Ad5/52s fiber substitution alters Ad5 tropism, bioavailability, and plasma sensitivity.

Adenovirus type 5 (Ad5) is a well-established platform for gene transfer but relies on the coxsackie and adenovirus receptor (CAR), limiting efficacy in CAR-deficient tissues and influencing biodistribution through interactions with blood cells. Human adenovirus 52 (Ad52) encodes two fibers: a long CAR-binding fiber (F52L) and a short fiber (F52s) that engages sialylated glycoconjugates. Here, we engineered a chimeric Ad5/52s vector by replacing the Ad5 fiber with F52s. Ad5/52s production was initially inefficient due to delayed replication, but optimization of harvest timing markedly improved infectious titers and particle quality. In vitro, Ad5/52s efficiently transduced different Schwann cell lines and other Ad5-refractory lines. A CAR-independent tropism was demonstrated for Ad5/52s, along with the ability to transduce all tested CAR-positive cell lines. In vivo, systemic delivery revealed strikingly reduced liver tropism and rapid loss of infectivity in circulation, despite comparable genome persistence. Fractionation experiments implicated plasma factors as mediators of Ad5/52s inactivation. Bioinformatic analysis identified a putative thrombin-sensitive cleavage motif within F52s, and mutation (R67N) conferred enhanced viral stability in blood. The Ad5/52s platform highlights both opportunities for targeted local applications and the potential to rationally modulate systemic safety profiles.