Targeted delivery of suicide gene to tumor cells using PD-L1-targeted extracellular vesicles for cancer therapy

Herpes simplex virus-thymidine kinase (HSV-TK) gene, a well-known suicide gene, causes cell death in rapidly dividing cells when treated with ganciclovir (GCV). The HSV-TK activates GCV and makes GCV-triphosphate. Insertion of activated GCV-triphosphate in elongating DNA by cellular DNA polymerases interferes DNA duplication and eventually leads to cell death. The suicide gene therapy, however, has been limited by non-specific cytotoxicity in off-target cells such as normal cells. In this study, we explored PD-L1-targeted extracellular vesicles (EVs) carrying the HSV-TK mRNA to deliver the HSV-TK selectively into tumor cells. HEK 293FT cells were transduced using lentivirus with the GFP-tagged, HSV-TK gene containing a sequence that is recognized by EV-trafficking RNA-binding protein. Stable HEK 293FT cells expressing the HSV-TK gene were established. EVs were isolated from the stable cells and labeled with a PD-L1-binding peptide (CVRARTR) using click chemistry to target PD-L1-high tumor cells. Delivery of the HSV-TK mRNA after incubation of cells with the EVs was validated by observing the fluorescence of GFP tag under a microscope. The PD-L1-targeted EVs carrying the HSV-TK mRNA exerted an efficient cytotoxicity in PD-L1-high MDA-MB-231 tumor cells. On the other hand, the EVs showed low levels of cytotoxicity in PD-L1-low tumor cells and normal cells. Systemic administration of the EVs inhibited the growth of MDA-MB-231 tumor xenografts in mice. These results demonstrate that the PD-L1-targeted EVs containing the HSV-TK mRNA provide a new avenue for the targeted delivery of a suicide gene. Keywords: HSV-TK; Peptide; PD-L1; Suicide gene therapy