We recently described a non-viral gene therapy paradigm offering long-term resolution of established neuropathic pain in several animal models. Here, the requirements for long-term therapeutic effects are described, and evidence is provided for a mechanism of action based on immunological priming of the intrathecal (i.t.) space. Long-term pain reversal was achieved when two i.t. injections of various naked plasmid DNA doses were separated by 5 h to 3 days. We show that an initial DNA injection, regardless of whether a transgene is included, leads to an accumulation of phagocytic innate immune cells. This accumulation coincides with the time in which subsequent DNA injection efficacy is potentiated. We show the ability of non-coding DNA to induce short-term pain reversal that is dependent on endogenous interleukin-10 (IL-10) signaling. Long-term efficacy requires the inclusion of an IL-10(F129S) transgene in the second injection. Blockade of IL-10, by a neutralizing antibody, either between the two injections or after the second injection induces therapeutic failure. These results show that this gene therapy paradigm uses an initial 'priming' injection of DNA to induce accumulation of phagocytic immune cells, allowing for potentiated efficacy of a subsequent 'therapeutic' DNA injection in a time- and dose-dependent manner.

Neuropathic pain remains a major unresolved problem, necessitating the identification of effective novel therapeutics. Adenosine is a neuromodulator regulating neuronal & non-neuronal cell function, and an immunomodulator acting as an anti-inflammatory agent on immune cells. Adenosine acts on four different subtypes of adenosine receptors, where agents selective for adenosine 2A receptors (A2AR) found on circulating immune cells decrease pro-inflammatory cytokine release & increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Microglia within the spinal cord are the primary resident immune cells & are involved in the induction & maintained production of mediators involved in chronic pain. Therefore, we evaluated A2AR agonists to determine if they reduce neuropathic pain. Sprague-Dawley rats underwent chronic constriction injury (CCI) of the sciatic nerve or sham surgery. Once CCI-induced allodynia was stable (10-14 days after surgery), as tested by von Frey filaments, we injected an A2AR agonist (ATL313) intrathecally. Behavioral testing occurred before and again after injection at 4, 24 & 72h & then weekly for 6wk. A single intrathecal injection of ATL313 produced a remarkably enduring reversal of allodynia for at least four weeks. No dose produced analgesia in sham-operated controls. An A2A antagonist (ZM241385) co-administered with ATL313 (i.t.), in rats with neuropathy-induced allodynia, completely abolished the action of ATL313, but had no effect on the allodynia alone. Neutralizing interleukin-10 (IL-10) IgG had no effect when co-administered intrathecally with ATL313. When the neutralizing IL-10 antibody was administered 1 wk after ATL313, there was a transient reversal of the effects obtained by ATL313 to full allodynia for 2 days after administering neutralizing IL-10 IgG. Activation of A2ARs within the spinal cord may be a novel, non-steroidal therapeutic approach for the treatment of neuropathic pain. (Support: APS Future Leaders in Pain Small Grant, Adenosine Therapeutics Group/PGxHealth (A Division of Clinical Data, Inc.) & NIH Grants DA024044 & DA017670.)