Chronic pain is a leading cause of disability, affecting about one-third of adults worldwide, with a prevalence greater than heart disease, cancer, and diabetes combined. Misuse and abuse of opiates have led to a nationwide addiction and overdose crisis. Thus, there is an urgent need for alternative, non-addictive analgesics. Non-selective voltage-gated sodium channel (NaV) blockers are among existing non-addictive FDA-approved drugs which can sometimes provide symptomatic relief for patients. However, their utility is limited by CNS and cardiac side effects. Genetic and functional studies of human pain disorders and animal models of pain have validated NaV1.7, a voltage-gated Na Channel that is preferentially expressed in peripheral neurons, as an attractive target for therapy. Isoform-selective Nav blockers, however, are difficult to generate and those that have been tested are rapidly cleared from the body, limiting their effectiveness. We propose a novel, non-addictive approach to treat pain by editing mRNA for NaV1.7 in order to alter its electrophysiological properties. By changing a single lysine codon to arginine in the ion selectivity filter, the channel will go from being Na+ selective to both Na+ and K+ selective, effectively creating a counter-current shunt that will dampen excitability. Site-Directed RNA Editing (SDRE) relies on the ADAR (Adenosine Deaminase that Acts on RNA) enzymes, which are endogenously expressed in human cells, including sensory neurons. Directed by a guide RNA (gRNA), SDRE systems convert precisely selected adenosines to inosine, a translational mimic for guanosine, which can recode specific amino acids. For use as an analgesic, editing mRNA is preferable to DNA because it is transient, thus limiting potential off-target effects, and ADARs are endogenous thus SDRE will not be as immunogenic. Compared to small molecule NaV blockers, SDRE can be more specific, because it relies on base-pairing of gRNAs for targeting, and its effects are likely to be longer lasting as long as the edited channels are expressed. We propose to use SDRE to edit NaV1.7 K1395R to render the channel permeable to both Na+ and K+. Work in RC2 will generate the Scn9ahSF mouse producing mNaV1.7hSF so that human-specific gRNA/NDD combinations could be tested in vivo. We will characterize acute sensory thresholds of WT and Scn9ahSF mice and will test their behaviors in the SNI, post-surgical, and headache models used throughout RC5. Recordings of NaV1.7 currents will be conducted on DRG/TG neurons from Scn9ahSF mice compared to WT mice to determine whether replacement of nucleotides influences mNaV1.7hSF gating properties and current density. Experiments will also test the ability of SDRE reagents developed in RC3 to modify the mNaV1.7hSF ion selectivity and excitability of fully mature DRG neurons where the full complement of NaV channels is present. Since these mice will be used in RC5 to test efficacy of the NaV1.7 SDRE reagents, these ele...