A customized synthetic gene circuit selectively engages an immune-related transcriptional program in p53-negative LLC cells

Purpose: Cancer immunotherapy has shown promising results in both mouse models and clinical trials. However, to specifically reprogram the immune microenvironment of advanced tumor lesions remains a huge challenge. Therefore, we assembled a synthetic gene circuit aimed at specifically targeting p53-negative LLC cells, but not their p53+ derivatives, for immune activation. Methods: Firstly, we established an LLC-derived knock-in line (p53+), where the WT p53 coding region was re-introduced into the endogenous locus to rescue the bi-allelic point mutations (a nonsense mutation at E32, and a R334P mutation) in the parental LLC cells. Next, both the parental LLC cells and p53+ LLC cells were introduced with the AND-NOT gene circuits which contains the tumor-specific survivin promoter-driven dCas9 (Psuv-dCas9), U6 promoter-driven Ifng-activating sgRNA (or control sgRNA), and p53-responsive promoter PM2-driven AcrIIA4max. 48 hours after transfection, the total RNA was isolated and subjected to RNAseq services. Results: The p53+ LLC cell showed differences of hundreds of genes compared with the parental LLC cells. This group of genes contain a number of classical p53 targets. Notably, the logic circuit drove selective induction of a different gene set in the parental, but not the p53+ cells. Importantly, the vast majority of the circuit-induced genes are known downstream targets of IFN, confirming the specificity of a CRISPRa-driven transcriptional rewiring. Conclusion: Overall, this dataset provides evidence that the AND-NOT gene circuit selectively targets p53-negative LLC cells, but not their p53+ derivatives, and subsequently programs a highly specific immune-rewiring output. Overall design: mRNA profiles of p53-negative LLC cells and their p53+ derivatives which introduced with the customized synthetic gene circuit