Plasmid constructions

The helper viral vector pmCherry-HV plasmid was generated by replacing the natural packaging signal of the pAdEasy-1 vector with a loxP-flanked modified encapsulation signal derived from Sandig et al.35 and a mCherry reporter under the control a PGK promoter. The plasmid pmCherry-HVR7 was derived from pmCherry-HV by inserting four mutations into the hypervariable loop 7 of the hexon (I421G, T423N, E424S and L426Y) as previously described.43 Further deletion of the RGD region of the fiber protein generated the plasmid vector pmCherry-HVR7-ΔRGD. All cloning steps involving helper viral plasmids were carried out by homologous recombination as reported in previous studies.43 The pUniversal plasmids were generated de novo using Gibson assembly47 by combining the pUniversal backbone fragment that encodes the origin of replication and ampicillin resistance derived from the pcDNA3.1(+) plasmid (Invitrogen) with gene fragments containing expression cassettes synthesized by GeneArt (Thermo Fisher Scientific). Each expression cassette consists of a unique pair of promoters and terminators (e.g., SV40 promoter + SV40 terminator, CMV promoter + bovine growth hormone terminator, EF1-α promoter + human growth hormone terminator, or PGK promoter + human β-globin terminator), each with a set of unique restriction sites for payload insertion (e.g., EcoRI and BamHI or EcoRI and XbaI).