DeLIVER: Epigenome-Sensitive CRISPR Therapy for Hepatocellular Carcinoma

Objective

CRISPR-Cas technology has revolutionized genome editing, and Cas9-based systems have already enabled successful ex vivo and in vivo gene therapies. We have discovered and engineered a unique Cas9 variant, ThermoCas9, that combines attractive features – compact size, high stability, and exceptional specificity – with an unprecedented property: strict sensitivity to CpG methylation within its PAM recognition site. This property creates therapeutic opportunities that are not achievable with canonical Cas9 enzymes. In hepatocellular carcinoma (HCC), essential genes often become demethylated and transcriptionally active, while the same genes remain methylated and silenced in healthy hepatocytes. By exploiting this difference, ThermoCas9 can selectively disrupt essential genes only in the tumor context, eliminating cancer cells while sparing healthy tissue. We have recently demonstrated methylation-dependent discrimination in biochemical assays and in human cell lines (HEK293, HCT116) (manuscript under review). The DeLIVER project will now provide the first proof-of-concept for a therapeutic application. Specifically, we will test whether demethylated target genes in Huh7 liver cancer cells can be efficiently disrupted via ThermoCas9-mediated InDel formation, leading to selective killing of diseased cells while leaving healthy control cells (iPSC-derived hepatocytes) unaffected. The primary outcome will be in vitro validation of this cancer-selective editing strategy, followed by testing in Huh7-derived xenograft models. In parallel, we will develop a business and IP strategy to enable future commercialization. Our long-term vision is to pioneer a next-generation therapeutic strategy by delivering ThermoCas9 as an mRNA/sgRNA payload via lipid nanoparticles – harnessing tumor-specific methylation signatures to achieve safe, selective, and highly effective treatment of cancer.