60 was delivered via intraperitoneal (IP) injection at 0.1 mg/kg in 250 µl saline. The top panel shows the raw EMG recording (scale bar = 40 µV) and the bottom trace shows the integrated signal (iEMG, arbitrary units, au). B Average inspiratory genioglossus EMG response to a single dose of J60 or saline (n = 7, crossover design). Data are normalized to the peak phasic EMG amplitude at baseline. Inspiratory bursting showed a time × treatment statistical interaction (F_1,27 = 46.3, P < 0.001). *denotes a lower value in J60 vs. saline treated, p < 0.001.