Generation of in vitro and in vivo models to study pain perception disorders using genome editing

Several heritable disorders alter an individual's perception of pain. In the present work, two independent models for in vitro and in vivo study of pain-related genes were successfully generated using the CRISPR-Cas system for the exploration of pain mechanisms. First, novel putatively pathogenic mutations in the NTRK1 gene were identified in patients with hereditary sensory and autonomic neuropathy type IV (HSAN-IV). Functional characterisation of the mutations was done using CRISPR-Cas-edited PC12 cells in which Ntrk1 was disrupted. Western blot analysis of cells overexpressing the mutant NTRK1 proteins revealed altered activation of signalling pathways as well as transport defects. Moreover, neurite outgrowth was impaired in Ntrk1-KO PC12 cells overexpressing the mutant proteins, and localisation studies showed altered expression patterns in one of the mutants. Taken together, these results indicate that this cellular system is a valuable tool to investigate the pathogenicity of NTRK1 protein variants and suggests that different perturbations of the downstream signalling cascade can result in loss of nociceptor function. Second, a double-knockout (DKO) mouse model disrupting the genetically linked genes Scn10A and Scn11a, encoding the ion channels NaV1.8 and NaV1.9, respectively, was generated. This mouse model will be used shortly to address the function of these two sodium channels in pain perception. Moreover, DKO-dorsal root ganglion neurones (DRGs) can be used as a cellular model for the functional characterisation of NaV1.8 and NaV1.9 disease-causing protein variants