Figure 3.

Concentrated EVs transfer functional Cre mRNA in vitro and in vivo

(A) Cre EVs transfer functional Cre mRNA to FLEx reporter cells over time. FLEx reporter cells were incubated with Cre EVs and Nanoluc bioluminescence evaluated in culture medium 24 and 72 h after incubation. Cre activity is represented by bioluminescence signal relative to control (incubated with WT EVs). Data are presented as means ± SEM and compared by unpaired t test. ∗∗∗∗p < 0.0001. (B) Cre EVs transfer functional Cre exRNA to Ai9 cells in a dose-dependent manner. Schematic illustration of Ai9 reporter in which tdTomato expression is prevented by a stop cassette between the promoter and the coding sequence. Removal of the stop cassette by Cre activation results in tdTomato expression. Bar graphs represent tdTomato expression levels evaluated by RT-digital droplet PCR (ddPCR) post-incubation with three different doses of Cre-EVs (2.2, 4.4, and 13.1 × 10⁹ particles) for 72 h. Data are presented as means ± SEM and compared unpaired t test. ∗∗∗∗p < 0.0001. (C) Cre mRNA is functionally delivered to the brain of Ai9 mice. Schematic illustration of Cre EVs intracranially injected in Ai9 reporter mice. Three weeks post-injection, tdTomato mRNA levels in coronal brain sections were evaluated through ddPCR to detect the injection site of Cre EVs (n = 4). Data are presented as tdTomato copies/μL mean ± SEM and compared by one-way ANOVA followed by Tukey’s multiple comparisons test (F = 5.641). ∗p < 0.05; ns, not significant. (D) Cre activity of exogenous EVs in brain. Control EVs (from HEK293T) or Cre EVs injected intracranially into Ai9 mice were compared for Cre activity in the coronal sections at the injection site in the brain. tdTomato expression at the injection site in the striatum of animals were evaluated by ddPCR (control n = 3 and Cre EVs n = 4). Data are presented as mean ± SEM and compared by unpaired t test. ∗∗p < 0.01.