Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles.

Abstract

Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia-responsive element (HRE) sequences within the promoter of target genes. HRE-driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the cytomegalovirus (CMV) and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid-packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions while maintaining tight regulation with no significant changes to cell viability in normoxia.