Thursday, May 9, 2024
5/9/2024
ABSTRACT Current therapeutics, including the biologics, improve management of arthritic joints but often do not adequately address associated pain, suggesting multiple mechanisms. Delivery of antibodies/immune complexes in serum from a K/BxN mouse to a normal mouse yields a persistent, but reversible joint inflammation accompanied by early onset pain that persists long after resolution of inflammation. In the inflammatory phase, allodynia and the conditioned place preference respond to anti-inflammatory drugs and drugs that block spinal sensitization, while in the post-inflammatory phase, the pain phenotype responds to only to the latter agents. This profile is accompanied by changes in joint innervation, DRG and dorsal horn biology, revealing a transition over weeks in both sexes from an inflammatory to a polyneuropathic pain phenotype. The origin of this ongoing traffic and the appearance of a post-inflammatory pain phase in the preclinical rodent model reflects a major change in the phenotypic expression of channels and receptors within the DRG. Among the changes generated by inflammation and nerve injury are concurrent, time-variant, increases in nociceptive afferent expression of sodium channels (e.g., NaV 1.3,1.7,1.8,1.9) and down regulation of potassium channels (e.g., Kv1.4), changes which yield increased afferent excitability and ectopic activity. Here we will utilize the CRISPR-dCas9 system coupled with transcriptional activation domains or transcriptional repression domains to enable either activation or repression of the above genes epigenetically to systematically study their roles in modulating pain states. We have already extensively characterized the efficacy, duration, and safety profiles of epigenetic repression of NaV1.7 in DRG primary afferents via intrathecal (IT) AAV9 (CRISPR)-dCas9 delivery. Our work has shown, in vitro and in vivo after intrathecal delivery, titer dependent reduction in DRG NaV1.7 mRNA and allodynia in murine inflammatory and polyneuropathic pain models. We propose now, using this IT AAV CRISPR-dCas9 epigenome modifying platform to focus in male and female K/BxN mice on the inflammatory and poly-neuropathic joint pain component of repressing DRG NaV 1.3, 1.7,1.8,1.9 and increasing expression of Kv 1.4-channels. We will characterize these modifications in K/BxN and K/BxN- IT AAV9 (CRISPR)-dCas9 treated male and female mice on i) DRG target expression using RNA seq/RNA-FISH; ii) K/BxN driven allodynia / aversiveness (using conditioned place preference); iii) adverse event profile; iv) ectopic activity and membrane excitability (patch clamping) in primary DRG neuronal cell cultures and v) in vivo basal and evoked afferent substance P release. These aims provide mechanistic insights into the role of. these DRG-afferent channel populations in the inflammatory and post inflammatory arthritic pain phenotype accounting for the ongoing and evoked pain phenotype. These insights into regulation of multiple DRG channels will also guide improved engineering of potential therapeutic interventions.
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