260. ITRs Necessary for IL-10 Encoding Naked Plasmid Induced Reversal of Chronic Pain State in Rat

Abstract

Controlling chronic pain in humans is a major unresolved problem. Spinal cord astrocytes and microglia are critically involved in the creation and maintenance of diverse enhanced pain states via the release of proinflammatory cytokines. Interluekin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production and activity. Control of chronic neuropathic pain such as sensitivity to light touch (allodynia) requires chronic spinal delivery of IL-10. Chronic pain can be surgically induced in rats by loose ligature of chromic cat gut around the sciatic nerve in four places. This creates a chronic constriction injury (CCI) as the nerve bundle swells against the irritating ligature. The CCI model produces an enhanced pain phenotype for 3 months. We have demonstrated that this pain is reversed transiently (4hrs.) through intrathecal injection of recombinant IL-10 protein and that two temporally spaced injections of an IL-10 expression vector containing inverted terminal repeats (ITRs) reverses this pain long term (3+ months, Milligan et al., these proceedings). The role of ITRs in gene expression is not well known, however there is evidence that they possess transcription promoter and enhancer activity (Flotte et al., 1992 Am J Respir Cell Mol Biol. 7:p349-56 and 1993 J Biol Chem. 268: p3781-90). Based on this and other evidence in the literature we sought to determine if the ITRs in our expression vector, which are located 5|[prime]| of the CMV enhancer and Chicken |[beta]|-actin promoter (5|[prime]| ITR) and 3|[prime]| of the SV 40 poly A tail sequence (3|[prime]| ITR), influence reversal of pain in the CCI model. To this end we deleted both ITR's from the expression vector and found that it was incapable of producing pain reversal. We next asked if one or both ITRs are necessary for pain reversal. We found that plasmids containing just the 3|[prime]| or 5|[prime]| ITR are not as robust in reversing chronic pain. We have also found that ITRs are required on the first injection but not on a second injection for long-term pain relief. In other words, initial injection of a plasmid containing ITRs seems to |[ldquo]|prime|[rdquo]| the system making the second injection of the expression vector vastly more efficacious. Ongoing studies seek to further characterize the role of ITRs on gene expression and pain reversal in the CCI model.