Analysis of DSG2 expression on HSCs. (A) Flow cytometry for CD46 and DSG2 on human CD34⁺ cells from 2 different healthy G-CSF–mobilized donors. (B) Schematic of HDAd vector capsids and genomes. The chimeric capsids contain either affinity-enhanced Ad35 or Ad3 fiber knob domains. Both vector genomes contain an EF1α-mgmt^p140k/GFP expression cassette used before.²¹  Both proteins are linked via a self-cleaving picornavirus 2A peptide. One set of vectors has an additional CMV promoter–human cxcr4 expression cassette. The 4 vectors used in this study are HDAd5/35++GFP, HDAd5/3+GFP, HDAd5/35++GFP/cxcr4, and HDAd5/3+GFP/cxcr4. (C) In vitro CD34⁺ cell transduction studies. Cells were infected with HDAd5/35++-GFP and HDAd5/3+-GFP virus at a multiplicity of infection of 2000 vp/cell with and without pre-incubation with recombinant soluble CD46 or DSG2 (10 μg/mL) for 1 hour. GFP expression was analyzed 24 hours’ postinfection. (D) CD46 and DSG2 flow cytometry on human and rhesus peripheral RBCs stained with antibodies that recognize the receptors in both species (anti-human CD46 mAb clone M177 from Santa Cruz Biotechnology [Dallas, TX] and anti-human DSG2 polyclonal antibody AF947 from R&D Systems [Minneapolis, MN]). (E) Transduction of 293 cells with HDAd5/35++GFP and HDAd5/3+GFP in the absence and presence of human (huRBCs) and rhesus (rhRBCs) RBCs (details are provided in the Materials and methods). Shown is the percentage of GFP-positive cells measured 2 days after transduction.