Evaluation of 6SHG/EM1 as a treatment for spinal cord injury-induced neuropathic pain in a pig model

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Project Summary: The goal of this research is to validate a novel treatment for neuropathic pain after spinal cord injury (SCI-NP) using a novel and highly clinically-relevant pig model. This research is innovative as we will validate exciting new findings from research in rodents which discovered a highly efficacious treatment for SCI-NP in a clinically- relevant pig model. Specifically, the new therapy involves delivering a gene construct encoding 6 copies of the NMDA antagonist serine-histrogranin and 1 copy of the opioid agonist endomorphin 1 (6SHG/EM1) via intraspinal delivery as detailed in Jergova et al., 2017. Indeed in the study to be validated, rats with the 6SHG/EM1 gene therapy did not exhibit a return of allodynia for the duration of the study (12 weeks). Data also showed that the effects of 6SHG/EM1 were transiently blocked by intrathecal injection of anti-SHG and naloxone, suggesting that the effects were due to "on-target" mechanisms. The expression of the transgenes in the spinal cord was confirmed in neuronal cells near and around the dorsal horn in the vicinity of the injection location (Jergova et al., 2017). To accelerate translation of this new therapy, we have back-translated quantitative sensory testing (QST) methods used in human medicine to evaluate neuropathic pain for use in pigs. In conjunction with QST testing, we have also developed a pig pain scale that includes both reflexive and supraspinal responses, critically important for translation in pain research. We will employ a collaborative approach to test the overarching hypothesis that administration of 6SHG/EM1 after SCI ameliorates SCI-NP in a highly clinically-relevant pig model which closely mimics human SCI. Our goal is to optimize and scale-up this highly efficacious SCI-NP treatment by using a clinically-relevant pig model to define key variables needed for successful translation. We will test our overarching hypothesis and achieve these translational goals by accomplishing three specific aims. In aim 1 we will optimize the methodology for successful delivery of AAV gene therapy to the spinal cord after SCI in a pig model. In aim 2 we will test the hypothesis that post-SCI administration of 6SHG/EM1 in the sub-acute period after SCI will ameliorate SCI-NP in a pig model. The SCI model will be a midthoracic contusion/compression injury in pigs that is well established in our research laboratory and as well as used by others. Baseline quantitative sensory testing (QST) over multiple dermatomes will be conducted. QST includes assessments of thermal (hot and cold), tactile, pressure, and dynamic stimuli. Pain responses will be scored on the porcine evoked pain scale that we developed which includes both supra- spinal (affective) and spinal (reflexive) components. At weeks 6 after SCI (a time point when neuropathic pain is developed), pigs will receive intraspinal injection of 6SHG/EM1 construct in adeno-associated virus (AAV). Motor and QST responses will be evaluated for a subsequent 12 weeks. In aim 2, we will test the hypothesis that post-SCI administration of 6SHG/EM1 in the sub-acute period after SCI induces expression of SHG and EM1 transgenes and peptides in the spinal cord. At 12 weeks after SCI, pigs from aim 2 will be humanely euthanized and spinal cord tissue, dorsal root ganglia, and cerebrospinal fluid extracted. We will assess the presence and expression pattern of SHG and EM1 transgenes and peptides in these tissues using fluorescent- linked immunosorbent assays (FLISA) and immunohistochemical evaluations. Expression patterns will be correlated with pain outcome data. The results of this study are the critical next step in translation of this new therapy to clinical trials, and ultimately to provide relief to Veterans who suffer from neuropathic pain after SCI.