Rat Na_V1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers

Abstract

Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype-phenotype relationships for drug development. Na V 1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are nonaddictive resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of Na V 1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat Na V 1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized Na V 1.7 rat expressing a chimeric Na V 1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription, the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, Na V 1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia, sciatic nerve, and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter dorsal root ganglia neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric Na V 1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of Na V 1.7 for pain behavior in rats and indicates that sufficient inhibition of Na V 1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global Na V 1.7 knockout mice that require time-intensive hand feeding during early postnatal development.

Quantitative differences in neuronal subpopulations between mouse and human dorsal root ganglia demonstrated with RNAscope in situ hybridization

“…The tissues were sectioned, fixed and dehydrated as described above. Following PAP-pen labeling, IHC was performed as described above but using a knockout-validated [19] mouse monoclonal antibody (mouse-anti-Nav1.7; cloneN68/6; NeuroMab; RRID:AB_10672976; 1:250) and with secondary antibody (goat-anti-mouse IgG1 555; 1:2000).…”

Convergence of peptidergic and non‐peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn

“…According to the manufacturer's datasheet, the antibody recognizes the expected monomeric (230 kDa) band for Nav1.7 on cell lysates of transfected HEK cells and does not cross-react with other Nav channels. The antibody has been knockout-validated using immunocytochemistry on mouse cultured DRG neurons and using IHC on rat brain (Grubinska et al, 2019). We have also recently shown that this antibody robustly stains human DRG and shows a specific and similar expression pattern to its mRNA (Shiers et al, 2020).…”

Quantitative differences in neuronal subpopulations between mouse and human dorsal root ganglia demonstrated with RNAscopein situhybridization

“…The tissues were prepared, fixed and dehydrated as described above. Following PAP-pen labeling, IHC was performed as described above but using a knockout-validated [18] mouse monoclonal antibody (mouse-anti-Nav1.7; cloneN68/6; NeuroMab; RRID:AB_10672976; 1:250) and with secondary antibody (goat-anti-mouse IgG1 555; 1:2000).…”