Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina

Objectives: To reevaluate the longevity and intraocular safety of recombina nt adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic im munosuppressants. Methods: An rAd vector carrying green fluorescent protein (GFP) gene was de livered subretinally in the rat eye. The GFP expression was monitored in re al time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in vir us-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymp hocytes, and CD68(+) macrophages, histologic findings, and dark-adapted ele ctroretinography. Two synergistic immunosuppressants, cyclosporine and siro limus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. Results: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coin cident with cell infiltration in virus-contacted area. Immunostaining showe d that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocyte s and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wav e of dark-adapted electroretinogram were observed at 3 to 4 weeks after inj ection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being t he most efficient. The combined immunosuppression attenuated but did not re tard the rAd-induced retinal damage. Conclusions: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sir olimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. Clinical Relevance: The intraocular safety of rAd should be carefully consi dered before clinical trials are performed.