Friday, November 22, 2024
11/22/2024
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 sodium 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 in clinical trials are rapidly cleared from the body, limiting their effectiveness. Alternative approaches are needed. We propose a novel, non-addictive approach to treat chronic pain by editing the messages that encode NaV1.7 in order to alter its ion selectivity. By changing a single lysine codon in the ion selectivity filter to arginine, the Na selective channel will become both Na+ and K+ selective, effectively creating a counter-current shunt that will dampen excitability. Site-Directed RNA Editing (SDRE) refers to novel mechanisms to generate programmed edits within RNAs. It 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, including malignant transformations. In addition, ADARs are endogenous while enzymes for DNA manipulation (e.g., Cas proteins) are not, thus SDRE will not be as immunogenic. Compared to small molecule channel blockers, SDRE can be more specific, because it relies on Watson-Crick base-pairing of gRNAs for targeting, and its effects are likely longer lasting because they will remain 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+. We will generate efficient and specific reagents through an in vitro selection assay, and then test their efficacy in cells, human sensory neurons induced from pluripotent stem cells, and cultured mouse and human DRG neurons. For in vivo testing, we will construct a transgenic mouse model that is specifically designed to test SDRE reagents targeting human NaV1.7 with the goal of ameliorating inflammatory, migraine and neuropathic pain.
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