ELUCIDATING THE ROLE OF P-S10H3 IN THE DEVELOPMENT OF HEAT HYPERALGESIA BY CRISPR/CAS9-MEDIATED GENE EDITING
Based on our previous findings excitatory dynorphinergic (Dyn) dorsal horn neurons play a pivotal role in the response to burn-injury associated tissue injury, via p-S10H3-dependent signaling. We hypothesized that blocking S10H3 phosphorylation in spinal dorsal horn (DH) Dyn neurons of mice would alleviate heat hyperalgaesia in response to burn injury. In order to selectively block S10H3 phosphorylation in DH Dyn neurons we induced mutation of histone 3 (S10A phenotype) by using CRRISPR/cas9 genome editing technology and two transgenic strains (CAG-cas9-EGFP and Dyn-cre). CRISPR elements were introduced into the mouse genome using a recombinant AAV vector (rAAV) as DNA carrier. The rAAV construct (rAAV-mutH3) consisted of the mutant histone H3.1 flanked by loxP sites, three strains of guiding RNA and an mCherry-tag, that is expressed cre-independently so that transfected cells could be visualized. The rAAV-mutH3 vector was administered via intrathecal (IT) or intraspinal (IS) injections targeting lower lumbar spinal segments of cre/cas9 hybrid mice. Differences between delivery routes in transduction efficiency as well as in thermal nociceptive threshold (TNT) were compared. While we successfully detected the mCherry tag of the rAAV-mutH3 virus in some dyn-positive neurons following both administration routes, neither IT nor IS administration was effective to cause alteration in TNT. We conclude that higher titer and/or repetitive application of the virus will be necessary to boost transfection to achieve detectable behavioral effects. Supported by NKFIH (FK_125035), János Bolyai Research Scholarship of the Hungarian Academy of Sciences and the ÚNKP-19-4.