Helper viral vectors were generated in the human cell line HEK293 as previously described; they originate from the HV plasmids pmCherry, pmCherry-HVR7, and pmCherry-HVR7-ΔRGD.61 HCAdVs were amplified as described in detail by Ehrke-Schulz et al.72 Following three wash steps with PBS, the collected cells from 15 × 15-cm dishes were lysed by three freeze-thaw cycles using a 37°C water bath and liquid nitrogen. The cell lysate was cleared by 8 min of centrifugation at 500 × g, 4°C. The cell supernatant was applied on the first CsCl gradient consisting of two steps, 1.25 and 1.25 g/cm3 CsCl. The remaining cell pellet was washed with 2 mL of PBS, and the supernatant was additionally applied on the first CsCl gradient. The lower viral band of the first CsCl gradient was extracted after 2 h of centrifugation at 12°C, 226,000 × g with a syringe and transferred to a second, four-step CsCl gradient, consisting of four steps ranging from 1.29 to 1.35 g/cm3. The lower-density HCAdV particles formed an upper band and were extracted from the second gradient after 18–24 h of centrifugation at 12°C, 226,000 × g. Following dialysis in dialysis buffer of 20 mM HEPES (pH 8.1), 150 mM NaCl, and 1 mM MgCl2, glycerol was supplied to the viral solution reaching a final concentration of 10% prior to viral storage at −80°C.

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