Figure 1.

Assessment of promoter-modified vectors in B cells ex vivo

(A) Vector schematic. All vectors were based on first-generation E1/E3 deleted Ads and expressed eGFP from the E1 region. Ad5.CMV is HAdV-C5 with the ubiquitous cytomegalovirus (CMV) promoter. AdRGD contains the RGD4C peptide inserted in the virus fiber knob HI loop region to expand vector tropism toward integrins. A molecular model of this modification was generated using AlphaFold 2.0 and is shown in the inset.³⁷ Model visualization was carried out using UCSF ChimeraX.³⁸ (B) Assessment of promoter-modified vectors in murine B cells ex vivo. Vectors were first screened at 1,000 MOI (left panels), and standout promoters were then assessed at increasing MOIs and vector toxicity was assessed using flow cytometry viability dyes (right panels). For the left panels, six total technical replicates were performed across two separate experiments, except for AdRGD.CMV, which contained five total technical replicates. For the right panel, two replicates were carried out. Statistical analysis was carried out using ordinary one-way ANOVA with Tukey’s multiple comparisons test. (C) Assessment of vectors in human B cells ex vivo. Panels are as described in (B). Two total replicates were performed across two separate experiments. In all cases, eGFP expression is normalized to a PBS mock control. All data are expressed as means ± SD.