Fig 2. The systemic administration of Cas9/16E7 targeting gRNAs coated in PEGylated liposomes effectively cleared established HPV 16-driven tumours via apoptosis with minimal toxicity.

(A) TC1 (HPV 16 +ve) cells were subcutaneously inoculated in C57BL/6J and allowed to grow to ≈ 50 mm³ before treatments were injected via tail vein (a total of 10 μg of plasmid DNA expressing Cas9 and 16E7 targeting gRNA for treatment groups, or Cas9 and nonspecific gRNA for control, or PBS for untreated group). Injections were administered second daily. First treatment arm (16E7#1) received a total of seven 16E7 treatments, then another four control (Cas9+nonspecific gRNA) injections. The second treatment arm (16E7#2) received a total of eleven 16E7 injections. Tumour volume was measured second daily with digital caliper. The experiment endpoint was tumour volume reaching 1000 mm³. (B) The survival analysis of established TC1 xenografts after 16E7 targeting, with similar experimental groups as in A. (C) Immunohistochemical staining of tumour specimens with H&E staining (upper panel) or cleaved caspase-3 (lower panel) for control (nonspecific) or 16E7 treated mice. (D) The apoptotic cell counts in cleaved caspase-3 stained tumour samples from untreated, control, or 16E7 treated mice. (E) H&E staining of liver and spleen specimens from untreated, control, or 16E7 treated mice. N = 6 per group. Data were represented as mean ± SD. Statistical significance was assessed by ANOVA with post-hoc analysis. * p<0.05, ** p<0.01, *** p<0.001.