✍🏼 Sidharth Tyagi, Mohammad-Reza Ghovanloo, Matthew Alsaloum, Philip Effraim, Grant P. Higerd-Rusli, Fadia Dib-Hajj, Peng Zhao, Shujun Liu, Stephen G. Waxman, Sulayman D. Dib-Hajj
🏠 Medical Scientist Training Program, Yale School of Medicine, New Haven, CT, USA
📑 BioRxiv (2025)
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Abstract
Chronic pain and addiction are a significant global health challenge. Voltage-gated sodium channel NaV1.8, a pivotal driver of pain signaling, is a clinically validated target for the development of novel, non-addictive pain therapeutics. Small molecule inhibitors against NaV1.8 have shown promise in acute pain indications, but large clinical effect sizes have not yet been demonstrated and efficacy in chronic pain indications are lacking.
An alternative strategy to target NaV1.8 channels for analgesia is to reduce the number of channels that are present on nociceptor membranes. We generated a therapeutic heterobifunctional protein, named UbiquiNaV, that contains a NaV1.8-selective binding module and the catalytic subunit of the NEDD4 E3 Ubiquitin ligase. We show that UbiquiNav significantly reduces channel expression in the plasma membrane and reduces NaV1.8 currents in rodent sensory neurons. We demonstrate that UbiquiNaV is selective for NaV1.8 over other NaV isoforms and other components of the sensory neuronal electrogenisome. We then show that UbiquiNaV normalizes the distribution of NaV1.8 protein to distal axons, and that UbiquiNaV normalizes the neuronal hyperexcitability in in vitro models of inflammatory and chemotherapy-induced neuropathic pain. Our results serve as a blueprint for the design of therapeutics that leverage the selective ubiquitination of NaV1.8 channels for analgesia.
How the WOLF was used in this study
In this study, the WOLF G2 Cell Sorter (NanoCellect) was used to fluorescence-activate sort transfected cell populations based on dual fluorescent reporter expression, ensuring precise selection of cells expressing the desired constructs. HEK293 (Expi293F) and ND7/23 cells were transfected with NaV channel constructs linked to eGFP and either UbiquiNaV-mCherry or mCherry, then harvested 48 hours post-transfection. Cells were resuspended in ExCell media supplemented with DNase I and MgCl₂ and sorted on the WOLF G2 using matched sheath and sample fluids to maintain gentle handling and cell integrity. The sorter was used to isolate large, highly pure populations of doubly positive (green and red) cells, with at least 500,000 cells collected per condition. Post-sort inspection confirmed >95% dual-fluorescent purity, ensuring that downstream functional and molecular analyses were performed on well-defined, uniformly transfected cell populations.
