Fig. 4. Activation of tumour suppressor genes in vivo by targeted polymeric delivery of CRISPR/dCas9 variants. (a) MCF-7 luciferase tumours were inoculated at day 0 by subcutaneous injection into the right flank of BALB/c nude mice (N = 11 per group). Mice received a treatment regime of one i.v. injection every 72 h, for a total of 5 injections between day 7 and day 19. Mice were euthanized at day 23 for histological assessment (N = 3 per group), and remaining mice were imaged on days 25, 32 and 39 for tumour burden assessment (N = 8 per group), more mice were sacrificed for histological assessment on day 39 after imaging (N = 3 per group). Final imaging for tumour burden was conducted on day 46, and all remaining mice were sacrificed for histological assessment. (b) Bioluminescence images of mice treated with either dCas9 no effector (control) or dCas9-VPR for the activation of CCN6 from days 25 to 46. (c) Representative ex vivo MCF-7 luciferase tumours excised at day 46 of experiment. (d–f) Tumour growth by luciferase bioluminescence shown as a fold change in size comparing days 25 vs. 32 (d), 25 vs. 39 (e) and 25 vs. 46 (f) respectively. At day 39 MASPIN- and CCN6-treated tumours demonstrated significant tumour growth arrest compared to dCas9 no effector control. At day 46 significant tumour repression was observed in CCN6 tumours. Each point represents an individual animal, while colored points represent animals which remained for the entire experiment. (g) Hematoxylin and eosin stained tumour tissues demonstrating grade 3 MCF-7 tumours for dCas9 no effector and MASPIN, and regressed tumour area for CCN6. Data was analyzed with a Mann–Whitney U test, against the control (*p ≤ 0.05, **p ≤ 0.01, ns p > 0.05).