Escaping from a dominant‐negative KRAS using REPLACE

A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology. KRAS (S17N) cDNA was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and the resulting RNAs were electroporated into aforementioned host cells. Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. The remaining cells were partially cryopreserved as backup and partially utilized for RNA extraction and mutation analysis. After another 9 d of culture, cells were harvested for total RNA extraction, sequencing, and mutation analysis. As a control, analogous experiments were conducted utilizing wild-type KRAS.