Figure 1.
siFga controllably decreases plasma concentration of fibrinogen in mice and knocks down human FGA mRNA in vitro. (A-E) Mice were injected with a single dose of siRNA targeting mouse Fga mRNA (Seq-1, Seq-2, and Seq-3) or siLuc as control. (A) Hepatic Fga mRNA levels 1 and 3 weeks postinjection, normalized to a housekeeping gene (Ppia) and graphed relative to Fga mRNA levels in siLuc-treated mice (n = 3). (B) Seq-1-3 all significantly knocked down plasma fibrinogen, measured by ELISA (n = 5; 1 week) and (n = 3; 3 weeks). (C) Representative western blots of fibrinogen and PF4 in platelets from mice 1-week postinjection with siLuc, Seq-1, Seq-2, or Seq-3. (D) Quantifying densitometry of bands in panel C (n = 3). (E) Mice were treated with fresh (red) or frozen (blue) formulation of siFga and plasma fibrinogen was quantified 1 week later by ELISA (n = 5). (F) Mice were treated with 0.1 (red), 0.5 (blue), 1.0 (green), or 2.0 (purple) mg/kg of siFga, and blood was collected retro-orbitally weekly post injection to quantify fibrinogen levels by ELISA. Plasma fibrinogen at each week was compared with blood collected prior to siFga injection (week 0; n = 5). (G) HUH7 cells were transfected with human-specific siFga (hsiFga-1, -2, -3, -4, or -5), or empty LNPs as negative control. FGA mRNA was quantified a day later, normalized to a housekeeping gene (Act; n = 3). *P < .05, **P < .01, ***P < .001. ns, no significant difference. Error bars represent mean ± standard error of the mean (SEM).