Fiber shaft extension in combination with HI loop ligands augments infectivity for CAR-negative tumor targets but does not enhance hepatotropism in vivo

Recent studies demonstrate that the fiber shaft length, which ranges from six β-repeats to 23 β-repeats in human adenoviruses (Ads), influences viral tropism. We have previously shown that artificial extension of the shaft length inhibits infectivity in CAR (coxsackievirus and Ad receptor)-positive cell lines, but does not affect infectivity in a CAR-independent, integrin-dependent cell entry pathway. On the basis of these findings, we hypothesized that Ad vectors with shaft extension might display lower infectivity in liver, which expresses high levels of CAR. We also postulated that infectivity of Ad vectors with shaft extension in CAR-negative tumors could be increased by exploiting a CAR-independent cell entry by incorporation of an RGD4C motif into the fiber knob HI-loop. We thus compared gene transfer efficiencies of our Ad serotype 5 (Ad5) capsid-based ‘longer-shafted’ Ad vector with or without an RGD4C motif in the HI-loop of the fiber knob (Ad5long and Ad5RGDlong, 32 β-repeats) to wild-type Ad vector (Ad5, 22 β-repeats) in vitro and in vivo . In this study, Ad5long showed similar infectivity in CAR-negative tumors (69.7%, P = 0.098), but significantly reduced infectivity in CAR-positive tumors (19.1%, P = 0.000038) and in liver (12.5%, P = 0.0047) compared with Ad5. On the other hand, Ad5RGDlong demonstrated similar infectivity in CAR-positive tumors (70.5%, P = 0.012) and in liver (83.4%, P = 0.51), but significantly increased infectivity in CAR-negative tumors (327%, P = 0.0000042) compared with Ad5. Importantly, Ad5RGDlong demonstrated an augmented gene transfer capacity for CAR negative tumors, but no enhanced hepatotropism in vivo . We suggest that Ad vectors with artificial fiber shaft extension in combination with HI loop ligands may be useful for gene therapy applications.