Lentiviral–Vector Mediated Expression of Murine IL–1 Receptor Antagonist Reduces the Severity of Endotoxin–Induced Uveitis

Abstract

Purpose: : In order to determine the extent to which we might be able to ameliorate uveitis by blocking the proinflammatory cytokine, IL–1, we have investigated the effects of murine IL–1 receptor antagonist (IL–1ra) gene delivery in a murine model of uveitis.

Methods: : An HIV–1 based lentiviral vector was constructed that contained a murine IL–1ra cDNA driven by an SFFV promoter (Lenti.SFFV.IL–1ra). ELISA was used to demonstrate efficient production of recombinant protein following in vitro transduction of 293 cells. For the in vivo experiments, 4 µl of Lenti.SFFV.IL–1ra viral suspension was injected through the peripheral cornea under direct vision of an operating microscope in the anterior chamber of the right eye of 8 C57Bl/6 mice. The anterior chamber of the contralateral eye received an injection of 4 µl of Lenti.SFFV.GFP viral suspension. After 14 days both eyes received an intravitreal injection of LPS (1 ng/2 µl PBS) to induce endotoxin–induced uveitis. Twelve hours after the endotoxin injection fluorescein angiography and slit–lamp examination were performed. The eyes were then fixed, frozen in OCT and cut in serial sections using a cryostat. Infiltrating inflammatory cells in the anterior and posterior segments were counted under the microscope on HE stained sections.

Results: : The Lenti.SFFV.IL–1ra treated right eyes showed a significantly lower mean inflammatory cell count in the anterior eye segment (70.7 ± 18.0 vs 201.1 ± 31.4 cells/mm2, p=0.0078) and posterior eye segment (59.8 ± 15.7 vs 183.9 ± 25.3 cells/mm2, p=0.0078) compared with the Lenti.SFFV.GFP injected left eyes. Furthermore, the Lenti.SFFV.IL–1ra treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with the Lenti.SFFV.GFP injected eyes. GFP expression was found in corneal endothelial cells and to a lower extent in cells of the trabecular meshwork.

Conclusions: : Lentiviral murine IL–1ra gene delivery to the anterior chamber significantly reduces the cellular infiltration in the anterior and posterior segments in an experimental uveitis model. These data suggest that anterior chamber lentiviral mediated delivery of immunomodulatory genes offers an opportunity to protect against uveitis.

Keywords: uveitis-clinical/animal model • cytokines/chemokines • gene transfer/gene therapy