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. Overall design: 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.