654. Recombinant Gene and Cell Therapy With Serine-Histogranin and Endomorphin-1 for the Experimental Treatment of Chronic Neuropathic Pain

Abstract

The insufficient pain relief provided by current pharmacotherapy for chronic neuropathic pain represents a serious medical problem. The development of new treatment strategies is therefore of a high clinical interest. The mechanisms underlying development of chronic pain include decrease in spinal tonic inhibition due to the dysfunction of GABA signaling and increase in excitatory signaling through glutamate NMDA receptors. Previous studies showed beneficial effect of the intraspinal transplantation of GABAergic neuronal precursor cells (NPCs) and of the intraspinal delivery of serine histogranin (SHG), an NMDA receptor antagonist. Moreover, our studies showed that combined injection of SHG with another analgesic peptide, endomorphin-1 (EM-1), potentiated analgesic effect of SHG. Endomorphins are highly selective for μ-opioid receptors and have been shown to decrease neuropathic pain in previous studies.Cells and gene therapy provides suitable tools for targeted delivery of therapeutic peptides thus minimizing adverse effect of systemic delivery. Here we attempted to simultaneously target inhibitory and excitatory pathways in the spinal cord of injured animals by using either recombinant cells or recombinant AAV vectors. Recombinant plasmid encoding single- and multi-SHG and EM-1 was engineered and successful production of recombinant peptides by transduced HEK 293 cells was analyzed by immunocytochemistry and FLISA. AAV vectors were used as carriers of recombinant cDNA and used for either transduction of NPC or for direct spinal injection. Clip compression injury of the spinal cord was use as a model of central neuropathic pain. Animals showing clear sign of pain-related behavior were used for intraspinal injection of either recombinant NPCs or recombinant AAVs. Animals were tested weekly to evaluate analgesic effect of the treatment. Two week post injection animals treated with rAAVs showed significant attenuation of tactile sensitivity compared to controls and the antinociceptive effect sustained up to 6 weeks post injection. Enhanced analgesic effect was observed in the group with 6SHG-EM1 construct compared to 1SHG-EM1. Animals treated with rNPCs did not display significant pain relief compared to nonrecombinant NPCs group. However, these results may be masked by a low survival rate of the transplanted cells. Better strategies for rNPC engineering are currently examined. The presence of SHG and EM (above the levels of controls animals) was detected by FLISA methods and by immunohistochemistry in the spinal cord of treated animals suggesting successful production of recombinant peptides.This study demonstrates that the beneficial effect of combined therapy in the management of chronic pain may be enhanced by using gene engineering tools to deliver therapeutic substances into CNS in a single step and minimize the need for extensive surgical interventions.