Chronic pain is a common condition estimated to affect around 20% of people worldwide. Current treatments are limited by a lack of long-term efficacy, potentially dangerous side-effects, a significant risk of overdose and the requirement of regular administrations. Chronic pain is sustained by persistent synaptic transmission within nociceptive circuits. Synaptic vesicle endocytosis replenishes vesicle pools required for neurotransmission, but its nociceptor-specific contribution to pain and long-lasting therapeutic potential are unclear. We have identified SV endocytosis in nociceptors as a critical driver of ongoing pain and developed a mRNA-based strategy to target this mechanism.
A lipid nanoparticle-encapsulated CRISPR/dCas9-repressor system (dCas9-R) was employed to achieve sustained and reversible transcriptional and epigenetic repression of synaptic vesicle endocytic mediators following intrathecal delivery. dCas9-R mRNA was packaged into LNPs with sgRNA targeting the promoters of adaptor-associated kinase 1 (AAK1) and dynamin 1 (Dnm1). Multiple preclinical mouse models of acute and chronic pain were utilised to assess the analgesic potential of this strategy, including models of postoperative, inflammatory, neuropathic and osteoarthritic pain.
Endogenous expression of AAK1 and Dnm1 in lumbar dorsal root ganglia (DRG) was reduced by around 50% 48 hours after intrathecal administration of the dCas9-R construct. Administration of dCas9-R targeting AAK1 and Dnm1 eliminated both mechanical allodynia and thermal hyperalgesia throughout the course of a model of postoperative pain (7 days), and induced a significant level of analgesia in an inflammatory pain model (10 days). In longer-term models of neuropathic and osteoarthritic pain, administration of dCas9-R targeting either AAK1 or Dnm1 provided significant pain relief over a period of 28 days, suggesting the potential for this approach to induce chronic analgesia. Importantly, a single administration prevented or reversed pain hypersensitivity for extended durations without impairing normal nociception or locomotor activity.
These findings establish synaptic vesicle endocytic regulation as a mechanism underlying chronic pain and demonstrate the translational potential of LNP-delivered dCas9-R as a durable, non-opioid pain therapy.