527. Reductions in Tissue Transduction and Toxicity after Systemic Administration of Adenovirus Vectors Containing Fiber-Shaft Exchange in Combination with both CAR- and av Integrin-Binding Ablation

Abstract

Adenovirus (Ad) vectors derived from Ad type 5 are widely used in gene therapy and infect a broad range of cells. The development of systemically deliverable Ad vectors that target a specific site requires a significant reduction of this broad tropism. The Ad receptors (coxsackievirus and adenovirus receptor (CAR), |[alpha]|v-integrins, heparin sulfate glycosaminoglycans (HSG)) are the tropism determinants of Ad vectors in vivo. We have developed Ad vectors with ablation of CAR and av integrin and HSG binding by deletion of four amino acids in the FG loop in the fiber knob as well as the RGD motif of the penton base and substitution of the fiber shaft domain for that derived from Ad type 35 (J Virol. 2003, 77, 13062-13072). The fiber shaft of Ad type 35 does not contain the HSG binding motif. We have reported that triple-mutant Ad vectors (Ad-3|[Delta]| (FG)) mediate more than 1,500-fold lower mouse liver transduction by intravenous administration than the conventional Ad vectors. In order to ablate CAR binding, mutation of the AB loop as well as deletion of the FG loop in the fiber knob are widely used. In the present study, we utilized a mutation of the AB loop in the fiber knob for the triple-mutant Ad vectors (Ad-3|[Delta]| (AB)) instead of a deletion of the FG loop, and compared gene transfer activity of Ad-3|[Delta]| (AB) with that of Ad-3|[Delta]| (FG). In the in vitro experiment, Ad-3|[Delta]| (AB) showed 2-log orders lower gene transfer activity than Ad-3|[Delta]| (FG). Ad-3|[Delta]| (AB) and Ad-3|[Delta]| (FG) mediated approximately 0.005% and 0.5%, respectively, of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors. In the in vivo experiment, Ad-3|[Delta]| (AB) mediated approximately 10,000-fold lower mouse liver transduction by intravenous administration than conventional Ad vectors, while Ad-3|[Delta]| (FG) mediated approximately 1,500-fold lower mouse liver transduction by intravenous administration. Interestingly, in the intraperitoneal administration, liver transduction by Ad-3|[Delta]| (FG) was not significantly different than that by conventional Ad vectors. In contrast, Ad-3|[Delta]| (AB) mediated approximately 5,000-fold lower mouse liver transduction by intraperitoneal administration than conventional Ad vectors. Ad-3|[Delta]| (AB) also exhibited lower transduction to heart, lung, kidney, and spleen by both intravenous and intraperitoneal administration. Inclusion of the RGD peptide in the HI loop of the fiber knob of Ad-3|[Delta]| (AB) and Ad-3|[Delta]| (FG) restored gene transfer activity both in vitro and in vivo. We also examined liver serum enzymes (aspartate transferase (AST) and alanine transferase (ALT)) and serum interleukin (IL)-6 levels after administration of each Ad vector. The levels of both AST/ALT and IL-6 in the mouse serum after administration of Ad-3|[Delta]| (AB) and Ad-3|[Delta]| (FG) were similar to those in the non treatment mouse. These results suggest that Ad-3|[Delta]| (AB) exhibits little tropism to specific organs and low toxicity by systemic administration and appears to be a fundamental vector for the targeted Ad vectors.