Optimizing loading and expression of HChrR6 mRNA in extracellular vesicles (EVs) for side effect-free prodrug-mediated treatment of HER2+ve breast cancer

Background: Lack of specific targeting and insufficient genetic material delivery has hampered gene-directed enzyme prodrug (GDEPT) therapies. We have developed EXO-DEPT/CNOB regimen that specifically targets and completely arrests the growth orthotopic implanted HER2 +ve tumours in mice. These EVs specifically deliver HchrR mRNA to tumours to generate the HChrR6 enzyme, which converts the prodrug CNOB into cytotoxic MCHB; MCHB can be quantified from its fluorescence. mRNA is superior to DNA for gene delivery, being directly translated upon delivery to the cytosol. To enhance the efficacy of this regimen, enhancement of mHChrR6 loading and expression were undertaken. Methods: Use of plasmids employed previously for loading mRNA can potentially introduce harmful genetic material in the EVs. We have therefore shifted to using in vitro transcribed (IVT) HchrR mRNA to load HEK293FT cells. Cholesterol-Teg-oligos, complementary to the HchrR mRNA coding region, were tested to facilitate loading into the EVs. Functionality was assessed by measuring MCHB fluorescence after CNOB addition; MTT assay measured cell viability. Results: Use of the IVT HchrR6 mRNA instead of the plasmid (XPort/ HChrR6) improved the loading of mHChrR6, decreasing the number of EVs required to deliver one mRNA copy from 5000 to 20. BT474 cells receiving the mRNA from these EXO-DEPTs retained the ability to convert CNOB into MCHB for up to 4 days. Whether this is because of stability of the mRNA or the HChrR6 protein is under investigation. Use of Cholesterol-Teg oligos permitted loading of HChrR6 IVT mRNA in EVs without using transfection reagents; this likely occurred via the endosomal pathway. The latter were able to induce caspase3-mediated cell killing. Summary/conclusion: We improved EXO-DEPT EV engineering by increasing their HchrR mRNA copy number without using plasmids and transfection reagents. Work is in progress to further enhance mRNA loading in the EXO-DEPTs using Cholesterol-Teg oligos complementary to the 3' and 5' mRNA regions. These measures can also stabilize mRNA expression in the recipient cells. Whether the zipcode sequence (believed to facilitate mRNA loading into EVs) we have so far used is essential, and whether stable expression of the mRNA can be enhanced by incorporation of the 3'UTR of Beta-globin mRNA are under investigation.