Shielding with retargeting reduces off-targeting and increases tumor–liver ratio. a 1.5 × 106 HAdV5HVR7 particles were injected into subcutaneous HER2+ SKOV3.ip tumors in Rag1-/- mice. Gene delivery was analyzed 48 hpi by luciferase activity, normalized to total protein amount. Fiber knob blocking of a shielded virus reduces viral gene delivery to the tumor only slightly compared to virus with free knob. In contrast, HER2 retargeting significantly increases gene delivery within the tumor by a factor of around 40 compared to the fiber knob-blocked virus. Shielding in combination with a blocked fiber knob significantly reduces gene delivery to the liver and all other organs, compared to non-modified virus. Presented data are representative for two independent experiments. b Shielding and retargeting significantly increases the tumor-to-liver ratio of gene delivery from ca. 1300 to ca. 1.1-million-fold, an improvement by a factor of ca. 900. Tumor-to-liver ratios of individual mice from two independent experiments are presented. c Retargeting of a shielded virus increased viral gene delivery to the tumor after intratumoral injection in A431 xenografts. In addition, off-targeting to liver and spleen was reduced. d The ratio of transgene activity between tumor and liver was increased from 300 for the unmodified virus to 9200 for the retargeted and shielded virus. Tumor-to-liver ratios of individual mice from two independent experiments are presented. e Intravenous delivery: in comparison to unmodified virus, shielding of an EGFR-targeted virus decreased the gene delivery to the tumor slightly upon intravenous virus injection, but drastically reduced off-targeting to all organs analyzed. f Systemic biodistribution was changed by the shield. The tumor–liver ratio in the A431 xenograft was increased by more than 2500-fold (each symbol represents (a, c, e) one organ analyzed with two-way ANOVA of log-transformed data or (b, d, f) the ratio of an individual with two-sided, unpaired Welch’s t-test of log-transformed data, *P < 0.05, **P < 0.01, ****P < 0.0001). Background signals from control injections with PBS are indicated by dashed lines