Fig. 7: Engineered lipidation of transcription factors enables loading into EVs and functional delivery to recipient cells.

a Schematic of workflow for evaluating the loading into EVs and functional activity of lipidated synthetic transcription factors (synTFs). Reporter cells express a red fluorescent protein, dsRed-Express2 (dsRedExp2), when activated. b Activity of synTF variants when plasmids were directly transfected into reporter cells. Left, log transform of reporter expression measured by flow cytometry in absolute units of mean molecules of equivalent phycoerythrin-texas red (PE-TR) (MEPTRs). Each symbol is an independent biological replicate, and error bars are the SEM (n = 3). A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (****p < 0.0001). synTFs were compared to the Sol condition; comparisons not shown were not significant. Right, scatter plot for the experiment on the left plotted against band intensity of synTF expression in those cells as measured by a single western blot (n = 1) (Supplementary Fig. 21). c Western blot band intensities of synTFs loaded into EVs, normalized to each individual blot’s PPF condition. Each symbol represents a biologically independent EV preparation and western blot, and the error bars represent SEM (n = 3). The numbers above each bar are the average fold increase of synTF loading compared to the Sol synTF condition. A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (****p < 0.0001). SynTFs were compared to the Sol condition; comparisons not shown were not significant. d Representative micrograph of reporter cells activated via EV-mediated delivery of lipidated synTFs (here, the PM variant). Scale bar is 100 µm. e Flow cytometry dot plots for a representative sample of reporter cells treated with either EVs isolated from Mock-transfected cells (no synTF) or from cells expressing PM-tagged synTF. Annotations state the percentage of reporter cells that expressed dsRedExp2. f Activation of reporter cells treated with synTF-containing EVs (evaluated by flow cytometry). Each symbol represents an independent biological replicate, and error bars represent SEM (n = 3). A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (****p < 0.0001). SynTFs were compared to the Mock condition; comparisons not shown were not significant. Throughout the figure, “Mock” refers to conditions where an empty backbone plasmid was transfected into a cell in lieu of a SynTF-encoding plasmid. Data in (c, e, and f) are representative of two independent experiments. Data in panel (b) were collected from one experiment. Source data are provided as a Source Data file.