Benevides, E. S. | Search bioRxiv

bioRxiv2024

Neonatal systemic gene therapy restores cardiorespiratory function in a rat model of Pompe disease

Absence of functional acid--glucosidase (GAA) leads to early-onset Pompe disease with cardiorespiratory and neuromuscular failure. A novel Pompe rat model (Gaa-/-) was used to test the hypothesis that neonatal gene therapy with adeno-associated virus serotype 9 (AAV9) restores cardiorespiratory neuromuscular function across the lifespan. Temporal vein administration of AAV9-DES-GAA or sham (saline) injection was done on post-natal day 1; rats were studied at 6-12 months old. Whole-body plethysmography showed that reduced inspiratory tidal volumes in Gaa-/-rats were corrected by AAV-GAA treatment. Matrix- assisted laser desorption/ionization mass spectrometry imaging (MALDI) revealed that AAV-GAA treatment normalized diaphragm muscle glycogen as well as glycans. Neurophysiological recordings of phrenic nerve output and immunohistochemical evaluation of the cervical spinal cord indicated a neurologic benefit of AAV-GAA treatment. In vivo magnetic resonance imaging demonstrated that impaired cardiac volumes in Gaa-/- rats were corrected by AAV-GAA treatment. Biochemical assays showed that AAV treatment increased GAA activity in the heart, diaphragm, quadriceps and spinal cord. We conclude that neonatal AAV9-DES-GAA therapy drives sustained, functional GAA expression and improved cardiorespiratory function in the Gaa-/- rat model of Pompe disease.

physiology↗

bioRxiv2024

Chemogenetic stimulation of phrenic motor output and diaphragm activity

Impaired respiratory motor output contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We investigated if expressing designer receptors exclusively activated by designer drugs (DREADDs) in the mid-cervical spinal cord could effectively stimulate phrenic motor output to increase diaphragm activation. Two primary questions were addressed: 1) does effective DREADD-mediated diaphragm activation require focal expression in phrenic motoneurons (vs. non-specific mid-cervical expression), and 2) can this method produce a sustained increase in inspiratory tidal volume? Wild type (C57/bl6) and ChAT-Cre mice received bilateral intraspinal (C4) injections of an adeno-associated virus (AAV) encoding the hM3D(Gq) excitatory DREADD. In wild-type mice, this produced non-specific DREADD expression throughout the mid-cervical ventral horn. In ChAT-Cre mice, a Cre-dependent viral construct was used to drive neuronal DREADD expression in the C4 ventral horn, targeting phrenic motoneurons. Diaphragm EMG was recorded in isoflurane-anesthetized spontaneously breathing mice at 4-9 weeks post-AAV delivery. The DREADD ligand JHU37160 (J60) caused a bilateral, sustained (>1 hour) increase in inspiratory EMG bursting in both groups; the relative increase was greater in ChAT-Cre mice. Additional experiments in ChAT-Cre rats were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume (VT) during spontaneous breathing, assessed using whole-body plethysmography without anesthesia. Three-to-four months after intraspinal (C4) injection of AAV driving Cre-dependent hM3D(Gq) expression, intravenous J60 resulted in a sustained (>30 min) increase in VT. Subsequently, phrenic nerve recordings performed under urethane anesthesia confirmed that J60 evoked a > 200% increase in inspiratory output. We conclude that targeting mid-cervical spinal DREADD expression to the phrenic motoneuron pool enables ligand-induced, sustained increases in phrenic motor output and VT. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.

neuroscience↗