Chemogenetic inhibition of sensory neuron excitability attenuates pain-like behaviours

Thesis

Chronic pain, particularly neuropathic pain and migraine, represents a significant clinical challenge, underpinned by the hyperexcitability of peripheral sensory neurons. Existing therapies offer limited efficacy and substantial side effects, creating an urgent need for novel strategies that can precisely and reversibly modulate pathological neuronal activity. This thesis aimed to develop and validate a chemogenetics-based gene therapy platform for targeted silencing of the peripheral sensory system, offering a new therapeutic avenue for chronic pain.

The research followed a logical progression from foundational validation to translational application. Initial proof-of-concept was established by silencing Nav1.8-positive sensory neurons using the inhibitory chemogenetic tool, GluCl.CreON. This strategy effectively alleviated nociceptive pain while preserving normal tactile sensation, confirming the theoretical viability of subtype-specific analgesia.

However, this success highlighted the limitations of conventional adeno-associated virus (AAV) delivery. To overcome bottlenecks in coverage and invasiveness, we pioneered a neonatal subcutaneous (nSC) injection strategy. This method exploits the transient permeability of the neonatal blood-nerve barrier to achieve simultaneous, efficient (~50% transduction), and stable gene delivery to multi-level dorsal root ganglia (DRG) and the trigeminal ganglia (TG) with a single, minimally invasive injection.

This efficient delivery platform enabled the application of a clinically promising, fully humanised chemogenetic tool, PSAM⁴-GlyR, in a nitroglycerin-induced model of chronic migraine. The combined strategy successfully reversed established mechanical allodynia and orofacial hypersensitivity, providing the first causal evidence for using a humanised chemogenetic tool to treat craniofacial pain.

Finally, to bridge the species translational gap, we performed human-centric validation using sensory neurons derived from human induced pluripotent stem cells (hiPSCs). We systematically evaluated 13 AAV serotypes, revealing a clear trade-off between efficacy and safety. While the engineered AAV-PHP.S was most efficient, it exhibited significant neurotoxicity. In contrast, the natural serotype AAV9 offered a superior safety profile with good transduction efficiency, suggesting a wider therapeutic window.

In summary, this thesis establishes a complete technological pipeline, from tool optimisation and delivery innovation to in vivo validation and preclinical human cell assessment. This work provides a powerful methodological toolkit for sensory neuroscience and lays a robust foundation for developing precise, reversible gene therapies for chronic pain and migraine.

Authors: Hu, H

• DOI: 10.5287/ora-kzvndowqo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: AAV; gene therapy; chronic pain; migraine; chemogenetics
• Subjects: Neurosciences