1013. Complement Component 3 Facilitates Ad5-Mediated Liver Transfection
Authors: Kurt R. Zinn, Tandra R. Chaudhuri, Amanda Stargel, Victor Krasnykh, Alex J. Szalai
Journal: Molecular Therapy (2004)

Replication incompetent adenoviruses (Ad5), now under development as vectors for gene therapy, activate innate immunity and promote inflammation. This evokes cellular and humoral host defense responses, contributing to acute toxicity of Ad5 vectors and ultimately reducing transgene expression in the desired target tissues. To assess the precise contribution of complement in this process, repeated bioluminescence imaging was performed on wildtype versus complement deficient mice after intravenous delivery of Ad5 encoding firefly luciferase (Luc1). Freshly obtained complement sufficient and complement deficient mouse sera were used for in vitro assays. Four animal experiments were conducted (8-10 mice/experiment) using complement component 3 deficient mice (C3−/−) and sex-, age- and strain-matched wildtype controls. Each mouse received i.v. 2.3×109, 4.0×109 or 1.3×1010 viral particles (v.p.) of replication incompetent Ad5 encoding Luc1 (Ad5Luc1; with normal fibers), or 4.0×109 v.p. of fiber-fibritin Ad5 encoding Luc1 (Ad5FFLuc1) which does not support CAR-dependent pathways of infection. Repeated, non-invasive bioluminescence imaging studies (9 total) of mice were conducted for 35 days post-infection. For in vitro studies (n=3), Ad5Luc1 was incubated for 5 min with PBS or with mouse serum (0.1 mL) from wildtype, C−/−, C1q−/−, or factor B deficient (FB−/−) mice, before dilution and 1-hr incubation with adherent mouse liver TIB76 cells. Bioluminescence imaging of TIB76 cells was performed 22 hours later. Overall in C3−/− mice, both short- and long-term hepatic expression of luciferase was significantly dampened compared to wildtype. C3−/− mice showed significantly less (p<0.05) luciferase expression in their liver than treatment-matched wildtype mice when 2.3×109 (Ad5Luc1) and 4.0×109 (Ad5Luc1 or Ad5FFLuc1) v.p. were infused. The maximal difference in luciferase activity between C3−/− and wildtype mice was 99-fold difference at 3 days post-infusion for the 2.3×109 v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0×109 v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0×109 v.p. dose (Ad5FFLuc1). In vitro, preincubation of Ad5Luc1 with C3−/− sera resulted in luciferase levels in the TIB76 cells that were not significantly different from the levels in TIB76 cells treated with Ad5Luc1 preincubated with PBS. However, preincubation of Ad5Luc1 with C3−/− serum resulted in significantly less (p<0.05) luciferase expression (threefold) compared to preincubation with wildtype serum. Remarkably, preincubation with C1q−/− and FB−/− sera achieved intermediate levels of transduction. The combined in vivo and in vitro results suggest that C3, and likely its activation via either a FB- or C1q-dependant pathway, enhances Ad5 mediated transduction of the liver or liver cells. Gene targeting approaches using Ad5 vectors could be improved by recognition of the importance of complement activation in general and C3 in particular. Improved targeting and reduced inflammation are potential benefits.