111. HSV Vectors Retargeted for Infection of TrkA Receptor-Bearing, Pain-Sensing C-Fibers

Abstract

Chronic pain represents a major cause of morbidity and effective pain therapy remains a significant unmet medical need. The standard of care relies primarily on systemic drug therapies that do not target the site of pain sensation. These therapies often have limited effectiveness, deleterious side effects, and ,induce tolerance. Herpes simplex viruses (HSV)-based gene therapy vectors offer an attractive alternative to drug therapy since therapeutic genes can be delivered to sensory nerve afferents where pain is arising. Our goal is to develop a transductionally retargeted HSV vector to selectively deliver therapeutic genes to those neurons activated during chronic pain states. NGF/TRKA signaling mediates the pain response associated with inflammatory hyperalgesia and neuropathic pain conditions, making TrkA-expressing cells an important target for chronic pain gene therapy. To obtain a fully retargeted HSV, the virus attachment/entry component glycoprotein D (gD) can be modified to eliminate recognition of its cognate receptors (HVEM and nectin1) and introduce a new ligand into the N-terminus of gD to allow entry through its corresponding cellular receptor. Therefore, we replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp) NGF to create a TrkA targeting protein, gD:ppNGF(Y38), that can still bind nectin1. Virus expressing gD:ppNGF(Y38) was propagated on cells expressing nectin1 and purified virus was shown to enter J1.1-2, nectin1-deficient cells, only when transduced with TrkA receptor (J/TrkA cells). To enhance the propagation of these vectors on complementing cells, we developed genetic selection methods to isolate retargeted virus variants that display enhanced entry and spread on J/TrkA cells. We found that a selected variant (J4H) had acquired mutations in other HSV envelope glycoproteins, including one glycoprotein involved in envelope-cell fusion events (gH) and two that were previously shown to contribute to virus spread (gE and gI). Moreover, we show that the gH mutation alone, when introduced into the parental virus backbone, enhances entry and minimally improves virus spread. We are currently investigating the specific phenotypes of the individual gE and gI mutations. A fully TrkA-retargeted J4H-based virus (J4HΔ38), further modified to completely eliminate nectin1-binding, also displays enhanced entry and spread on J/TrkA cells. We are currently testing the J4HΔ38 virus in primary sensory neurons in culture and in infections of animals in vivo. We suggest that the TrkA-vector retargeting will provide a means for transduction of pain sensing C-fibers to more precisely introduce pain-relieving gene products.